To Elucidate Pharmacological Profile of Bioactive Principles of some Himalayan

Medicinal and Aromatic Plants

A Thesis Submitted for the Degree of DOCTOR OF PHILOSOPHY

in

CHEMISTRY KUMAUN UNIVERSITY

2009

by

SANGEETA PILKHWAL M. Pharm

DEPARTMENT OF PHARMACY

KUMAUN UNIVERSITY, NAINITAL UTTARAKHAND

CC EE RR TT II FF II CC AA TT EE

This is to certify that the work incorporated in this thesis

entitled “To Elucidate Pharmacological Profile of Bioactive Principles

of some Himalayan Medicinal and Aromatic Plants” has been carried

out by Mrs. Sangeeta Pilkhwal, under our supervision. She has fulfilled

the requirement of prescribed period for the Degree of Doctor of

Philosophy in Chemistry of Kumaun University, Nainital.

Unless stated otherwise, the work included in this thesis is

original and has not been submitted for any other degree.

DR. KANWALJIT CHOPRA PROF. C. S. MATHELA Co-supervisor Supervisor Head, Pharmacology Division Professor & Head UIPS, Panjab University Department of Chemistry Chandigarh. Kumaun University, Nainital.

DDeeddiiccaatteedd TToo

MMyy FFaammiillyy MMeemmbbeerrss

AACCKKNNOOWWLLEEDDGGEEMMEENNTT

As I sit back to write this little piece, I think of the innumerable hands that

have steered me through this arduous journey and put me on the right path of

learning and enlightment.

At the outset, I deem it my prime duty and obligation to express my

sagacious sense of gratitude and indebtedness to my esteemed teacher and guide

PPrrooffeessssoorr CC.. SS.. MMaatthheellaa,, HHeeaadd,, DDeeppaarrttmmeenntt ooff CChheemmiissttrryy,, for gracious initiation,

valuable guidance, congenial discussion and constant encouragement throughout the

course of this study. My inner conviction does not only allow me to express heartfelt

gratitude but something else which is inexpressible for my co guide Dr.((MMrrss..))

KKaannwwaalljjiitt CChhoopprraa, Head, Pharmacology Division, University Institute of

Pharmaceutical Sciences, Panjab University, Chandigarh. Her inspiration, moral

support and valuable suggestions goes a long way in the successful execution of the

research protocols.

I wish to express my sincerest thanks to PPrrooff.. KKaattaarree,, Chairman, University

Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, for providing

the necessary facilities for pharmacological studies. I am also thankful to PPrrooff.. SS.. KK..

KKuullkkaarrnnii for his generous help and constant encouragement.

Words fail to express my feelings towards my B. Pharm teachers MMrr.. SS..CC..

SShhaarrmmaa who has always been a great source of inspiration for me. I wish to express

my sincere thanks to Prof. Vijay JJuuyyaall aanndd Mr. B.K. Singh, Reader in Department

of Pharmacy for tthheeiirr ccoonnssttaanntt hheellpp aanndd ssuuppppoorrtt dduurriinngg mmyy wwoorrkk

I am elated with delight to express my deep sense of gratitude to all my

seniors and colleagues especially DDrr.. KKuummuudd UUppaaddhhaayyaayy,, DDrr.. AAnniittaa SSiinngghh,, MMrrss..

DDiivvyyaa JJuuyyaall,, MMrrss.. AArrcchhaannaa NNeeggii SSaahh,, MMrr.. MMaahheennddrraa SSiinngghh RRaannaa,, MMrr.. TTiirraatthh

KKuummaarr,, MMrr.. RRaajjeesshhwwaarr KKaammaallkkaanntt who have always lend a helping hand during the

hours of need.

Batch mates and friends are always the pacemakers of life as they polish you

in proper shape of personality. It is my pleasure to acknowledge my special thanks to

my friends and classmates AAnnuurraagg,, MMaahheennddrraa aanndd AAsshhiisshh for their constant

cooperation, day to day help, love, affection and care.

I would like to extend my heartiest gratitude to DDrr.. RRaajjeennddrraa PPaaddaalliiyyaa and

other co-workers for GC-MS screening of the samples and help in chemical

investigations and spectral interpretation.

I am also thankful to Dr. L.S. Rautela, Lab technician, Department of

Pharmacy and others in nonteaching staff MMrr.. GG..CC.. BBhhaatttt, MMrr.. UUmmeesshh,, MMrr..

JJaaggddiisshh,, MMrr.. MMaanniisshh aanndd MMrr.. BBhhuuppeennddrraa for their help and support throughout the

project duration. I also thank, Mr. S.S. Mehra and Mr. Mukesh Chandra in the

library staff for their help and support.

I also express my special thanks to Ms. Maninder Kaur of CPEBS and Mr.

Kiran of Pharmacology division, UIPS, Panjab University, Chandigarh for their

help and cooperation. I would also like to acknowledge the laboratory staff of

Pharmacology division of UIPS, Panjab University for their help and support.

It is beyond my capacity to express my gratitude to my people and my family

who are pillars of my strength. My vocabulary fails in expressing my indebtness to

all my family members for their unending love, inspiration and moral support. I wish

to reciprocate the feeling of thanks to my husband MMrr.. MMuukkeesshh LLaall ssaahh who has

always encouraged me with constant help and valuable suggestions throughout the

study.

I find myself devoid of words in my lexicon to acknowledge my indebtness to

MMuummmmyy,, PPaappaa aanndd mmyy mmootthheerr--iinn--llaaww for their love, affection, inspiration,

unquestionable patience, encouragement and unparallel sacrifice.

I thank all beloved and respected people around me who directly and

indirectly helped me during my degree program whose names could not be mentioned

here.

AApprriill 22000099 SSAANNGGEEEETTAA PPIILLKKHHWWAALL

ABBREVIATIONS

A1, A2A Adenosine receptors

ATP Adenosine triphosphate

BDZs Benzodiazepines

BHA Butylated hydroxyanisole

BHT Butylated hydroxytoluene

CBR Conditioned behavior response

cGMP Cyclic guanosine monophosphate

COX Cyclooxygenase

DCM Dichloromethane

DNA Deoxyribose nucleic acid

DPPH 1,1 diphenyl 2 picryl hydrazyl

ECG Electrocardiogarm

ED50 Median lethal concentration

EEG Electroencephalogram

EPM Elevated plus maze

FST Forced swim test

GABA Gamma amino butyric acid

GC-MS Gas chromatography-Mass spectroscopy

5HT 5 Hydroxytryptamine

GSH Reduced glutathione

H-NMR Proton nuclear magnetic resonance

HPLC High performance liquid chromatography

HPTLC High performance thin layer chromatography

IL Interleukin

iNOS Inducible nitric oxide synthase

IR Infrared

IVHD Isovaleroxy hydroxyl didrovaltrate

LC50 Median lethal concentration

L-NAME N-omega-nitro-l-arginine methyl ester

LPS Lipopolysaccharide

LSD Lysergic acid diethylamine

MDA Malondialdehyde

MES Maximal electro shock seizures

ML Melatonin

MPO Myeloperoxidase

NBT Nitrobluetetrazolium

NF-κB Nuclear factor kappa B

NMDA N-methyl-D –aspartate

NMR Nuclear magnetic resonance

NO Nitric oxide

NOS Nitric oxide synthase

NSAIDs Non steroidal anti-inflammatory drugs

OD Optical density

PMS Post mitochondrial supernatant

PTZ Pentylenetetrazol

Rf Retention factor

ROS Reactive oxygen species

SOD Superoxide dismutase

TLC Thin layer chromatography

CONTENTS Introduction................................................................................... Aims and objectives.................................................................................... CHAPTER 1

Review of literature.................................................................................... CHAPTER 2 Chemical screening of Valeriana wallichii collected from different regions of Kumaun Himalaya

Section A: Isolation and identification of constituents from Valeriana wallichii essential oils 2.1.a. Introduction …………….......................................................... 2.2.a. Material and methods............................................................... 2.3.a. Results..................................................................................... Section B: Isolation and analysis of valepotriates of Valeriana wallichii chemotypes 2.1.b. Introduction………………………………………………………………… 2.2.b Material and methods……………………………………………………… 2.3.b Results…………………………………………………………………………

CHAPTER 3

Psychopharmacological profile of Valeriana wallichii chemotypes

3.1. Introduction …………………………………………………………. 3.2. Material and methods ………………………………………………. 3.3. Results ………………………………………………………………………. 3.4. Discussion ...…………………………………………………………

CHAPTER 4

Screening of Valeriana wallichii chemotypes for antidepressant effect

4.1. Introduction ………………………………………………………… 4.2. Material and methods ……………………………………………… 4.3. Results ……………………………………………………………… 4.4. Discussion …………………………………………………………..

CHAPTER 5

Pharmacological and neurobiochemical evidence for antidepressant like effect of Valeriana wallichii chemotypes

5.1. Introduction ………………………………………………………… 5.2. Material and methods ……………………………………………… 5.3. Results ……………………………………………………………… 5.4. Discussion …………………………………………………………..

CHAPTER 6

Studies on analgesic activity of Valeriana wallichii chemotypes

6.1. Introduction ……………………………………………………… 6.2. Material and methods …………………………………………… 6.3. Procedure ………………………………………………………… 6.4. Results …………………………………………………………… 6.5 Discussion………………………………………………………………..

CHAPTER 7

Evaluation of in vitro antioxidant profile of Valeriana wallichii chemotypes

7.1. Introduction ……………………………………………………… 7.2. Material and methods …………………………………………… 7.3. In vitro antioxidant assays ………………………………………. 7.4. Results …………………………………………………………… 7.5. Discussion ………………………………………………………..

CHAPTER 8

Modulation of antioxidant defense system in mice brain by Valeriana wallichii chemotypes

8.1. Introduction ………………………………………………………. 8.2. Material and methods ……………………………………………. 8.3. Results ……………………………………………………………. 8.4. Discussion …………………………………………………………

Conclusion ……………………………………………………………………. Bibliography ………………………………………………………………….

INTRODUCTION

Medicinal plants are the nature’s gift to human being to make disease free

healthy life. India is one of the most medico culturally diverse country in the world

where the medicinal plant sector is part of a time-honored tradition that is respected

even today. Medicinal plants are believed to be much safer and in our country more

than two thousand medicinal plants are recognized. Chemical and biological diversity

of aromatic and medicinal plants depending on factors, such as cultivation area,

climatic conditions, vegetation phase, genetic modifications and others is an important

impetus to study flora present in different growing sites, countries and geographical

zones. A great number of aromatic, spicy and other plants contain chemical

compounds exhibiting antioxidant and psychopharmacological properties. Numerous

studies were carried out on some of these plants, e.g. rosemary, sage, oregano, which

resulted in a development of natural antioxidant formulations for food, cosmetic and

other applications. However, scientific information on biological properties of various

plants, particularly those that are less widely used in culinary and medicine, is still

rather scarce. Therefore, the assessment of such properties remains an interesting and

useful task, particularly for finding new sources for natural antioxidants, functional

foods and neutraceuticals. Besides this, efficacy of medicinal plants is more evident in

problems related to the nervous system; as stress, anxiety, tension and depression are

intimately connected with most illnesses.

Valerianaceae is a family of about 13 genera and more or less 400 species,

chiefly confined to temperate regions with exception of Andes of South America.

Plants under this genus are mostly herbs or shrubs, annual to perennial; with or

without basal aggregation of leaves. Many members of the Valerianaceae have a

distinctive odor due to the presence of valerianic acid and its derivatives. Several

species have medicinal properties, and root and leaf extracts are used in treating nerve

complaints. A few species are used for perfumes and dyes. The major constituent

present in Valeriana is iridoids. Iridoids are found in many medicinal plants and may

be responsible for some of their pharmaceutical activities. Isolated and purified,

iridoids exhibit a wide range of bioactivities including cardiovascular,

antiphepatotoxic, chlorectic, hypoglycemic, anti-inflammatory, antispasmodic,

antitumor, antiviral, immunomodulator and purgative (Didna et al., 2007). A

considerable number of investigations on Valeriana species have yielded other

compounds like sesquiterpenoids, lignans and alkaloids with pharmacological

properties, including sedative, cytotoxic, antitumor, antioxidant, and vasorelaxant

(Piccinelli et al., 2004; Thies et al., 1981; Bounthanh et al., 1981).

This work, therefore is aimed at evaluating psychopharmacological profile and

preliminary screening of free radical scavenging activities of the extracts and oils

isolated from Valeraina wallichii chemotypes found in Himalayan region.

AIMS AND OBJECTIVES

Herbal medicine is the oldest and most natural form of medicine whose history

of efficacy and safety spans centuries and covers every country on the planet. Because

herbal medicine is holistic medicine, it is, in fact, able to look beyond the symptoms

to the underlying systemic imbalance when skillfully applied by the trained

practitioner. Herbal medicine offers very real and permanent solution to problems,

many of them seemingly intractable to pharmaceutical intervention. The efficacy of

herbal medicine is more evident in problems related to the nervous system as stress,

anxiety, tension and depression are intimately connected with most illness. So there is

great scope for the development of herbal medicine in the area of nervous diseases

and of its application in mental illness. Another area where medicines from herb can

be used is in the treatment of disorders caused by oxidative stress. The majority of

disease conditions like atherosclerosis, hypertension, ischemic diseases, Alzheimer’s

disease, parkinsonism, cancer, diabetes mellitus and inflammatory conditions are

considered to be primarily due to the imbalance between prooxidant and antioxidant

homeostasis. Antioxidant principles from natural resources possess multifacetedness

in their magnitude of activities and provide enormous scope in correcting the

imbalance. Hence, there is no doubt that phytochemicals deserve a proper position in

the therapeutic armamentarium.

Herbal medicine can justifiably boast of Valeriana, the ideal "tranquillizer".

Valeriana is an important medicinal genus in many Pharmacopoeial systems. The

Valeriana wallichii DC. has long been used in Ayurveda (Charak Samhita, Susruta

Samhita) and Unani systems of medicine. The rhizomes of this plant contain volatile

oil (which includes valerianic acid), volatile alkaloids (including chatinine), and

iridoids (valepotriates) which have been shown to reduce anxiety and aggression and

even to counteract the effects of ethanol. Valeriana is effective in treating anxiety

while maintaining normal mental awareness as it enables the patient to continue the

most complicated mental exercise without drowsiness, loss of consciousness or

depression.

Thus the aim of the present study was to compare the

psychoneuropharmacological and antioxidant effects of Valeriana wallichii

chemotypes growing in different areas of Kumaun hills and to correlate it with

interspecific variation in chemical constituents under the following experimental

protocols:

A) Chemical profiling of Valeriana wallichii collected from different regions of

Kumaon:

• Chemical screening for terpenoids.

• Chemical screening for valepotriates.

B) Bioactivity evaluation of Valeriana wallichii extracts and oils:

• Evaluation of psychopharmacological profile.

• Screening of antidepressant effect.

• Pharmacological and neurobiochemical evidence for antidepressant like effect.

• Preliminary studies on the analgesic activity.

• Evaluation of in vitro antioxidant profile.

• Modulation of antioxidant defense system in mice brain.

CHAPTER-1

Review of Literature

1.1. INTRODUCTION

India is one of the 12 mega biodiversity centre having 45,000 plant species

and diversity is unmatched due to 16 different agroclimatic zones, 10 vegetative

zones, and 15 biotic provinces. The country has a rich floral diversity, about 500

plants with medicinal use are mentioned in ancient texts and around 800 plants have

been used in indigenous systems of medicine. Indian subcontinent is a vast repository

of medicinal plants that are used in traditional medical treatments (Chopra et al.,

1956), which also forms a rich source of knowledge. The various indigenous systems

such as Siddha, Ayurveda, Unani and Allopathy use several plant species to treat

different ailments (Rabe and Staden, 1997). In India around 20,000 medicinal plant

species have been recorded recently (Dev, 1997), and more than 500 traditional

communities use about 800 plant species for curing different diseases (Kamboj,

2000). Currently 80% of the world population depends on plant-derived medicine for

the first line of primary health care for human alleviation because it has no side

effects.

In the last century, roughly 121 pharmaceutical products were formulated

based on the traditional knowledge obtained from various sources. The estimation of

total phytomedicine sale reported in country wise European Union was about US $ 6

billion in 1991 and $ 4 billion in 1996, of which almost half were sold in Germany $ 3

billion, in France $ 1.6 billion, in Italy $ 0.6 billion and in Japan $ 1.5 billion. The

present global market is said to be US $ 250 billion. In India the sale of total herbal

products is estimated at $ 1 billion and the export of herbal crude extract is about $ 80

million, of which 50% is contributed by Ayurvedic classical preparations.

A number of scientific investigations have highlighted the importance and the

contribution of many plant families used as medicinal plants and they have played a

vital role for the development of new drugs. Plant derived drugs are used to cure

mental illness, skin diseases, tuberculosis, diabetes, jaundice, hypertension and

cancer. Medicinal plants play an important role in the development of potent

therapeutic agents. Plant derived drugs came into use in the modern medicine through

the uses of plant material as indigenous cure in folklore or traditional systems of

medicine. Determining the biological (activity) properties of plants used in traditional

medicine is helpful to the rural communities and informal settlements. Several

attempts are currently being undertaken to isolate the active compound(s) by

bioassay-guided fractionation from the species that showed high biological activity

during screening. Therefore, these scientific investigations may be utilized to develop

drugs for these diseases. Further research is deserved to isolate the compounds

responsible for the observed biological activity. So the present study also aims at

determining the biological activity of certain plant species found in Himalayan region

with special emphasis on evaluation of psychopharmacological and antioxidant

activity.

1.2. VALERIANA

The genus Valeriana constitutes a group of perennial herbs or undershrubs,

with a short, often strong-smelling rootstock, distributed chiefly in the temperate and

cold region of the northern hemisphere. Several species are however also found in

Andean Chile, and some in Brazil, South Africa, and South and South-east subtropical

Asia. The genus is scarce in the mountain of the tropics and about 12 species occur in

India.

The dried rhizomes and roots of V. jatamansi as well as those of V. officinalis

were official in the earlier editions (pre 1966) of the Pharmacopoeia of India (The

Wealth of India, 2005). In view of pharmaceutical and perfumery value, the Valeriana

species of various global regions have been investigated in the recent years. Three

distinct classes of compounds have been associated with the medicinal properties of

valerian: a) mono- and sesquiterpenes, b) iridoid triesters (valepotriates), and c)

pyridine alkaloids. The composition of the volatile oil varies markedly between

cultivars and species, as does the amount and relative proportion of valepotriates,

making chemical standardization difficult but highly desirable (Moore et al., 2003;

The Wealth of India, 2005).

1.2.1. Historical uses

The valerian oil in large amounts is said to produce dullness of intellect,

drowsiness ending in deep sleep, reduced frequency of pulse and increased urine flow.

It is useful in cases of irregular nervous action, when not connected with

inflammation, or an excited condition of the system. It has also been used in

intermittents, combined with Peruvain bark and in acute rheumatism. It is said that the

virtues of valerian reside chiefly in the volatile oil. By 1923, there was the first

indication that the action of valerian could also act through an odourous pathway

(BPC, 1923). The overall action of valerian rhizome is virtually because of its volatile

oil and valepotriates. It has been used as carminative and antispasmodic in hysteria

and similar nervous manifestations and as the perfect herbal tranquillizer. It was used

for this purpose in the First World War to treat soldiers suffering from shell shock

(Howard, 1987).

1.2.2. Chemical constituents of Valeriana officinalis

The roots of V. officinalis contain several compounds with demonstrable

pharmacological activity. These include the essential oil and its sesquiterpenoids

(valerenic acid), epoxy iridoid esters (valepotriates) and their decomposition products

such as baldrinal and homobaldrinal, amino acids (arginine, GABA, glutamine,

tyrosine) and alkaloids. Valerian also possesses small amounts of phenolic acids,

flavonoids, valerosidatum, chlorogenic acid, caffeic acid, choline, β-sitosterol, fatty

acids, and various minerals (Herbalist, 1999).

1.2.3. Modern uses

Smooth muscle relaxant

Valeriana compounds probably relax stimulated smooth muscle cells by

acting as musculotropic agents and not by interacting with receptors of the autonomic

nervous system (Hazelhoff, 1982). Valeriana officinalis var. latifolia has property to

relieve smooth muscle spasms and is a powerful vasodilator (Yang, 1994). A

preparation of a volatile oil fractionated from the root was used to treat patients with

angina pectoris, among whom ST-T ischemic changes appeared on ECG in 50 cases

before treatment. Its effective rate in simple angina was found to be 87.8% while in

angina with ischemic findings the rate was 88%. The mild myorelaxant action of

valerian is attributed to the valepotriate component of the herb (Dunaev, 1987). The

valepotriates, isovaltrate and valtrate and the essential oil compound valeranone were

observed to suppress the rhythmic contraction in a closed part of the guinea-pig ileum

in vivo (Hazelhoff, 1982). The same compounds and didrovaltrate relaxed potassium

stimulated contractions and inhibited BaCl2 contractions in guinea pig ileum

preparations in vitro. Potassium stimulated smooth muscle cells were also relaxed by

the Valeriana compounds even when autonomic receptors were blocked by

appropriate antagonists thus suggesting that the effects are not mediated through

receptors of the cholinergic or adrenergic nervous system, but rather demonstrated a

direct effect on the muscle tissue. The inhibition of muscle contractions by the valium

chemicals valeranone and didrovalrate were as potent as papaverine (Hazelhoff,

1982).

Neuroprotective effect

V. officinalis was studied against the toxicity induced by amyloid beta peptide

25-35 Abeta. Cultured rat hippocampal neurons were exposed to Abeta (25-35) (25

microM) for 24-48 h. Valerian extract prevented Abeta evoked neuronal injury which

is characterized by a decrease in cell reducing capacity and associated neuronal

degradation. It also partially inhibited ascorbate /iron-induced peroxidation and

inhibited excess influx of Ca2+ following neuronal injury. Neuroprotective properties

of Valerian against Abeta toxicity at long term can contribute to introduction of

Valerian extract to prevent degeneration in aging or neurodegenerative disorders

(Malva et al., 2004).

Sedative effect

Sedative and sleep enhancing effects were found with Valeriana officinalis

and Valeriana wallichii. Studies from around the world have demonstrated that

Tagara taken before bedtime can improve the quality of sleep, reducing the time to

fall asleep, number of awakenings during the night producing minimal or no

drowsiness in the morning and did not affect anterograde memory (Leathwood and

Chauffard, 1982; Leathwood and Chauffard, 1985). Bicuculline antagonized the

inhibitory effects of both the valerian extract and valerenic acid depicting GABAergic

mechanism in sedative effect. Thus valerian may potentiate the effects of anesthetics

that act on GABA receptors and presurgical valerian use may cause a valerian-

anesthetic interaction (Yuan et al., 2004). Recently, it has been suggested that

valepotriates could be useful in improving the condition of animals (Andreatini and

Leite, 1994) and humans (Poyares et al., 2002) during benzodiazepine withdrawal.

The sedative action could be attributed to the essential oil and valepotriate fractions

(Hendricks et al., 1981). Valerenic acid also produced a dose-related increase in

pentobarbital-induced sleep with 50 and 100 mg/kg i.p. (Hendricks et al., 1985).

Hesperidin present in valerian has sedative and sleep enhancing properties but is not a

ligand for BDZ’s (Fernandaz et al., 2004).

Attempts have been made using in vitro assay techniques to delineate the

mechanism of action for the sedative effects of valerian. Several researchers have

linked the effects of valerian extracts and/or its components with an effect on the

inhibitory neurotransmitter GABA (Mennini et al., 1993; Santos et al., 1994a, 1994b).

GABA mediates sedation in the central nervous system and benzodiazepines exert

their actions via this system. Valerenic acid and acetylvalerenic acids have been

reported to inhibit GABA transaminase, thereby prolonging the inhibitory effect of

GABA (Riedel et al., 1982). However, the effect was small and required mM

concentrations. An aqueous extract of valerian, containing 55 mg valerenic acids/100

g extract, was recently shown to displace radiolabeled GABA from its binding sites

on synaptosomes isolated from rat brains (Santos et al., 1994a). Analysis of the

content of the extract for amino acids revealed that the extract itself contained GABA

in sufficient quantity (4.6 mM) to account for the displacement activity (Santos et al.,

1994b) however, it is unlikely to account for the activity of valerian in vivo because

GABA does not cross the intact blood-brain barrier. Analysis also showed that the

extract contained high amounts of glutamine (13 mM) which is able to cross the

blood-brain barrier. The usual concentration of glutamine in the brain extracellular

fluid is in the range of 0.2 to 0.5 mM. Glutamine has been shown in vitro to stimulate

GABA synthesis in synaptosomes and brain slices, and the authors are investigating

whether valerian extracts have any effect on rat brain amino acid levels in vivo.

Anxiolytic effect

Clinical research demonstrates that standardized valerian extract from

Valeriana officinalis effectively relieves anxiety-related insomnia and suggests that

valerian extract may be comparable to some prescription anti-anxiety drugs for

relieving anxiety. Unlike many drugs, however, valerian is not addictive or habit-

forming when taken in recommended doses. SEREDYN a formulation contains the

optimal dose of standardized Valerian extract (minimum 0.8% valerenic acid) to

provide maximum relief without side effects or drowsiness and only SEREDYN

combines valerian with Passion Flower extract and L-theanine to provide a complete

anti-anxiety supplement. Valeriana produces anxiolytic effects via gamma-

aminobutyric acidergic mechanism i.e inhibition of GABA-transaminase, interaction

with GABA/benzodiazepine receptors and interference in uptake and release of

GABA in synaptosomes (Morazzoni and Bombardelli, 1995; Houghton, 1999).

Apigenin derivatives (6 methylapigenin) isolated from Valeriana officinalis

also exhibited medium-high affinity for the benzodiazepine-binding site and has

anxiolytic effects in mice (Fernandaz et al., 2004). In one study the effect of a mixture

of valepotriates was evaluated on the elevated plus maze performance of diazepam

withdrawn rats and both the diazepam and valerian 12 mg/kg reversed the anxiogenic

effect (Andreatini & Leite, 1994).

Antioxidant effect

In one study efficiency of valerian as antioxidant was tested by the influence

of its extract on the yield of photochemiluminescence of Gly-Trp solutions.

Antioxidant property was examined under conditions when its own absorption was

minimized. Riboflavin as additional sensitizer was used for superoxide generation

(Bol’shakova et al., 1997). The aglycones 8 hydroxypinoresinol and prinsepiol were

isolated from valerian and were found to display powerful antioxidant activity.

Anti-inflammatory effect

In traditional European medicine valerian has also been reported as an

antiinflammatory remedy. A study reports that the ethanolic (EtOAc) extract of the

underground parts of V. officinalis showed inhibitory activity against NF-kappaB at

100 µg/mL in the IL-6/Luc assay on HeLa cells and provided protection against

excitotoxicity in primary brain cell cultures at micromolar concentrations. Bioassay-

guided fractionation of the EtOAc extract led to the isolation of three known

sesquiterpenes: acetylvalerenolic acid, valerenic acid and valerenal. The first two

were active as inhibitors of NF-kappaB at a concentration of 100 µg/mL.

Acetylvalerenolic acid reduced NF-kappaB activity to 4%, whereas valerenic acid

reduced NF-kappaB activity to 25% (Jacob- Herrera et al., 2006).

Muscle relaxant action

The essential oil of valerian and the isolated components valerenal, valerenic

acid, valeranone, and isoeugenyl-isovalerate were screened for central nervous system

effects on mice upon intraperitoneal administration (Hendricks et al., 1985). The

essential oil showed muscle relaxant activity with the oxygenated components

exhibiting more activity than the hydrocarbon fraction. Valerenal and valerenic acid

were more active than valeranone, producing ataxia at 50 mg/kg. In a subsequent

study on valerenic acid, the authors reported a decrease in rotorod and traction

performance in mice given 100 mg/kg ip.

Other actions

Fixed valerian-hops extract combination Ze91019 which is used as a sleep aid

was studied for in vitro binding study at 14 subtypes of five classes of system

receptors (dopamine, serotonin, melatonin, MCH and neuropeptide Y). Binding

affinities were demonstrated at some of the screened melatonin (ML1 and ML2) and

serotonin (5-HT4, 5-HT6 and 5-HT7) receptor subtypes. However the nature of affinity

(agonist/antagonist) of Ze91019 to the respective receptors is yet to be determined.

Serotonin (5-HT4, 5-HT6, 5-HT7) receptor has its role in cognitive performance,

depression and sleep disorders. It was found that valerian extract (V. officinalis) has

affinity to ML1 and ML2 receptors (Abourashed et al., 2004; Fauteck et al., 1996). In

other study polar extracts of valerian roots (V. officinalis) activated A1 receptors

(partial agonistic activity) while nonpolar extracts showed antagonistic or inverse

agonistic activity at A1 receptors as demonstrated by GTPγs binding assays at human

recombinant A1 receptors stably expressed in Chinese hamster ovary cells. Isovaltrate

was characterized as a potent, highly efficacious inverse agonist at adenosine A1

receptors (Lacher et al., 2007). Adenosine appears to be one of the main sleep

inducing substances in the brain, which accumulates during wake time, and its

sedative effects may be mediated by both adenosine A1 and A2A receptors. Activation

of adenosine A1 receptors results in sedative, anticonvulsive, analgesic, antidiuretic,

negative inotropic and antiarrhythmic effects (Yan et al., 2003). Selective antagonists

for A2A receptors are promising novel therapeutics for Parkinson’s disease and may

also exhibit neuroprotective and antidepressive activities (Chen, 2003). In one

radioligand binding study it was found that valerian extracts (DCM and petroleum

ether) were found to have strong affinity to 5HT (5a) receptor, and only weak binding

affinity to the 5HT (2b) and the serotonin transporter. 5HT (5a) receptor is located in

suprachiasmatic nucleus of the brain, which is implicated in the sleep-wake cycle.

Petroleum ether extract inhibited [(3) H] lysergic acid diethylamide (LSD) binding to

the human 5-HT (5a) receptor (86% at 50 µg/ml) and DCM extract inhibited LSD

binding by 51%. These results indicate that valerian and valerenic acid are new partial

agonists of the 5HT (5a) receptor (Dietz et al., 2005).

1.3 VALERIANA WALLICHII Synonym: Valeraina jatamansi (Indian valerian)

Hindi- Muskhbala, Tagar Bengal- Mushkbala, Tagar

Kashmir- Mushkbala Punjab- Balamushk, Bala, Mushkwali

Garhwal- Sumaiya Bombay- Tagarganthoda

Kumaon- Samyo

Valeriana wallichii commonly known as Indian valerian is one of the

important plant species of commerce, which belongs to the family Valerianaceae. It is

native to Indian Himalayas.

1.3.1. Distribution

It is found in the temperate zones of Himalayas, from Kashmir to Bhutan at an

altitude of 1300-3300 m, in the Khasia Hills (India) at an altitude of 1300-2000 m,

and in Afghanistan and Pakistan (Bos et al., 1997).

1.3.2. Characteristics

V. wallichii is a perennial, erect herb; 60-100 cm high. The roots are yellowish

brown, 1.5-7 cm long and 1-2 mm thick. The rhizome is yellowish to brownish, 4-7

cm long and 1 cm thick, sub-cylindrical. Leaves are radical, persistent, stalked,

cordate-ovate, acute, toothed. The flowers are white or tinged with pink in a terminal

corymb, 2.5-8 cm across, often unisexual, and the male and female on different plants.

Fruits are small, smooth, without hairs (Kapoor, 1990).

V. jatamansi is abundant in the Western Himalayas whereas V. hardwickii and

V. officinalis are rather scarce. It prefers deep rich soil and flourishes in shady and

moist localities. The plant flowers during April-June and the rhizomes and roots are

dug out in the autumn. They are cleaned with water and dried in the sun on mats, or

sometimes by spreading them in trays, supported over wood-fire. Considerable

quantities of the drug are transported from the hills, especially from Kashmir and

other places in North-West Himalayas, to the plains. Some quantities of this drug are

also imported from Afghanistan. A survey in Pakistan showed that drug is sometimes

adulterated with other miscellaneous rhizomes upto extent of 27%. The dried

rhizomes and roots of V. jatamansi have been recognized as Indian valerian in the

IPC, distinct from those of V. officinalis recognized as valerian.

Dried Indian Valerian

Indian valerian occurs in the market in dull yellowish brown pieces of

rhizome, 4-8cm long × 5-12 mm thick, sub cylindrical, somewhat flattened usually

slightly curved and unbranched, upper surface bearing numerous raised leaf scars and

the under having prominent circular root scars, with a few roots attached; fracture and

horny; taste bitter. The drug according to official specification shall contain ash not

more than 12%, foreign organic matter not more than 12%; and alcohol (60%),

solvent extractive not more than 30%. On steam distillation, the dried rhizomes and

roots of both the forest and cultivated types yield a sweet smelling essential oil. The

yield of the oil from the fresh rhizomes and roots of cultivated types was found to be

higher (1.8%) whereas the dried ones of the forest origin have been found to yield

only 0.5-0.7 %. The physicochemical characteristics of the oils obtained from the

cultivated and wild rhizomes and roots are significantly different (Sood, 1965). The

oil is a pale-brown or amber-yellow coloured liquid with root-like colour with a

distinct note of valeric acid, more or less pronounced according to the age of the oil.

Musk-like and patchouli-like camphoraceous notes are quite characteristic. The oil

distilled from shade-dried roots, not too old, has a finer odour reminiscent of violet

leaf oil. The odor and flavor of Indian valerian oils are considered poor as compared

to those of V. officinalis oils. Solvent extraction of Indian valerian roots by benzene,

petroleum ether or alcohol yields semisolid resinoids which also have a fair demand

because of the relative low price and the ease with which it can blend with other

perfumes.

1.3.3. Chemical constituents

Valepotriates

These compounds are chemically unstable iridoid triesters in which the

various hydroxy groups are esterified with acetic, isovaleric, hydroxyisovaleric, and

α-methylvaleric acids. The valepotriates are divided into the monoene valepotriate

didrovaltrate and several diene valepotriates (valtrate, isovaltrate, homovaltrate, and

acevaltrate). Valtrate is the most abundantly present diene valepotriate alongwith 1-

acevaltrate and dihydrovaltrate in V. wallichii (Bounthanh et al., 1981). The

valepotriates content varies greatly among the species with higher percentage in

underground parts but lower quantities are reported from aerial parts. Because of their

lipophilicity and instability in aqueous solutions, these compounds are present in only

small amounts in commercially available root extracts. Several active degradation

products of valepotriates (baldrinal, 11-ethoxyviburtinal, and homobaldrinal) and an

iridoid ester glycoside designated as valersidatum (isovaleryl glucoside, m.p. 78-

80°C) has been identified. V. wallichii contains IVHD-valtrate, valerosidate (Yu et al.,

2006) and five new iridoids, 1-homoacevaltrate, 1-homoisoacevaltrate, 11-

homohydroxyldihydrovaltrate, 10-acetoxy-1-homovaltrate hydrin, and 10-acetoxy-1-

acevaltrate hydrin, were isolated from the rhizomes and roots of V. jatamansi (Tang et

al., 2002). A new iridoid, 11-methoxyviburtinal (Chen et al., 2005), 8-

methylvalepotriate and 1,5-dihydroxy-3,8-epoxyvalechlorine were also isolated from

the roots of V. wallichii.

O

O

O

H

O

O

CH3CH3

O

O

O

O

O

O

O

O H3C

CH3

H

O

O

CH3

CH2OCOCH2CH(CH3)2 Acevaltrate Didrovaltrate

O

O

O

H

O

CH3

O

CH3

O

O

CH3

O

O

O

O H3C

CH3

HO

O

O

CH3

O

CH3CH3

O

O

O

1-Homoisovaltrate Isovaleroxyhydroxy didrovaltrate (IVHD)

O

O

O

HO

O

CH3

O

CH3

O

O

CH3

O

O

O

O H3C

CH3

H

O

O

CH3

CH2OCOCH2CH(CH3)2

11-Homohydroxyldidrovaltrate Didrovaltrate

O

O

O

O

O

O

O

OH

H3CO

OH

H

O

OH

O

O

H

OH

Cl

O

8-methylvalepotriate 1,5-dihydroxy-3,8-epoxyvalechlorine A

O

CH2O CH3

O

CHO

O

CH2O

O

CHO

CH3

CH3

Baldrinal Homobaldrinal

Volatile oil

The essential oil from root contains calarene, β-bargamotene, α-santalene, α-

curumene, xanthorrhizol, valeranone, curcumene, α, β and γ-patchoulenes, α-

fenchene, patchouli alcohol, maaliol, β-sitosterol, valeranone, maali-oxide, valerenic

acid, isovaleric and β-methylvaleric acids (chief constituents) (Khare, 2007), formic,

propionic, butyric, palmitic acid and stearic acids, and isovaleryl ester of D(-)α-

hydroxyisovaleric acid. The oil also contains valerianian, α-pinene, camphene and

terpineol (Arora & Arora, 1963). From the rhizome, citric acid, malic acid, maliol,

succinic acid and tartaric acid have been isolated (Kapoor, 1990).

A study revealed that the chemical compositions of the oils show two

chemotypes within V. wallichii. The type-I was characterized by presence of maaliol

(64.3%), viridiflorol (7.2%) and sesquiterpene hydrocarbons (19.2%). The type-II

contained patchouli alcohol (40.2%), viridiflorol (5.2%), 8-acetoxy-patchouli alcohol

(4.5%) and sesquiterpene hydrocarbons (34.5%). Viridiflorol and 8-acetoxy-patchouli

alcohol have been isolated from V. wallichii for the first time. Didrovaltrate was

major compound in the dichloromethane extract in both the chemotypes. 11-α-

acevaltrate was present in both varieties but was in greater percentage in V. wallichii

(maaliol type). Isovaleroxyhydroxy didrovaltrate (IVHD) was common in both

varieties. Homoisovaltrate is present in V. wallichii (patchouli alcohol type), and is

absent in V. wallichii (maaliol type) (Sammal, 2005; Mathela et al., 2005a; Mathela et

al., 2005b).

H

OH

H

H

OH

H

H Maaliol Patchouli alcohol

H

COOH

H

O

Valerenic acid Valeranone

H

OHH

OH

Viridiflorol Terpineol

Flavonoids

Two new flavone glycosides, acacetin 7-O- -sophoroside and acacetin 7-O-

(6”-O-α-l-rhamnopyranosyl)-β-sophoroside were isolated from the rhizomes and roots

of V. jatamansi Jones (Tang et al., 2003). Plant contains 6-methylapigenin and

hesperidin (Marder et al., 2003).

OOH

HO O

OH

6-methylapigenin

Other constituents

Rhizomes and roots contain cyclopentapyrans, acacetin-7-O-rutinosides,

linarin iso-valerinate (Thies, 1968), 4-methoxy-8-pentyl-1-naphthoic acid (Pandey

and Shukla, 1993), lignan prinsepiol-4-omicron-beta-D-glucoside, coniferin,

hexacosanic acid, limonene, choline, chatinine, valerianine, actinidine, tannins and

resins. 4-methoxy 8-pentyl-1-napthoic acid and methyl eicosanoate (Pande et al.,

1994), cubenol, caryophyllene oxide, cadinol and aristolene (Dua et al., 2008) are

other constituents isolated from this plant.

N

CH3

H3C

N

CH2CH

H3C

O Actinidine Limonene Valerianine

1.3.4 Characterization of V. wallichii by TLC

a) Under 254 nm UV V. wallichii is characterized by two dominating

bands at Rf 0.37 and 0.53, and the bands at the higher Rf are missing

(Fig. 1).

b) Under visible light, the band at Rf 0.25 in V. wallichii is blue. The

most intense band of V. wallichii is a brown band at Rf 0.53. There is

another intense brown band at Rf 0.37. No bands appear at Rf 0.55 and

0.8 and V. wallichii shows a sharp violet band at Rf 0.48 between two

broad brown bands (Fig. 2), (Herbalist, 1999).

Fig. 1. HPTLC plate viewed under 254 nm UV light

Fig. 2. HPTLC plate viewedafter application of the HCl-acetic acid reagent in visiblelight

1.3.5. Ethnomedical uses

Locally it is being used for medicinal purpose especially for headache and eye

trouble. In Ayurvedic medicine, it is used as aromatic, stimulant, diuretic (Said,

1970), carminative, and antispasmodic. It is also used for the treatment of epilepsy,

hysteria, chorea, shell shock and neurosis (Sharma, 2003). The crushed leaves are

rubbed on the forehead in extreme headache (Bhattacharjee, 1998). Powdered drug,

mixed with sugar is used in urinary troubles. It is useful in jaundice (Awan, 1990),

ulcers, wounds, cardiac debility, dry cough, asthma, chronic and intermittent fever. A

decoction of the drug is reported to be given in Nepal to mother after parturition

probably as sedative. The extract showed antimicrobial effect against Micrococcus

pyogens var. aureus and Entamoeba histolytica. It is also used as an insect repellent.

1.3.6. Other uses

V. wallichii is mainly used in different pharmaceutical or medical

manufacturing for the proper cure of migraine. In India, the dried rhizomes are used in

perfumes, hair preparations and as incense (Bhattacharjee, 1998). The oil is used as

adjunct of certain flavours for tobacco, honey, root-beer types, etc. In perfumery, it

can be employed for blending in high-grade perfumes and as a fixative. The oil finds

use as a tonic and stimulant in certain medicinal preparations.

1.3.7. Actions of chemical constituents

Valerenic acid

• It reduces reactive oxygen species production and enhances the cell viability.

• Valerenic acid inhibits the enzyme responsible for the central catabolism of

GABA (Riedel, 1982).

• Valerenic acid has also been shown to depress CNS activity (Hendricks et al.,

1985).

• It causes activation of K+ ATP channel, thus act as hypotensive.

• It enhances sedative and sleeping properties.

• It causes increase in short-term memory.

• Valerenic acid (VA) antagonized picrotoxin induced convulsions in mice at

12.5 and 25 mg/kg i.p. (Hiller and Zetler, 1996).

• Valerenic acid makes substantial contribution to the sedative and spasmolytic

activity of the essential oil and extract of V. officinalis (Singh et al., 2006).

• The valerenic acids (val. acid, AcO-val. acid) inhibit the enzymatic

inactivation of GABA in the brain by 20-38% (accumulation of GABA may

result in a decreased central activity and increased sedation).

Valepotriates

• The valtrates (especially dihydrovaltrate) have specific affinity for barbiturate

and peripheral benzodiazepine receptors.

• In one study valtrate was found to be a new Rev-transport inhibitor (a HIV-1

viral regulatory protein) with anti-HIV activity. Valtrate also inhibited the p-

24 production of HIV-1 virus without any cytotoxicity against the host MT-4

cells (Murakami et al., 2002).

Glutamime

• It may also contribute to increasing the GABA concentrations, because in the

brain glutamine (which easily passes the blood-brain-barrier) can be converted

into GABA.

Actinidine

• It appears to affect the GABA neurosystem.

Cyclopentapyrans

• It exhibit sedative, tranquillising and bacteriocidal properties.

1.3.8. Pharmacological actions of V. wallichii

Anxiolytic effect

V. wallichii extract is useful in the treatment of human stress related disorders.

It not only significantly reduces stress and anxiety, but also significantly improved

depression and enhanced the willingness to adjustment (Bhattacharyya et al., 2007).

Sedative effect

It has been observed that defatted extract of V. wallichii is much more

effective than aqueous one. The former (100 mg/kg) reduces the motility to about

30% and the animal remains drowsy. 50 and 100 mg/kg of the defatted extract

showed a marked potentiation on thiopental sleep. EEG shows a reduction in voltage

and frequency of the fast waves. The animal is less alert and responsive to pricking.

While neither of the extract was effective against electro- and chemo-convulsion.

Conditioned behavior response (CBR) remains unaffected. Valeriana wallichii +

Humulus lupulus + Passiflora incarnate +Trifolium pretense + Momordica charantia

is a combination used for treating insomnia.

Neuroprotective effect

Bilateral carotid artery occlusion followed by reperfusion produce significant

cerebral infarction and impair short-term memory, motor co-ordination and lateral

push response. In a study done on ischemia-reperfusion induced cerebral injury,

extracts of V. wallichii markedly attenuated cerebral injury in terms of decreased

infarct size, increase in short-term memory, motor incoordination and lateral push

response (Rehni et al., 2007).

Antiprotozoal activity

A root extract of V. jatamansi (code BAL-O) exhibited larvicidal and

adulticidal activity against different mosquito species. The median lethal

concentration (LC50) of BAL-O against larvae of Anopheles stephensi, Anopheles

culicifacies, Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus were 68.1,

42.8, 51.2, 53.8, and 80.6 mg/liter, respectively. The LC50 and the 90% lethal

concentration against adult Anopheles stephensi, Anopheles culicifacies, Anopheles

aegypti, Anopheles albopictus, and Culex quinquefasciatus were 0.14, 0.16, 0.09,

0.08, and 0.17 and 0.24, 0.34, 0.25, 0.21, and 0.28 mg/cm2, respectively (Dua et al.,

2008).

Nematicidal action

In a study, essential oil of V. wallichii exhibited good nematicidal activity

against the pine wood nematode, Bursaphelenchus xylophilus (Kim et al., 2008).

Anxiolytic action

A study was done on V. wallichii, for its role in stress disorders in hospital

based clinical set up. The observations exhibited that, V. wallichii not only

significantly (p < 0.001) attenuated stress and anxiety, but also significantly (p <

0.001) improved depression and also enhanced the willingness to adjustment.

Nevertheless it did not alter memory, concentration or attention of the volunteers. The

results suggest that V. wallichii may be useful in the treatment of stress related

disorders in human and may be a promising anti-stress agent in near future

(Bhattacharyya et al., 2007). Apigenin derivatives (6 methylapigenin) isolated from V.

wallichii and V. officinalis also exhibited medium-high affinity for the

benzodiazepine-binding site and has anxiolytic effects in mice (Fernandaz et al.,

2004). In one study the effect of a mixture of valepotriates was evaluated on the

elevated plus maze performance of diazepam withdrawn rats. Both the diazepam and

valerian 12 mg/kg reversed the anxiogenic effect (Andreatini and Leite, 1994).

Anticonvulsant effect

The water extract (i.p or i.g) of V. jatamansi together with pentobarbital

sodium can antagonize convulsive action induced by thiosemicarbazide. Although it

is ineffective on the convulsion induced by picrotoxin, it can rather prolong the latent

period of convulsion induced by picrotoxin in mice (Cao and Hong, 1994). It was

shown that pure valerenic acid (VA) antagonized picrotoxin induced convulsions in

mice at 12.5 and 25 mg/kg ip (Hiller and Zetler, 1996). VA was assumed to be the

most important active component in Valeriana.

Analgesic effect

There’s a study which depicts that a substance isolated from the rhizomes and

roots of Indian valerian has marked effect in the Haffner test (Schultz and Eckstein,

1962). Dried leaves of V. jatamansi at a dose of 2 mg acts as central analgesic

(Shrivastava and Sisodia, 1970).

Antispasmodic effect

The valepotriates and sesquiterpenes are reported earlier for their

antispasmodic activity (Hazelhoff et al., 1982) and they might have a role in the

relaxant effect of the extract. Spasmolytic effect of the V. wallichii has been used in

different gastrointestinal disorders such as diarrhea and abdominal spasm. Crude

extract of V. wallichii rhizomes (0.1-3 mg/ml) and its fractions caused relaxation of

spontaneous contractions in rabbit jejunum preparations. When tested against high K+

(80 mM)-induced contractions it produced weak inhibitory effect, while caused

complete relaxation of the contractions induced by low K+ (20mM). These results

indicate that the antispasmodic and hypotensive effects of V. wallichii are mediated

possibly through KATP channel activation, which justify its use in gastrointestinal and

cardiovascular disorders (Anwar et al., 2005). 8 hydroxypinoresinol also showed a

higher vasorelaxant activity.

1.4. VALERIANA HIMALAYANA

Synonyms: Valeriana dioica C.B. Clarke, Valeriana pusilla Royle

1.4.1. Distribution

It is distributed in N.W. Asia, Kashmir and the Karakorums and is fairly

common in marshy or moist places, also in shade, among rocks or meadows from

3500-5000 m.

1.4.2. Characteristics

A dioecious or polygamo-monoecious plant, 11-38 cm tall. Rootstock with

slender roots. Scape solitary or 2-3, glabrous, striate. Nodes minutely pilose. Radical

leaves entire, crenulate or subserrate, oblong to suborbiculate, 1-2.5 cm x 0.8-1.6 cm;

nerves prominent; petiole up to 6.5 cm long. Lower cauline leaves pinnati-partite,

with 1-3 lateral lobes, the terminal one largest, ovate-oblong to elliptic-ovate, entire or

slightly toothed; the uppermost cauline leaves with narrower segments. Flowers in

dichotomous corymbs or compact heads, pink or white. Upper bracts lanceolate, 4

mm long, acute or obtuse. Ovary glabrous; stigma 2-3-fid. Achene 2.5-3 mm long,

glabrous, shorter than the upper bracts; pappus segments 10-13. Flowering period is

from June-August. The female flower can be recognized by its smaller size and 2-3-

fid stigma; the male flowers are broader with more or less included anthers.

1.4.3. Uses

Roots of this plant are used for psychiatric problems, for increasing memory,

and in cough, asthma, paralysis and nerve problems (Khan and Khatoon, 2008).

1.4.4. Chemical constituents

It contains maaliol, valeranone, kessane and α-kessyl acetate as major

compounds (Mathela et al., 2005b).

1.5. VALERIANA PYROLAEFOLIA. DECNE

1.5.1. Distribution

It is found in the temperate Himalayas from Kashmir to Bhutan.

1.5.2. Characteristics

Valeriana pyrolaefolia Decne is closely related to V. jatamansi in morphology

and both these species occur more or less interspersed. A preliminary chemical

investigation of the former species has indicated that it may form a substitute for the

valerian. It is therefore not unlikely that the rhizomes and roots of both the species

may be mixed at the time of collection of the drug. Pharmacognostic study of V.

pyrolaefolia has, however, shown that it has smaller rhizome than that of V.

jatamansi. V. pyrolaefolia can also be differentiated from the latter species by

differences in the dimensions of cells in various tissues (The Wealth of India, 2005).

V. pyrolaefolia is a less known Himalayan species within the genus Valeriana. The V.

pyrolaefolia is a 5-25 cm sub-succulent herb that grows along waysides, hilly slopes

and shrubberies at an altitudinal range of 2,100-3,200 m. The distinguishing

morphological feature of the herb V. pyrolaefolia from V. wallichii is in having 1-3

pairs of stalkless broadly ovate entire stem leaves, white flushed pink flowers, hairless

fruits and other characteristics like the dimension of the cells in various tissues. The

roots are highly aromatic and the fresh leaves have a strong odor though weaker than

that of the roots. No popular uses are referred to in the literature examined, but it is

mentioned that based on closely related morphology with V. wallichii, V.

pyrolaefolia is also collected along with V. wallichii for commercial purpose and has

been reported as a substitute for V. wallichii (Prakash, 1999).

1.5.2. Chemical constituents

The chemical composition of root and leaf oils of V. pyrolaefolia Decne. was

determined by GC and GC/MS, and 45 components were identified. Both oils were

rich in sesquiterpenoids and characterized by high contents of valeranone (31.8-

37.9%) and patchouli alcohol (8.6-12.9%). The leaf oil composition showed

remarkable presence of a non-terpenoid, 2,6-dimethoxyphenol (19.8%), among the

major compounds. The presence of valeranone and patchouli alcohol suggests that it

has commercial potential (Mathela et al, 2005b).

O

O

OH

2, 6 dimethoxyphenol

1.6. VALERIANA HARDWICKII

Hindi - Tagger, Shumeo Bengali- Tagger, Bal, Chur, Ushusr, Saru tagar

Punjab-Taggar, Balachar Kumaun- Asarun, Shumeo

Lepcha- Chammaha Bombay- Tagger-genthoda

1.6.1. Distribution

It is found from Easren Asia - China to the Himalayas. Usually found amongst

herbaceous vegetation on humus-rich soils, 1900 - 3100 metres in Kashmir, Bhutan

(Singh and Kachroo, 1976). Also found in the Khasi and Jaintia hills between 1500

and 1800 m.

1.6.2. Characteristics

The herb is common along road sides, in shady areas of forests, hilly slopes

and flowers during rainy season. An erect herb, 0.3-1.6 m high. Rootstocks are

descending. Radical leaves long petiolate, ovate, soon disappearing, cauline leaves

opposite, odd-pinnate and very deeply pinntified; leaflets 3-9, ovate to oblong-

lnaceolate usually entire, terminal segments largest, petiolate, lateral ones subsessile.

Flowers in compound corymbs forming a terminal lax panicle corolla white about 1.5-

2mm long. The flowers are dioecious (individual flowers are either male or female,

but only one sex is to be found on any one plant so both male and female plants must

be grown if seed is required) and are pollinated by insects. Fruit ovate-oblong,

compressed with 3-dorsal, 1 ventral and 2 marginal ribs, hairy on one side or both

crowned with long, plumose calycinal bristles (Pandey, 1995). The plant prefers light

(sandy), medium (loamy) and heavy (clay) soils and requires moist soil. Taxonomists

have classified two distinct varieties of Valeriana hardwickii viz. V. hardwickii var.

hardwickii and Valeriana hardwickii var. arnottiana. Both these subspecies occur in

the Kumaun (Uttarakhand) in higher altitude region (8,000- 10,000 ft).

The rhizomes and roots of V. hardwickii possess more or less the same

properties as those of V. jatamansi and V. officinalis and are, therefore a good

substitute for the drug valerian. Their tincture also has shown to be equivalent in

pharmacological activity to that of valerian. The rhizomes and roots of V. hardwickii

are transported to the plains and not infrequently they are mixed with the official drug

valerian. Besides the use in medicine they are also employed as an incense and to

scent the hair.

1.6.3. Medicinal Uses

Used as V. jatamansi and V. officinalis. The root is bitter, carminative,

diuretic, expectorant, nervine and stimulant (Usher, 1974) It is used as a nerve tonic

and in the treatment of conditions such as epilepsy and hysteria. It is also used in the

treatment of rheumatism and low blood pressure. The pounded root or leaves are used

as a poultice to treat boils. The plant is antispasmodic, aphrodisiac, emmenagogue,

diaphoretic and stimulant (Duke and Ayensu, 1985). This plant is an effective

substitute for V. officinalis (Chopra et al., 1986).

1.6.4 Chemical constituents

Roots contain a crystalline acid, α-eudesmol, edemo, angelicin etc (Sharma,

2003). Epoxysesquithujene, a new sesquiterpene epoxide has been characterized in

the essential oil of Valeriana hardwickii var. hardwickii on the basis of chemical

reactions and extensive NMR data (Mathela et al., 2007). In case of V. hardwickii var

hardwickii total analysis revealed that the sesquiterpenoids constitute 75.1% out of

which 18.19 % are the sesquiterpene hydrocarbons and 56.91% as oxygenated

sesquiterpenes while monoterpenoids are 19.77 %, out of which monoterpene

hydrocarbons constitute 1.9% and oxygenated are 17.87%. Alpha-agarofuran

(46.52%), bornyacetate (14.46%), 1,7-di-epi alpha-cedrene (8.62%) and alpha-

gurjunene (2.45%) are among the major compounds identified. While in case of V.

hardwickii var arnotianna the analysis of oil revealed dominance in oxygenated

sesquiterpenoids (50.95%) followed by sesquiterpene hydrocarbons (22.43%),

oxygenated monoterpenes (16.35%) and monoterpene hydrocarbons (2.43%). The

chief components of the oil are valeracetate (17.26%), bornyl acetate (15.25%) methyl

linoleate (11.73%), cuprarene (10.37 %) and alpha-cedrene (6.22%) (Sati, 2002; Sati

and Mathela, 2004). Most interesting feature in the composition of V. hardwickii var

arnotianna is the presence of sesquiterpene ester, valeracetate which is used for

sedation and antispasmodic purposes.

V. hardwickii var. arnottiana was found to exist as two independent

chemotype i.e. type I contains α-kessyl acetate, valeracetate and 8-epi-kessylglycol

diacetate whereas chemotype-II contains maaliol and kessyl acetate as major

constituents.

O

H

H

AcO

O

H

H

OAc

α-kessyl acetate Valeracetate

O

H

H

AcO

OAc

O 8-epi-kessyl glycol diacetate Epoxysesquithujene

OO

H

OH

H

H Bornyl acetate Maaliol

1.6.5. Uses

It is used as insecticide for scenting hair and is useful in mental disorder,

scenting hair (Sharma, 2003).

Chemosystematic markers within the genus Valeriana

S. No. Name of the species Major/characterstic compounds

1. Valeriana wallichii chemotype I Maaliol, virdifloral

2. Valeriana wallichii chemotype II Patchouli alcohol, 8-acetoxy-patchouli

alcohol, virdifloral

3. Valeriana pyrolaefolia Patchouli alcohol, valeranone

S. No. Name of the species Major/characterstic compounds

4. Valeriana himalayana Valeranone , maaliol, α-kessyl acetate

5. Valeriana hardwickii var.

arnottiana chemotype I

Valeracetate, 8-epikessyl glycol diacetate,

α-kessyl acetate

6. Valeriana hardwickii var.

arnottiana chemotype II

Maaliol, kessanyl acetate

7. Valeriana hardwickii var.

hardwickii

Bornyl acetate

1.7. BIOACTIVITY EVALUATION

The extracts (steam distilled oils and solvent extracts) from above plant

species have been studied for biological activity with major emphasis on following

activities.

1.7.1. Analgesic activity

Analgesics are the drugs which increase the threshold for pain either by acting

on opioid receptors (e.g morphine) or by inhibiting the synthesis of prostaglandins

(aspirin) which are responsible for pain production. Painful reactions in animals can

be produced by intraperitoneal injection of chemicals like phenylquinone, bradykinin

or acetic acid and also by thermal method (application of heat).

1.7.2 Antiepileptic activity

Epilepsy is a heterogeneous symptom complex- a chronic disorder

characterized by recurrent seizures. Seizures are finite episodes of brain dysfunction

resulting from abnormal discharge of cerebral neurons. The causes of seizures are

many and include full range of neurologic diseases, from infection to neoplasm and

head injury. Experimentally epilepsy can be produced in mice by electroshock, by

administration of chemicals like pentylenetetrazole, strychinine and kainic acid.

1.7.3. Anxiolytic activity

Anxiety is a physiological and psychological state characterized by cognitive,

somatic, emotional, and behavioral components (Seligman et al., 2001). These

components combine to create an uncomfortable feeling that is typically associated

with uneasiness, apprehension, or worry. The elevated plus-maze is commonly used

to assess anxiety-like behaviour in laboratory animals (rats/mice). The maze is usually

a cross shaped maze with two open arms and two closed arms, which is elevated

above the floor.

This task exploits the conflict between the innate fear that rodents have of

open areas versus their desire to explore novel environments. Security is provided by

the closed arms whereas the open arms offer exploratory value. When anxious, the

natural tendency of rodents is to prefer enclosed dark spaces to opened brightly lit

spaces. In this context, anxiety-related behaviour is measured by the degree to which

the rodent avoids the unenclosed arms of the maze.

1.7.4. Antidepressant activity

Major depression is one of the most common psychiatric disorders. Despite

intensive research, the mechanisms of action of various pharmacologic treatments are

still not understood, though most are believed to involve effects on two monoamine

neurotransmitters: serotonin and norepinephrine. Antidepressant activity of a drug can

be evaluated using forced swim test in mice.

1.7.5. Antioxidant activity

Living systems have evolved to survive in the presence of molecular oxygen

and for most biological systems; life depends upon its presence. However, oxygen has

double-edged properties i.e. it is essential for life, but it can also provoke damaging

oxidative events within cells. Free radicals are highly reactive substances with one or

more unpaired electrons in its outer orbital and are formed in the body's cells as a

result of metabolic processes (Niki, 1992; Niki, 2001). Free radicals react rapidly with

adjacent molecules via a variety of reactions including: hydrogen abstraction

(capturing), electron donation and electron sharing (McCord, 2000). Reactive oxygen

species (ROS) are found intracellularly and extracellularly and may be produced

endogenously or arise from exogenous sources, i.e. taken in from the environment

(Sies and Cadenas, 1985; Sies, 1997). Important sources of endogenous free radicals

include prooxidative enzyme systems (e.g. lipoxygenase), drugs and their metabolites,

pollutants, and other chemicals and toxins (Halliwell, 1996; Spiteller, 2001). External

sources such as sunlight and other forms of radiation can generate endogenous ROS,

which can lead to a number of diseases (Halliwell, 1996; Stief, 2003). ROS can also

be formed in food through lipid oxidation and photosensitizers exposed to light (Fang

et al., 2002). ROS can be classified into oxygen-centered radicals such as superoxide

anion (O2•−), hydroxyl radical (OH•), alkoxyl radical (RO•), peroxyl radical (ROO•)

and oxygen-centered nonradical derivatives such as hydrogen peroxide (H2O2) and

singlet oxygen (1O2). Other common reactive species are nitrogen species such as

nitric oxide (NO•), nitric dioxide (NO2•), and peroxynitrite (OONO –).

ROS cause lipid oxidation, protein oxidation, DNA strand breaks, and

modulation of gene expression (Evans and Halliwell, 1999; Halliwell, 2000). ROS are

involved in many diseases such as atherosclerosis, cancer, stroke, asthma, arthritis and

other age related diseases. (Halliwell et al., 1992). Because antioxidant defense in the

human body is not completely efficient, increased free radical formation may produce

a continuous level of oxidative damage. So there is a continuous need of antioxidants

from outside source to prevent oxidative stress which refers to a severe disturbance in

the prooxidant-antioxidant balance in favor of the prooxidant, leading to potential

damage. Primary antioxidants most often act by donating a hydrogen atom, while

secondary antioxidants may act by binding metal ions able to catalyse oxidative

processes, by scavenging oxygen, by absorbing UV radiation, by inhibiting enzymes

or by decomposing hydroperoxides (Schwarz et al., 2001). It is known that different

natural phenolic compounds function as both primary and secondary antioxidants by

different mechanisms. Monitoring of either the decrease of the radical or the

antioxidant, or the formation of products can be used for assessing the antioxidant

activity (Decker et al., 2005).

CHAPTER-2

Chemical Screening of Valeriana wallichii Collected from Different Regions of Kumaun

Himalaya

Section A

Isolation and Identification of Constituents from Valeriana wallichii

Essential oils

2.1. a. INTRODUCTION

Valeriana wallichii has long been used in Ayurveda and Unani systems of

medicine (Gupta and Shah, 1981). Roots of V. wallichii are aromatic, possess sedative

activity and are useful in hysteria, epilepsy and neurosis (Kapoor, 1990). The roots of

V. wallichii have been investigated for terpenoids and valepotriates with α-pinene, β-

pinene, camphene and β-cymene as the prinicipal monoterpene hydrocarbons in the

essential oil (Bos et al., 1997). Major sesquiterpenoids reported in the essential oil

V.wallichii are α-curcumene, β-farnesene, α patchoulene, β-patchoulene,

cryptomeridiol, konokonol, maaliol, xanthrorizol and patchouli alcohol (Naryanan et

al., 1964). Present study deals with chemical screening of V. wallichii natural samples

for comparison of their constituents.

2.2. a. MATERIAL AND METHODS

2.2.1. a. Plant material and its identification

V. wallichii (Voucher specimen No. Chem/DST/V.II) and (Voucher specimen

No. Chem/DST/V.01) were collected from Kumaun region during August to October.

The plant materials were identified from Botanical Survey of India, Dehradun and

were coded as VW-I (Voucher specimen No. Chem/DST/V.01) and VW-II (Voucher

specimen No. Chem/DST/V.II) .

2.2.2. a. Extraction of essential oil

The collected plant material was first freed from other mixings and washed

with a stream of water. The fresh plant material (roots 2 kg) each time was subjected

to steam distillation in a copper electric still fitted with spiral glass condensers. The

distillate was saturated with NaCl and the oil was extracted with n-hexane and

dichloromethane. The organic phase was dried over anhydrous Na2SO4 and the

solvent was evaporated under reduced pressure in a thin film rotatory evaporator at

30°C. The oil yield was 0.60% (v/w) for plant VW-II and 0.16% for VW-I. The oil

obtained from VW-II was coded as V-PA and oil isolated from VW-I was coded as

V-MA.

Valeriana wallichii essential oil

(4.0 g)

Column chromatography (Silica gel)

n-hexane 5% Et2O in 10% Et2O in 20% Et2O in 30% Et2O in EtOAC n-hexane n-hexane n-hexane n-hexane

A B C D E Hydrocarbon mixture (1.0 g)

Scheme 2.1.a: Isolation of components from V. wallichii (VW-II) root oil

Valeriana wallichii essential oil

(4.0 g)

Et2O: n-hexane (5% to 10%)

Et2O: n-hexane (10% to 20%)

VW-IIC VW-IID 210 mg 180 mg

n-hexane 5% Et2O in 10 n-hexane

A B C (1 Hydrocarbon Et2O: n-hexane Emixture (1.0 g) (5% to 10%) (

VW-IB VW-IC

(50 mg) (80 m

Scheme 2.2.a: Isolation of compon

Column chromatography (Silica gel)

% Et2O in 30% Et2O in EtOAC n-hexane n-hexane

.0 g) D (0.2 g) E

t2O: n-hexane 8% to 20%)

g)

ents from V. wallichii (VW-I) root oil

2.2.3.a. GC-MS analysis

GC-MS was done using fused silica capillary column (30m x 0.25mm) liquid

phase DB-5 with helium as a carrier gas in Thermoquest Trace GC 2000 interfaced

with Finnigan MAT Polaris Q mass spectrometer. The column temperature was

programmed at 3°/min from 60° to 210 °C. The mass spectra corresponding to GC

peaks were scanned at 70 eV under EI conditions.

2.2.4.a. Isolation of compounds

The oil isolated from the roots of plant VW-II was chromatographed over

silica gel (75 g, 230-400 mesh, Merck). The column was eluted with n-hexane

followed by a mixture of n-hexane: ether (5% to 30% ether in n-hexane) and finally

washed with ethyl acetate. Similar fractions were mixed to give a total of five

workable fractions among several others. The fractions were concentrated and

examined by TLC, IR, GC and GC-MS under isothermal and column temperature

programmed conditions. They were subjected to repeated column chromatography to

isolate pure compounds.

VW-IIC was isolated from fraction C (5-10 % Et2O in hexane) by repeated

column chromatography and VW-IID from Fraction D with 10-20 % Et20 in hexane

(Scheme 2.1.a). Similar procedure was repeated for sample VW-I. Repeated column

chromatography of the fractions gave compound VW-IB and VW-IC (Scheme 2.2.a)

2.3.a. RESULTS

2.3.1.a. GC- MS screening of the essential oil of Valeriana wallichii (VW-II)

The essential oil of the Valeriana wallichii (VW-II) was analysed using GC-

MS. The aim was to identify minor constituents which could not be isolated in the

pure form. The constituents were identified using NIST Library Search Programme

and compared with literature reports. The gas chromatogram showed the presence of

nearly 28 peaks of which 20 have been identified (Fig. 2.1.a). The results of the

analysis are summarized in Table 2.1.a. The total identified constituents comprise

approximately 95% of the total, which is dominated by sesquiterpenoids followed by

monoterpenes. V. wallichii possesses patchouli alcohol (30.72%) as the major

constituent followed by the presence of δ-guaiene (10.69%), seychellene (10.29%),

acetoxyl patchouli alcohol (10.45%), α-guaiene (4.87%), α-humulene (4.22%) and α-

patchoulene (4.28%). Among minor constituents are kessane, δ-selinene,

caryophyllene, bornyl acetate and valencene. The major oxygenated sesquiterpenes in

the previous reports are cryptomendiol, kanokonol, maaliol, xanthrorhizol and

patchouli alcohol but except for patchouli alcohol, other oxygenated sesquiterpenoids

were found to be totally absent in V. wallichii material under investigation.

Fig. 2.1.a. Gas Chromatogram of V. wallichii (VW-II)

Table 2.1.a. Chemical composition of the essential oil V. wallichii (VW-II)

S.No. Content

(% oil)

M+ Major Fragments Compound identified

1. 1.44 136 93,91,77, 121,136, 105 α-pinene

2. 2.87 136 93,121,107,80,79,41 β –pinene

3. 1.75 136 93,91,121,79,77,94,107 Camphene

4. 1.83 164 149,163,134,105,119,150 thymol methyl ether

5. 2.50 164 149,164,134,105,119,150,135 carvacrol methyl ether

6. 1.92 196 95,121,136,154,108,80 bornyl acetate

7. 4.87 204 105,91,147,133,161,189,204 α-guaiene

8. 1.17 204 122,105,79,133,147,161,189,204 γ- patchoulene

9. 4.22 204 93,41,107,121,133,147,204 α-humulene

10. 4.28 204 107,93,119,135,189,147,175 α- patchoulene

11. 10.29 204 91,105,119,133,147,161,175,189 Seychellene

12. 1.09 204 105,91,161,79,119,133,189,204 Valencene

13. 0.92 204 204,105,91,161,189,119,133,175 δ-selinene

14. 10.69 204 187,93,135,119,161,204 δ- guaiene

15. 1.60 204 108, 93,81, 149,189,204 Unidentified

16. 1.73 204 105,161,119,133,147,189,204 α-murrolene

17. 1.62 204 93,107,91,108,189,204 Caryophyllene

18 0.41 204 161,107,105,91,147,189,204 β-gurjunene

19. 1.19 222 108,55,67,126,135,149,189,205 Kessane

20. 2.35 222 107,91,176,187,205,220 Unidentified

21. 30.72 222 138,205,81,95,109,125,161,189,222 patchouli alcohol

22. 10.45 280 125,81,107,177,159,79,205,220,282 8-acetoxyl patchouli alcohol

2.3.2.a. GC-MS screening of essential oil of Valeriana wallichii (VW-I)

The essential oil of VW-I was analyzed using GC-MS with the primary aim of

identification of the major and minor constituents of the essential oil. The gas

chromatogram (Fig. 2.2.a) showed the presence of 30 compounds, out of which 26

compounds have been identified constituting about 92.41% of the total oil. The

separation of solid compound of the root oil was done through the process of

decantation i.e. separation of crystalline part from that of the mother liquor. The

patchouli alcohol which is considered as the characteristic of the V. wallichii was

separated in the form of crystalline part and traces of it were found present in the

mother liquor part giving minor peaks in the GC-MS analysis which was also

confirmed through Co-TLC. Maaliol (36.82%) was found to be the major component

followed by β-gurjunene (21.28%), guaiol (8.60%) and α-santalene (5.42%). The

detailed analysis is given in the Table 2.2.a. The present results show maaliol as the

chief component of the oil (36.79 %) while patchouli alcohol being present as trace

constituent. The present chemical investigation has also shown the presence of

bicyclogermacrene (2.87%), α-longipinene and α-trans-bergomotene (trace) in the

root oil. The results are shown in Table 2.2.a.

Fig. 2.2.a. Gas Chromatogram of VW-I

Table 2.2.a. GC-MS data of essential oil of Valeriana wallichii (VW-I) t= trace percentage

S.No. Content(% oil)

Major Fragments Compound identified

1. 0.65 93,91,79,94,67,107,92,60,105,41,43, 65,121,122,136 α-pinene

2. t 119,91,117,134,120,115,77,41,105,55 p-cymene

3. t 67,93,91,94,79,77,107,121,65,105,53, 51,136 limonene

4. 0.42 91,93, 77,92,121,136,79,105,119,107,65,51,41 γ-terpinene

5. t 93,91,121,79,136,105,77,107,119,92, 67,51,41 α-terpinolene

6. 0.41 95,67,93,91,121,41,77,136,65,105,51, 121 borneol

7. 0.11 149,164,91,134,117,119,150,77,93, 105,51,121 thymol methyl ether

8. 0.68 149,164,91,117,119,150,134,115,105,77,135,65,79 carvacrol methyl ether

9. 0.79 93,95,121,67,91,81,79,136,108,107,43,41,154 bornyl acetate

10. 1.12 105,119,161,204,189,133,91,147,107,81,93 α-longipinene

11. 5.42 91,93,94,79,105,95,189,119,161,77,133,67,148,41,65,204,51 α-santalene

12. 21.28 161,105,119,133,91,189,147,162,93,79,77,204,175,55,41 β-gurjunene

S.No. Content (% oil)

Major Fragments Compound identified

13. t 119,91,93,105,77,79,92,120,41,67,65,135,149,204 α-trans-bergomotene

14. 9.80 105,119,91,161,93,79,133,67,107,77,81,121,41,189,147,175,204 acoradiene

15. 2.87 121,93,91,119,105,107,161,79,77,81, 189,133,162,67,147,204,175 bicyclogermacrene

16. 0.60 91,93,105,79,119,161,107,67,77,133,189,204 trans-β-guaiene

17. 0.62 119,105,161,204,91,162,133,134,81,189,79,67,145,65,51 δ-cadinene

18 36.82 105,107,119,161,91,93,133,79,67,189,147,43,204,41,175,222 maaliol

19. 8.60 105,161,107,91,93,189,133,67,79,147,121,175,41,43,204,55,222 guaiol

20. 0.59 131,144,159,187,128,117,115,202,91,104,77,63,43,51 desmethoxyencecalin

21. 0.72 95,204,121,137,161,162,205,137,148,105,79,71,43,67,222 α-cadinol

22. t 138,222,83,207,125,161,105,81,95,81,67,179,189,55,41 patchouli alcohol

23. 0.62 105,107,91,93,119,161,67,81,147,135,189,41,204,222 bulnesol

24. 0.29 119,67,109,93,91,107,161,120,133,79,43,189,204,222 α-bisabolol

25. t 109,161,105,176,185,119,91,147,133,203,177,121,93,67,79,204,222 drimenol

26. t 107,119,121,67,79,105,133,257,145,187,173,201,272,243,229,216,41,55 laurenene

2.3.3.a. Isolation of compounds from Valeriana wallichii (VW-II)

Two compounds VW-IIC and VW-IID were isolated from the plant.

Characterization of compound VW-IIC

IR film (cm-1): 3500, 2925, 1450 and 1370

MS m/z (%): 222 (M+, 32), 207 (24), 204 (20), 189 (21), 161(33), 138 (100), 121

(29), 98 (107), 83 (38), 81(25)

1H-NMR (δ ppm): δ 0.76 (d, 3H), 0.83 (s, 3H), 1.03 (s, 3H), 1.06 (s, 3H), 1.09(t,

2H), 1.27 (q, 2H), 1.40 (q, 2H), 1.92 (t, 2H), 1.70 (t, 2H), 1.20 (m, 1H), 1.50 (m, 1H),

1.80 (t, 1H).

13C-NMR (δ ppm): δ 18.4, 20.5, 24.2, 24.2, 24.4, 26.7, 28.0, 28.5, 28.7, 32.6, 37.5,

39.0, 40.0, 43.7 and 75.6.

The IR spectrum (Fig. 2.3.a.) and mass spectral data with M+ at 222

corresponding to C15H26O and fragment ion (M-18) suggest the compound to be a

sesquiterpene alcohol. The 13C-NMR spectrum with 15 carbon signals and 1H-NMR

spectral signals at δ 0.76, 0.83, 1.03 and 1.06 showed the presence of 4 methyls. The

signals at δ 1.09, 1.27, 1.40, 1.92 and 1.70 showed the presence of five –CH2- and

signals at δ 1.21, 1.50 and 1.85 showed the presence of three –CH-. The spectral data

suggest the compound as patchouli alcohol and the NMR values of compound VW-

IIC match well with those given in literature for the proposed structure (Nishiya et al.,

1995; Sati, 2002; Tewari, 2002; Sammal, 2005).

OH

H

H

VW-IIC (Patchouli alcohol)

Fig. 2.3.a. IR spectrum of compound VW-IIC

Fig. 2.4.a. MS spectrum of compound VW-IIC

Fig. 2.5.a. 13C-NMR spectrum of compound VW-IIC

Fig. 2.6.a. 1H NMR spectrum of compound VW-IIC

Charcterization of compound VW-IID

IR film (cm-1): 1275, 1735, 3500

MS m/z (%): 280 (M+, 2), 222 (2), 220 (19), 205 (22), 177 (54), 159 (39), 125 (100),

107 (71), 81(76), 79 (21), 67(16), 55(8).

1H-NMR (δ ppm): δ 0.8 (d, 3H), 0.9 (s, 3H), 1.10 (s, 3H), 1.15 (s, 3H), 2.02 (s, 3H),

148 (t, 2H), 1.52 (t, 2H), 1.64 (q, 2H), 1.76 (t, 2H), 1.34 (m, 1H), 1.43 (q, 1H), 1.96

(m,1H), 4.8 (t, 1H).

13C-NMR (δ ppm): δ 18.6, 19.8, 21.5, 23.3, 24.9, 27.7, 27.7, 28.4, 32.5, 35.8,

38.9, 39.9, 42.3, 43.1, 74.5, 75.0, 170.9

The mass spectrum showed, M+ peak at 280 corresponding to C17H28O3 (Fig

2.7.a) while the IR spectrum showed a hydroxyl (3500 cm-1) and acetoxyl group

(1735 and 1275 cm-1). The 1H-NMR spectrum showed methyl signals at (δ 0.80, 0.90,

1.10, 1.15 and an acetoxyl methyl group (δ 4.80) (Fig. 2.8.a.). The 13C-NMR

spectrum indicated the presence of 17 carbons (Fig. 2.9.a.). The compound with 4

degree of unsaturation is a tricyclic sesquiterpene with one hydroxyl and the other as

acetoxyl group. The positions of –OH and –COCH3 groups at C1 and C8 is supported

by comparison of 1H and 13C-NMR data of closely related structures given in

literature (Nishiya et al., 1995; Arantes et al., 1999). On the basis of above spectral

data the compound was identified as 8-acetoxyl patchouli alcohol (Tewari, 2002,

Sammal, 2005).

OH

H

H

OAc

W-IID (8-acetoxyl patchouli alcohol)

Fig. 2.7.a. MS spectrum of compound VW-IID

Fig. 2.8.a. 1H-NMR spectrum of compound VW-IID

Fig. 2.9.a. 13C-NMR spectrum of compound VW-IID

2.3.4.a. Isolation of compounds from Valeriana wallichii (VW-I)

Characterization of compound VW-IB

White needle shaped crystals

IR (Nujol) (cm-1): 3507, 3007, 2917, 1457, 1387, 1373, 1325, 1174, 1103.

MS (m/z) (%): 222 (M+) (3), 207 (2), 204 (16), 189 (36), 175 (13), 161(74), 62(18),

147(35), 133(48), 119(75), 107(80), 105(100), 93 (68), 91(69), 79(45), 67(45),

55(10), 43 (17), 41(14).

1H-NMR δ (ppm): 0.48 (1H, dd, J=5.1, 7.2 Hz), 0.59 (1H, dd, J=7.2, 7.2 Hz),0.76

(1H, m), 0.88 (3H, s), 1.08 (3H, s), 1.27 (3 H, s)1.47 (2H, s), 1.49 (2H, s), 1.74 (2H,

s),1.79 (2H, m),1.80 (2H, s) (Table 2.9.a).

13C-NMR δ (ppm): 72.84, 49.63, 42.70, 41.25, 39.61, 33.02, 29.15, 23.02, 20.14,

19.78, 19.17, 18.76, 17.54, 15.99 and 15.49.

Compound VW-IB was the major component (36.82%) of the oil. The IR

absorption band at 3507 cm-1, m/z at 204 through loss of H2O from M+ confirms the

hydroxyl (-OH) in the molecule [222 (M+)- H2O].

The 1H-NMR exhibits two singlets at δ 0.88 and δ 0.93 ascribed to -CH3 and

CH3-C(OH)- respectively. Two singlets at δ 0.59 (1H, dd, J=7.2, 7.2 Hz) and δ 0.76

(1H, m) must be due to two methine protons fused on cyclopropane ring. Two singlets

at δ 1.08 (3H) and δ 1.27 (3H) are showing a gemdimethyl group. The mass spectrum

with molecular ion peak (m/z) at 222 (M+) corresponds to C15H26O. The carbon

attached to –OH showed up at δ 72.8 ppm. The spectral data show the compound to

be maaliol which is also supported by the literature reports (Nishiya et al., 1992, Sati,

2002, Sammal, 2005).

H

OH

H

H VW-IB (Maaliol)

Fig. 2.10.a. Infra Red spectrum of compound VW-IB

Fig. 2.11.a. Mass spectrum of compound VW-IB

Fig. 2.12.a. 13C-NMR spectrum of compound VW-IB

Fig. 2.13.a. 1H-NMR spectrum of compound VW-IB

Characterization of compound VW-IC

White needle crystals with pleasant odour.

IR (Nujol) (cm-1): 3507, 3007, 2980, 2917, 2856, 1457, 1387, 1373, 1325, 1174,

1141, 1103, 953.

MS (m/z, %): 222 (M+) (65), 207 (32), 205 (5), 204 (15), 189 (17), 179 (20), 161

(30), 138 (100), 125 (32), 105 (24), 95 (22), 91 (20), 83 (34), 81 (22), 67 (18), 55 (7),

41 (12).

1H-NMR δ (ppm): 0.79 (3H, d, J=6.7 Hz), 0.82 (3H, s), 1.02 (3H, s), 1.05 (3H, s),

1.16 (2H, t), 1.22 (2H, q), 1.25 (1H, q), 1.40 (2H, t), 1.45 (1H, m), 1.48 (2H, q), 1.73

(1H, m), 1.83 (2H, t).

13C-NMR δ (ppm):72.87, 49.57, 42.66, 41.22, 39.57, 32.98, 29.12, 22.97, 20.10,

19.76, 19.12, 18.73, 17.49, 15.46 and15.49

Compound VW-IC was another major component. The IR absorption band at

3507 indicates the compound VW-IC possesses an alcoholic group (Fig. 2.14.a). The 1H-NMR spectral data δ 0.82 (3H, s), δ 1.02 (3H, s) and δ 0.79 (3H, d) showed –CH3,

(CH3)2C-OH and >C-CH3, respectively (Fig. 2.15.a). The mass spectrum showed

molecular ion peak (m/z) 222 corresponding to C15H26O which is for a tricyclic

sesquiterpene alcohol (Fig 2.16.a). The carbon attached to –OH showed up at δ 72.8

ppm. Comparison of the data with literature report ((Nishiya et al., 1995; Sati, 2002;

Tewari, 2002; Sammal, 2005) confirms the compound VW-IC as patchouli alcohol.

OH

H

H VW-IC (Patchouli alcohol)

Fig. 2.14.a. Infra Red spectrum of compound VW-IC

Fig. 2.15.a. 1H-NMR spectrum of compound of compound VW-IC

Fig. 2.16.a. Mass spectrum of compound VW-IC

2.4.a. DISCUSSION

The percentage yield of the essential oil was found to be 0.60% (v/w) in case

of plant VW-II. Gas chromatogram revealed 28 peaks out of which 20 compounds

have been identified. The major constituents were separated by column

chromatography followed by their purification. The minor constituents were identified

with the help of GC-MS. Patchouli alcohol (VW-IIC), an isolated compound was

found to be major constituent. 8-Acetoxyl patchouli alcohol (VW-IID) constituted

10.45 % of the oil. The percentage yield of the essential oil of plant VW-I was found

to be 0.16% (v/w). The GC-MS analysis showed the presence of 30 compounds, out

of which 26 compounds constituting 92.41% of the total oil have been identified. An

interesting feature in the mother liquor was the presence of oxygenated

sesquiterpenoid like maaliol (36.82%) followed by β-gurjunene (21.28%), guaiol

(8.60%), α-santalene (5.42%), bicyclogermacrene (2.87%). The major component was

found to be maaliol. The results showed that the composition of the essential oil

isolated from roots and rhizomes of V. wallichii varied depending on the origin. The

plant VW-I was found to be chemotype maaliol of V. wallichii and VW-II was found

to be patchouli alcohol chemotype.

Section B

Isolation and Analysis of Valepotriates of Valeriana wallichii Chemotypes

2.1. b. INTRODUCTION

Same as in section 2.1.a. of chapter 2.

2.2.b. MATERIAL AND METHODS

2.2.1.b Plant material

Roots and rhizomes of Valeriana wallichii (Maaliol type), Valeriana wallichii

(Patchouli alcohol type) were collected from regions as mentioned in section 2.2.1.a.

2.2.2.b. Extraction

After collection, the plant materials were immediately stored at cool and dry

place. The plant material (1kg) were dried in shady place and subjected to

dichloromethane (CH2Cl2) extraction. The extract was concentrated to dryness with

rotary evaporator at 35°C to yield a brown dry mass (2 g). So the yield being 20% for

both the chemotypes. Extract from plant VW-II (Patchouli alcohol type) was coded as

VVDR03 and that from VW-I (Maaliol type) was coded as VVW02. Extraction of the

plant material for HPLC screening was carried out by homogenizing 10 g of dried

material of each Valeriana plant with 50 ml of dichloromethane for 3 days. The

homogenate was filtered and the residue was washed with 5 ml portion of

dichloromethane. Evaporation of the solvent was done below 35°C by rotary

evaporator, and the residue was again dissolved in dichloromethne (3 ml).

2.2.3.b HPLC Analysis

Thermoelectron Quaternary HPLC system with an injection valve system and

a 20µl sample loop and stainless steel column (Thermoelectron C-18) 25 cm x 4.6 mm

i.d was used. The column outlet was connected to UV detector (UV-1000) and RI-

detector (RI-150). The detection wavelengths were 254 nm for diene valepotriates and

210 nm for monene valepotriates.

2.2.4.b. Solvents

Dichloromethane and acetonitrile used were of HPLC grade (Qualigens). The

water used for solvent system was deionised and the HPLC system was isocratic with

binary solvent system, acetonitrile: water (70:30).

2.2.5.b. Analytical separation of valepotriates

Diene valepotriates show absorption maximum at 254 nm, due to their two

conjugated double bonds, and can be detected with high sensitivity at this wavelength.

Monene valepotriates were detected at 210 nm. Acetonitrile: water (70:30) was used

as eluent with 1ml/ min flow rate (Fig. 2.1.b, 2.2.b, 2.3.b & 2.4.b).

Fractionation

The residue (oily mass) was fractionated on column chromatography silica gel

(60-120 mesh) in Et2O: n-hexane (8% to 30%). Repeated column chromatography of

the column fractions followed by purification afforded three compounds (VW-I≠01,

VW-I≠02 and VW-I≠03) from Valeriana wallichii (Maaliol type) and three

compounds (VW-II≠04, VW-II≠05 and VW-II≠06) from Valeriana wallichii

(Patchouli alcohol type).

Valeriana wallichii (Maaliol type) Extract (0.5 g)

Column Chromatography

(n-hexane: Et2O) Silica gel (50 g)

n-hexane 8% Et2O 15% Et2O 20% Et2O 30% Et2O

(A) (B) (C) (D) (E)

Recolumn (n-hexane:Et2O) (5-10%) Si gel (10g)

VW-I≠01 VW-

10 mg 8 m

Scheme 2.1.b: Isolation of components fro

Recolumn (n-hexane: Et2O) (15-20%) Si gel (10g)

I≠02 VW-I≠

g 10mg

m V. wallichii (VW-I

Recolumn (n -exane:Et2O) (25-30%) Si gel (10g)

Hydrocarbonmixture

03

)

Valeriana wallichii (patchouli alcohol type) Extract (0.5 g)

Column chromatography (n-hexane: Et2O) Silica gel (80.0g)

n-hexane 8% Et2O 10% Et2O 15% Et2O 30% Et2O

Hydrocarbon mixture

VW-II≠04 VW-II≠05 VW-II≠06 F

8 mg 10 mg 12mg

(A) (B) (C) (D) (E)

Scheme 2.2

Fig. 2.1.b. HPLC of C

Recolumn (n-hexane:Et2O) (0-5%) Si gel (10g)

.b: Isolation of components from V.

H2Cl2 extract of V. wallichii (Maali

Recolumn (n-hexane:Et2O) (5-10%) Si gel (15g)

wallichii (VW-II)

ol type) detected at 210 nm.

Fig. 2.2.b. HPLC of CH2Cl2 extract of V. wallichii (Maaliol type) detected at RI detector

Fig. 2.3.b. HPLC of CH2Cl2 extract of V. wallichii (Patchouli alcohol type) at 210 nm

Fig. 2.4.b. HPLC of CH2Cl2 extract of V. wallichii (Patchouli alcohol type) detected at RI detector

2.3.b. RESULTS

2.2.1.b. Compounds isolated from Valeriana wallichii (Maaliol type)

Three compounds were isolated from the plant viz. VW-I≠01, VW-I≠02 and VW-

I≠03.

Characterization of compounds

VW-I≠01

IRKBr υ max cm-1: 2925, 1766, 1733, 1250

1H-NMR (CDCl3, 300 MHz) δ ppm: 6.52 (1H, s), 5.84 (1H, d, J=0.5), 4.95 (1H, t,

J=5.5), 4.69 (2H, d, J=12.5), 4.69 (2H, d, J=12.5), 2.97-2.99 (1H, m), 2.72 (1H, d,

J=8.0), 2.05 (3H, s), 1.0 (3H, s).

13C-NMR (CDCl3, 75 MHz) δ ppm: C-1 (88.3), C-3 (145.4), C-4 (11.2), C-5 (60.8),

C-6 (83.0), C-7 (73.2), C-8 (62.2), C-9 (48.8), C-10 (48.2), C-11 (61.9), R1’-C-1’

(170.9), C-2’ (43.3), C-3’ (25.7), C-4’ (17.2), C-5’ (18.8), R2-OAc-(169.8), (20.8),

R3”-C”-1 (169.6), C”-2 (43.3), C”-3 (25.6), C”-4 (22.3), C”-5 (22.4).

The compound VW-I≠01 was obtained as a light brown viscous liquid. 13C-

NMR showed the presence of twenty-three carbons. Valtrate type skeleton was

confirmed by the presence of one epoxide group at the C-8 carbon, one methyl

protons at δ 4.74 adjacent to an oxygen function and one methine carbon δ 83.0

having an oxygen function with an acetate residue at C-7. 13C-NMR (Fig. 2.5.b) and 1H-NMR (Fig. 2.6.b) signals confirmed two isovaltrate groups at C-1 and C-11. The

structure of the compound VW-I≠01 was therefore characterized as didrovaltrate.

Finally, identity was confirmed by comparison of its spectral data with those reported

in literature (Tang et al., 2002; Thies et al., 1981, Sammal, 2005).

O

O

O

O H3C

CH3

H

O

O

CH3

CH2OCOCH2CH(CH3)2 VW-I≠01 (Didrovaltrate)

Fig. 2.5.b. 13C-NMR spectrum of VW-I≠01

Fig. 2.6.b. 1H-NMR spectrum of VW-I≠01

VW-I≠02

IRKBr υ max cm-1: 3500, 1766, 1633, 1255.

1H-NMR (CDCl3, 300 MHz) δ ppm: 6.65 (1H, s), 6.07 (1H, d, J=2.0), 4.92 (2H, d,

J=12.5), 4.93 (1H, d, J=10.0), 4.73 (1H, d, J=12.5), 3.11(2H, d, J=5.0), 2.91 (2H, d,

J=2.0), 2.83 (2H, d, J=5.0), 2.06 (3H, s), 1.99 (2H, t, J=13.0).

13C-NMR (CDCl3, 75 MHz) δ ppm: C-1 (88.1), C-3 (145.2), C-4 (111.2), C-5

(69.5), C-6 (40.25), C-7 (73.1), C-8 (62.1), C-9 (48.02), C-10 (48.2), C-11 (61.1), R1-

C’-1 (169.5), C’-2 (76.6), C’-3 (172.9), C’-4 (42.93), C’-5 (25.51), C’-6 (22.1), C’-7

(22.1), C’-8 (29.81), C’-9 (17.03), C’-10 (18.6), R2-(C”-1) 169.7, (C”-2) 20.64 (R3-

OAc, C”-1), (170.8), C”-2 (42.83), C”-3 (25.55), C”-4 (22.2), C”-5 (22.2).

Compound VW-I≠02 is a viscous light brown liquid. The 13C-NMR showed

27 signals, indicating 7 methyls, 5 methylene, 8 methine and 7 quaternary carbons

(Fig. 2.7.b). The chemical shift of two olefinic methine carbons at δ 145.2 and 111.2

assigned for C-3 and C-4 respectively. The quaternary carbon at δ 62.1 (C-8 and the

methylene carbon at δ 48.2 C-10) are characteristic for an epoxide ring at C-8 and C-

10 and corresponds to a valtrate-type iridoid. A singlet, at δ 2.06 in the 1H-NMR

spectrum was assigned to the methyl protons of an acetate residue (Fig. 2.8.b). 13C-

NMR signal at δ 169.7 was assigned to the carbonyl carbon of the acetate residue.

13C-NMR showed 10 carbon signals due to 4 methyl carbons (δ 22.2, 22.2, 18.6 and

17.03), methylene carbons (δ 42.93) linked to the carbonyl carbon (δ 172.9) three

methine carbons (δ 25.5, 29.8 & 76.6) linked to a carbonyl residue. All these data

were used to assign compound VW-I≠02 as isovaleroxyhydroxy didrovaltrate.

Finally, identity was confirmed by comparison of its spectral data with those reported

in literature (Tang et al., 2002; Becker et al., 1984, Sammal, 2005).

O

O

O

O H3C

CH3

HO

O

O

CH3

O

CH3CH3

O

O

O

VW-I≠02 (Isovaleroxyhydroxy didrovaltrate)

Fig. 2.7.b. 13C-NMR spectrum of VW-I≠02

Fig. 2.8.b. 1H-NMR spectrum of VW-I≠02

VW-I≠03

IRKBr υ max cm-1: 2925, 1766, 1733, 1640.

1H-NMR (CDCl3, 300 MHz) δ ppm: 6.72 (1H, s), 5.98 (1H, d, J=10), 5.89 (2H, d, J=

3.6), 5.39 (1H, d, J=2.5), 2.36-2.21 (2H, m), 2.08 (3H, s), 1.0 (3H, s).

13C-NMR (CDCl3, 75 MHz) δ ppm: C-1 (92.4), C-3 (148.6), C-4 (108.21), C-5

(141.1), C-6 (118.3), C-7 (83.3), C-8 (64.0), C-9 (42.9), C-10 (47.7), C-11 (60.7), R1-

C’-1 (169.5), C’-2 (79.2), C’-3 (170.8), C’-4 (22.1), C’-5 (26.6), C’-6 (26.5), C’-7

(26.5), R2-OAc- (170.3), (20.84), R3-C”-1 (170.4), C”-2 (43.9), C”-3 (25.5), C”-4

(22.1), C”-5 (22.2).

The compound VW-I≠03 was obtained as yellow viscous liquid. 13C-NMR

spectrum of the compound showed the presence of twenty four carbons. 1H-NMR

spectrum revealed signals for methyl groups at δ 0.99, 1.31, 1.52 and 2.06, one methyl

proton at δ 4.69 adjacent to an oxygen function and one methyl carbon δ 79.2 having

oxygen function. Furthermore , the 13C-NMR spectrum showed 7 carbon signal due to

2 methine carbons (δ 25.5, 79.2), 3 methyl carbons (δ 22.1, 26.5 and 26.5) and 2 ester

carbonyl carbons (δ 170.8 and 170.3). These spectral data suggested the presence of

acetoxylisovaleryl group and isovaleryl group present at C-11 and C-1, respectively.

The structure of the compound VW-I≠03 was therefore characterized as 1-α

acevaltrate. Finally, identity was confirmed by comparison of its spectral data with

those reported in literature (Thies et al., 1981; Sammal, 2005).

O

O

O

H

O

O

CH3CH3

O

O

O

O

VW-I≠03 (Acevaltrate)

2.2.2.b Isolation of compounds from Valeriana wallichii (Patchouli alcohol type)

Three compounds were isolated from the plant viz. VW-II≠04, VW-II≠05 and VW-

II≠06.

Characterisation of compounds

VW-II≠04

IRKBr υ max cm-1: 3500, 1770, 1640, 1250.

1H-NMR (CDCl3, 300 MHz) δ ppm: 6.65 (1H, s), 6.07 (1H, d, J=2.0), 4.92 (2H, d,

J=12.5), 4.93 (1H, d, J=10.0), 4.73 (1H, d, J=12.5), 3.11 (2H, d, J=5.0), 2.91 (2H, d,

J=2.0), 2.83 (2H, d, J=5.0), 1.99 (2H, t, J=13.0).

13C-NMR (CDCl3, 75 MHz) δ ppm: C-1 (88.2), C-3 (145.4), C-4 (111.2), C-5

(69.7), C-6 (40.3), C-7 (73.2), C-8 (88.2), C-9 (48.1), C-10 (48.8), C-11 (61.9), R1-C’-

1 (169.6), C’-2 (76.8), C’-3 (173.1), C’-4 (43.0), C’-5 (25.5), C’-6 (22.3), C’-7 (22.3),

C’-8 (29.6), C’-9 (17.1), C’-10 (18.7), R2-OAc- (169.8), (20.84), R3-C”-1 (170.9), C”-

2 (43.0), C”-3 (25.7), C”-4 (22.3), C”-5 (22.3).

Compound VW-II≠04 was obtained as a viscous light brown liquid. It’s 13C-

NMR showed 27 resonance lines. Analysis of the overall NMR spectral data revealed

the presence of an iridoid skeleton with ester units. The quaternary carbons at δ 88.2

(C-8) and the methylene carbon at δ 48.8 (C-10) were characterstic for an epoxide

ring at C-8 and C-10 and corresponds to a valtrate type iridoid. A methyl singlet,

which appeared at δ 2.06 in the 1H-NMR spectrum was assigned to the methyl protons

of an acetate residue. The 13C-NMR spectrum showed 10-carbon signals due to 4-

methyl carbons (δ 22.3, 22.3, 18.7 and 17.1), methylene carbon (δ 42.93) linked to the

carbonyl carbon (δ 173.1), three methine carbons (δ 25.5, 29.9 and 76.6) linked to a

carbonyl residue. All the data show the compound VW-II≠04 as isovaleroxy hydroxy

didrovaltrate. Finally its identity was confirmed by comparison of its spectral data

with literature reports (Thies et al., 1981; Sammal, 2005).

O

O

O

O H3C

CH3

HO

O

O

CH3

O

CH3CH3

O

O

O

VW-II≠04 (Isovaleroxyhydroxy didrovaltrate)

VW-II≠05

IRKBr υ max cm-1: 2925, 1766, 1740, 1640, 1610.

1H-NMR (CDCl3, 300 MHz ) δ ppm: 6.71 (1H, s), 5.98 (1H, d, J=10.0), 5.86 (2H, d,

J=2.5), 5.38 (1H, d, J=2.5), 2.06 (3H, s), 1.10 (3H, s)

13C-NMR (CDCl3, 75 MHz ) δ ppm: C-1 (92.4), C-3 (148.6), C-4 (108.2), C-5

(141.1), C-6 (118.3), C-7 (83.3), C-8 (64.0), C-9 (42.9), C-10 (47.7), C-11(60.7), R1-

C’-1 (169.5), C’-2 (79.2), C’-3 (170.8), C’-4 (22.1), C’-5 (26.6), C’-6 (26.5), C’-7

(26.5), R2-OAc-(170.3), (20.84), R3-C”-1 (170.4), C”-2 (43.9), C”-3 (25.5), C”-4

(22.1), C”-5 (22.2).

The compound VW-II≠05 was obtained as a pale yellow viscous liquid. 13C-

NMR spectrum of the compound showed the presence of twenty four carbons. 1H-

NMR spectrum revealed signals for methyl group at δ 0.99, 1.31, 1.52 and 2.06, one

methyl proton at δ 4.69 adjacent to an oxygen function and one methyl carbon δ 79.2

having an oxygen function. Furthermore, the 13C-NMR spectrum showed seven

carbon signal due to two methine carbons (δ 25.5, 79.2), three methyl carbons (δ 22.1,

26.5 and 26.5) and two ester carbonyl carbons (δ 170.8 and 170.3). These spectral

data suggested the presence of acetoxylisovaleryl group and isovaleryl group present

at C-11 and C-1, respectively. The structure of compound VW-II#05 was therefore

characterized as 1-α acevaltrate. Finally, identity was confirmed by comparison of its

spectral data with those reported in literature (Becker et al., 1984; Thies et al., 1981;

Sammal, 2005).

O

O

O

H

O

O

CH3CH3

O

O

O

O

VW-II≠05 (Acevaltrate)

VW-II≠06

IRKBr υ max cm-1: 1770, 1740, 1640, 1610, 1250.

1H-NMR (CDCl3, 300 MHz ) δ ppm: 6.69 (1H, s), 5.86 (1H, d, J=3.0), 5.96 (1H, d,

J= 10.0), 4.76 (2H, d, J= 12.0), 4.66 (2H, d, J= 12.0), 3.03 (1H, d, J=5.0), 2.91 (1H, d,

J=5.0), 2.06 (3H, s).

13C-NMR (CDCl3, 75 MHz ) δ ppm: C-1 (92.5), C-3 (148.5), C-4 (108.3), C-5

(140.9), C-6 (118.6), C-7 (83.0), C-8 (64.1), C-9 (43.0), C-10 (47.8), C-11(60.8), R1-

C’-1 (170.8), C’-2 (42.9), C’-3 (26.1), C’-4 (22.2), C’-5 (22.3), R2-C”-1(172.4), C”-2

(42.9), C”-3 (25.6), C”-4 (29.6), C”-5 (11.2), C”-6 (19.9).

The compound VW-II#06 was obtained as light brown viscous liquid. 13C-

NMR showed 23 carbons, indicating 5 methyls, 5 methylene carbons, 7 methine

carbons and 6 quaternary carbons (Fig 2.9.b). The signals at δ 6.69 and δ 92.5

respectively were assigned to H-1 and C-1. The chemical shifts of two olefinic

methine carbons at δ 108.3 and 140.9 respectively were assigned to C-3, C-6, C-4 and

C-5. The chemical shift of one methine carbon (δ 83.0), one quaternary carbon (δ

64.1) and one methylene carbon (δ 43.0) were assigned C-7, C-8 and C-9

respectively. The quaternary carbon at δ 64.1 (C-8) and the methine carbon at δ 47.8

(C-10) were characteristic for an epoxide ring at C-8 and C-10. 1H and 13C-NMR data

of compound showed the presence of acetate residue at C-7 and a β-me-isovaltrate

group at C-11. Therefore, compound VW-II#06 was identified as 1-homoisovaltrate.

Finally, the identity was confirmed by comparison of spectral data (Fig. 2.9.b) with

that reported in literature (Tang et al., 2002; Thies et al., 1981; Sammal, 2005).

O

O

O

H

O

CH3

O

CH3

O

O

CH3

VW-II≠06 (1-Homoisovaltrate)

Fig. 2.9.b 13C-NMR spectrum of VW-II≠06

CHAPTER-3

Psychopharmacological Profile of Valeriana wallichii chemotypes

3.1. INTRODUCTION

Although a multitude of pharmaceutical agents are available for the treatment

of mood disorders, anxiety, insomnia and epilepsy; many patients have difficulty

tolerating the side effects, do not respond adequately, or eventually lose their

response. Many therapeutic herbs and nutrients have far fewer side effects and may

provide an alternative treatment or can be used to enhance the effect of prescription

medications.

Epilepsy is one of the most common disorders of the central nervous system,

worldwide. Though, the advent of newer techniques in neurobiology has provided

some insight into the pathophysiology of epilepsies, many aspects of this phenomenon

still remain unknown. It is, therefore not surprising that the currently used

antiepileptic drugs fail to provide satisfactory seizure control for nearly 15 % to 20 %

per cent of patients with epilepsy. For such patients, the combinations of antiepileptic

drugs are often prescribed in attempts to improve seizure control. However, toxicities

associated with these drugs further compromise the quality of life, while drug-drug

interactions may complicate management. Thus there is an ever lasting need for

search of newer molecules for treating seizures.

Valeriana wallichii DC. (Syn: V. jatamansi Jones) is a plant that belongs to

family “Valerianaceae” commomly called as Indian valerian. It is a small perennial

herb of 15-45 cm height with rootstock, thick branching stem, sharply pointed leaves,

white or pink flowers in clusters and hairy fruit. It is indigenous to the temperate

Himalayas and found in Kashmir, Nepal, Bhutan, Burma and Afghanistan. The plant

is widely known for its use in anxiety, insomnia, epilepsy, failing reflexes, hysteria,

neurosis and sciatica (Nadkarni, 1976). It is also considered useful as a potent

tranquilizer, emmenagogue (Nadkarni, 1976), diuretic (Said, 1970) and

hepatoprotective (Awan, 1990). Its active principles are valepotriates (Becker and

Chavadej, 1985) (like dihydrovaltrate) (Bounthanh et al., 1981), linarin-isovalerianate

(Thies, 1968), sesquiterpenoids (Ron et al., 2000), 6- methylapigenin and hesperidin

(Marder et al., 2003). Valerian preparations yield isovaleric acid, a substance

analogous to valproic acid and likely to possess anticonvulsant properties (Eadie,

2004). It is well known that the content and composition of active compounds in

herbal medicines are strongly influenced by many factors, such as genotype, climate,

harvest and preparation process. For example, V. wallichii roots from different

sources were found to contain between 0.09 and 1.30% v/w of essential oil (Kapoor,

1990). In the present work the effect of variation of active compounds between V.

wallichii growing under different environmental conditions has been correlated with

their psychopharmacological profile

3.2. MATERIAL AND METHODS

3.2.1. Plant material and its extraction

Plants were collected and identified as mentioned in section 2.2.1.a. of chapter

2 (section A). The oil was extracted by steam distillation as described in section

2.2.2.a. of chapter 2 (section B). The oil obtained from VW-II (Patchouli chemotype)

was coded as V-PA and that obtained from VW-I (Maaliol chemotype) was coded as

V-MA. Similarly the extracts were obtained by the method as mentioned in section

2.2.2.b. and were coded as VVW02 (maaliol chemotype) and VVDR03 (patchouli

alcohol chemotype).

3.2.2. Animals

Animals used in the study were laca mice of either sex (20-40g) bred in

Central Animal House facility of Panjab University, Chandigarh housed in cages with

food and water ad libitum and maintained on a natural light and dark cycle. The

experimental protocols were approved by the Panjab University Animal Ethical

committee.

3.2.3. Drugs

The drugs used in the present study were Pentylenetetrazole (PTZ) (Sigma,

USA), Diazepam (Sigma, USA). PTZ was dissolved in normal saline. Diazepam was

suspended in a drop of Tween 80 and volume was made up with sterile water. The test

drugs VVW02, VVDR03, V-PA and V-MA were also suspended in Tween-80.

Vehicle treated group received a drop of Tween-80 dissolved in sterile water and

acted as control group.

3.2.4. Experimental protocol and procedure

3.2.4.1. Anticonvulsant activity

Pentylenetetrazol (PTZ, Metrazol) is a central nervous system stimulant and

was synthesized in 1924. In 1926, Hildebrant showed its convulsant action. It

produces jerky type of clonic convulsions in mice. Pentylenetetrazole has been used

experimentally to study seizure phenomenon and to identify pharmaceuticals that may

control seizure susceptibility. The convulsive effect of this drug is considered to be

analogous to petit mal type of convulsions in man. It is considered as a GABA

antagonist but the mechanism of the epileptogenic action of PTZ at the cellular

neuronal level is still unclear.

For evaluating the effect of V. wallichii chemotypes on PTZ-induced

convulsions, animals were divided into following groups, each group consisting of six

animals.

Group 1: Vehicle treated

Group 2-4: VVDR03 (20, 40 and 80 mg/kg p.o.)

Group 5-7: VVW02 (20, 40 and 80 mg/kg p.o.)

Group 8-10: V-PA (20, 40 and 80 mg/kg p.o.)

Group 11-13: V-MA (20, 40 and 80 mg/kg p.o)

Group 14: Diazepam (1 mg/kg, i.p.)

Animals were injected pentylenetetrazol 80mg/kg intraperitoneally (i.p.) one

hour after the drug treatment. Onset of seizures, delay in death and the mortality were

noted after 1 hr in all the groups (Kulkarni, 1999).

3.2.4.2 . Measurement of anxiety levels

Elevated plus maze, is a novel test to study the anxiogenic and anxiolytic drug

effects in rodents (Kulkarni, 1999). Exposure of the animals to novel maze alley

evokes an approach-avoidance conflict which is stronger in open arm as compared to

enclosed arm. Rodents (rats and mice) have aversion for high and open arm, they

freeze, become immobile, defecate and show fear like movements. The plasma

cortisol level is also reported to be increased, as a true reflection of anxiety. Major

advantages of this test procedure are a) it is simple, fast and less time consuming b)

no prior training or noxious stimuli is required and c) it is predictable and reliable

procedure for studying anxiety response as well as antianxiety action of drugs.

The plus maze apparatus for mice consist of two open (16x5 cm) and two

closed arms (16x5x12 cm) and is placed at a height of 25 cm. Anxiety reduction in the

plus-maze is indicated by an increase in the proportion of time spent in the open arms

(time in open arms/total time in open or closed arms), and an increase in the

proportion of entries into the open arms (entries into open arms/total entries into open

or closed arms). Total number of arm entries and number of closed-arm entries are

usually employed as measures of general anxiety in rodents (Hogg, 1996).

Animals were divided into following thirteen groups, each group consisting of

six animals.

Group 1: Vehicle treated

Group 2-4: VVDR03 (10, 20 and 40 mg/kg p.o.)

Group 5-7: VVW02 (10, 20 and 40 mg/kg p.o.)

Group 8-10: V-PA (10, 20 and 40 mg/kg p.o.)

Group 11-13: V-MA (10, 20 and 40 mg/kg p.o)

The animals were then placed individually at the center of the maze with head

facing the open arm one hour after the treatment. During the 5 min test, the number of

entries in closed arms and the time spent in closed arm were recorded (Kulkarni,

1999). Similarly in a separate protocol the effect of above treatments were noted in

elevated plus maze after 14 days of daily dosing.

3.2.4.3. Rota rod test

The test was carried out using an apparatus consisting of a horizontal rod of 3

cm diameter which was made to rotate at a speed of 20 rpm. A pretest was carried out

and only those animals who demonstrated the ability to remain on the revolving rod

for at least 1 min were selected. Animals were divided into following groups, each

group consisting of six animals.

Group 1: Vehicle treated

Group 2-4: VVDR03 (10, 20 and 40 mg/kg p.o.)

Group 5-7: VVW02 (10, 20 and 40 mg/kg p.o.)

Group 8-10: V-PA (10, 20 and 40 mg/kg p.o.)

Group 11-13: V-MA (10, 20 and 40 mg/kg p.o.)

Group 14: Diazepam (1 m/kg, i.p.)

The drugs were given 1 h before the test and the time for which mice remained

on the revolving rod was noted and compared with the control group. Mouse unable

to remain on the rod at least for three min was considered as a positive test and the

time of its fall was recorded (Viswanatha et al., 2006).

3.2.4.4. Statistical analysis

Results were expressed as mean±SEM. The intergroup variation was measured

by one way analysis of variance (ANOVA) followed by Tukey’s test. Statistical

significance was considered at P<0.05. The statistical analysis was done using the

Jandel Sigma Statistical Software version 2.0.

3.3. RESULTS

3.3.1. Acute toxicity

The oral administration of V-PA, V-MA, VVDR03 and VVW02 at doses

ranging from 10-80mg/kg did not produce any lethal effect. No adverse effect or

mortality was detected in albino mice up to 2000mg/kg, p.o dose of VVDR03 and

VVW02 during 24 h observation period. Similarly no adverse effect or mortality was

detected in albino mice up to 1000mg/kg dose of V-PA and V-MA.3.3.2. Effect on

PTZ induced convulsions

In PTZ induced convulsions both the extracts and oils were found to have no

effect on onset of convulsions when PTZ was injected to the mice (data not shown).

VVW02 and VVDR03 were found to produce dose dependent effect in delaying onset

of death but the effect was significant at 80 mg/kg in comparison to control and other

groups (Table 3.1.). VVDR03 delayed the time by 40.8 % at 80 mg/kg while VVW02

increased the time for onset of death by 30.4 %.

In case of valerian oils a dose dependent response was obtained with V-PA

and an inverted U shaped curve was obtained with V-MA but only 40mg/kg dose

significantly delayed the onset of death with respect to control group. When V-PA

was evaluated for effect on PTZ-induced convulsions then only 80 mg/kg dose of V-

PA produced significant delay in onset of death (73.9 %) (Table 3.2.).

The delay in onset of death is similar for VVDR03 and VVW02 as there is no

statisitical significant difference between them. Similarly V-PA was found to be more

effective in delaying the onset of death than V-MA. And out of all four V-PA was

most effective in delaying onset of death after PTZ injection.

3.3.3. Effect on elevated plus maze

Oral administration of both the extracts and oils at doses 10, 20 & 40 mg/kg

were found to have no anxiolytic effect in plus maze after 1 hr of administration

(Table 3.3. & 3.4.). Similarly no anxiolytic effect was found when the above drugs

were administered daily for 14 days and then tested in elevated plus maze (Table 3.5.

& 3.6.).

3.3.4. Effect on rota rod apparatus

All the doses (10, 20 and 40 mg/kg p.o) of VVDR03, VVW02, V-PA and V-

MA were found to have no effect on the time of fall in rota rod test suggesting that at

these doses valerian extracts and oils do not have muscle relaxant effect (Fig. 3.1.,

3.2., 3.3. & 3.4.). Diazepam the standard drug produced a significant decrease in time

of fall (49 sec) after intraperitoneal injection, depicting skeletal muscle relaxant

effect.

Table 3.1. Effect of different doses of VVDR03 and VW02 on convulsions induced by pentylenetetrazole. * denotes significance at p <0.05 in comparison to vehicle treated group

Treatment Dose (mg/kg) Onset of death (secs)

Vehicle 743 ±80

VVDR03 20 437±35

VVDR03 40 581 ±54

VVDR03 80 1257 ±140*

VVW02 20 566±50

VVW02 40 740.3±30

VVW02 80 1068±90*

Diazepam 1 No death

Table 3.2. Effect of different doses of V-PA and V-MA on convulsions induced by pentylenetetrazole. * denotes significance at p <0.05 in comparison to vehicle treated group

Treatment Dose (mg/kg) Onset of death (secs)

Vehicle 513±69

V-PA 20 323±67.9

V-PA 40 592.2±23

V-PA 80 1972±118*

V-MA 20 538±28

V-MA 40 735.7±39.6 *

V-MA 80 360±50

Diazepam 1 No death

Table 3.3. Effect of single administration different doses of VVDR03 and VVW02 on elevated plus maze

Treatment Dose Number of entries Time duration (secs) (mg/kg) (Closed arm) (Closed arm)

Vehicle 8±2 237.3±19.7

VVDR03 10 5±1.2 257±10.0

VVDR03 20 6±0.47 230±20.0

VVDR03 40 4±1 260±11.0

VVW02 10 3.8±1.39 265.2±14.7

VVW02 20 4±1.67 276±6.84

VVW02 40 4.4 ±1.83 248.4±42.4

Table 3.4. Effect of single administration of different doses of V-PA and V-MA on

elevated plus maze

Treatment Dose Number of entries Time duration (secs) (mg/kg) (Closed arm) (Closed arm)

Vehicle 5.3±0.3 211±42.8

V-PA 10 6.0±0.9 260±13

V-PA 20 5.3±1.7 262±23

V-PA 40 4.3± 1.3 251.3±31.4

V-MA 10 5.0±0.9 242±28

V-MA 20 4.6 ±1 237±48.5

V-MA 40 4± 1 271±3

Table 3.5. Effect of different doses of VVDR03 and VVW02 on elevated plus maze after 14 days of dosing.

Treatment Dose Number of entries Time duration (secs) (mg/kg) (Closed arm) (Closed arm)

Vehicle 8±2 237.3±19.7

VVDR03 10 4.2±1.7 222.6±13

VVDR03 20 6±2.1 211±9.8

VVDR03 40 9± 3.8 234±15.6

VVW02 10 5.2±0.47 209±14.3

VVW02 20 9.5±2.17 236±17.7

VVW02 40 6.6 ±1.8 242±16.1

Table 3.6. Effect of different doses of V-PA and V-MA in plus maze after 14 days of dosing Treatment Dose Number of entries Time duration (secs) (mg/kg) (Closed arm) (Closed arm)

Vehicle 3.6±1.2 230.7±19.7

V-PA 10 5±1.9 260±13.0

V-PA 20 3.4±0.7 278.2±9.8

V-PA 40 4.6±1.9 228.2±25.3

V-MA 10 6±1.2 222±28.0

V-MA 20 5±1.3 209.2±39.6

V-MA 40 7.2±2 257.8±10.6

0

50

100

150

200

250

300

350

Vehicle VVDR03(40)

VVDR03(20)

VVDR03(10)

Diazepam(1)

Tim

e of

fall

(sec

s)

0 min60 min

*

Fig. 3.1. Effect of single oral administration of VVDR03 (10, 20 & 40 mg/kg) on rota rod apparatus. * denotes significance at p <0.05 in comparison to 0 min reading

0

50

100

150

200

250

300

350

Vehicle VVW02(40)

VVW02(20)

VVW02(10)

Diazepam(1)

Tim

e of

fall

(sec

s)

0 min60 min

*

Fig. 3.2. Effect of single oral administration of VVW02 (10, 20 & 40 mg/kg) on rota

rod apparatus. * denotes significance at p <0.05 in comparison to 0 min reading

0

50

100

150

200

250

300

350

Vehicle V-PA(40) V-PA(20) V-PA (10) Diazepam(1)

Tim

e of

fall

(sec

s)

0 min60 min

*

Fig. 3.3. Effect of single oral administration of V-PA (10, 20 & 40 mg/kg) on rota rod apparatus. * denotes significance at p <0.05 in comparison to 0 min reading

0

50

100

150

200

250

300

350

Vehicle V-MA(40) V-MA(20) V-MA (10) Diazepam(1)

Tim

e of

fall

(sec

s)

0 min60 min

*

Fig. 3.4. Effect of single oral administration of V-MA (10, 20 & 40 mg/kg) on rota rod apparatus. * denotes significance at p <0.05 in comparison to 0 min reading

3.4. DISCUSSION

The present study reports some neuropharmacological activities of valerian

extracts and oils in mice. Epilepsy is a common neurological disorder affecting about

0.5-1 % of the world’s population (Hackinski, 1998). The most popular and widely

used animal seizure models are the traditional Maximal Electroshock Seizures and

PTZ tests. Prevention of seizures induced by PTZ in laboratory animals is the most

commonly used preliminary screening test for characterizing potential anticonvulsant

drugs. Though the PTZ test predicts activity against absence seizures, the underlying

neuronal abnormality is poorly understand. Diminution of brain GABA level has been

reported after sub convulsive dose of PTZ (Ha et al., 2000). The MES test is

considered to be a predictor of likely therapeutic efficacy against generalized tonic-

clonic seizures. By contrast, the PTZ assay has been used preliminarily to evaluate

antiepileptic drugs. A large body of experimental evidences supports the involvement

of γ-Amino butyric acid (GABA) in seizures. GABA is the principal inhibitory

neurotransmitter in the cerebral cortex maintaining the inhibitory tone that

counterbalances neurons excitation. When this balance is disturbed, seizures ensue.

Reduction in GABA-mediated inhibitory activity of glutamate decarboxylase has

been reported in studies of human epileptic brain tissues (David, 2001). Glutamate

concentration increases before seizure onset and is found to be highest in the epileptic

hippocampus than non-epileptic hippocampus while GABA levels increased during

seizures was greater in non-epileptic hippocampus than in epileptic hippocampus

showing decreased levels of GABA in epilepsy (During and Spancer, 1993). The

GABAA receptor is responsible for most fast inhibitory neurotransmission in the

central nervous system. Consequently, this receptor has been targeted for the

pharmacological control of anxiety, sleep, and epilepsy. Numerous natural and

synthetic compounds interact with the GABAA receptor at distinct, yet incompletely

defined, sites. These compounds include barbiturates, benzodiazepines, neurosteroids

and picrotoxin (Sieghart, 1992; Smith and Olsen, 1995). GABAA receptor agonists as

well as drugs, which allosterically modulate the GABA receptor channel complex, are

therapeutically active against convulsive seizures (David, 2001).

In our study valerian extracts and oils were found to have delayed onset of

death in comparison to vehicle treated group while no effect was produced on onset of

convulsions and it failed to produce protection against mortality. There is an assertion

also that most drugs with anticonvulsant activity do not counteract pentylenetetrazole

seizures but only retard them (Loscher et al., 1991). We found that both VVDR03 and

VVW02 produced a dose dependent effect in delaying onset of death and the effect

being significant at 80 mg/kg dose in comparison to control group. But none of the

dose prolonged the time of onset of convulsions as compared to control group. The

delay in onset of death produced by valerian extracts may result from the effect of its

components on inhibitory neurotransmitter GABA (Menini et al., 1993; Santos et

al.,1994a, 1994 b). The effect produced by VVDR03 and VVW02 is comparable. It is

suggested that different constituents of valerian extract interact with the GABA

system in the brain like inhibition of GABA transaminase, interaction with

GABA/benzodiazepine receptors and interference in uptake and release of GABA in

synaptosomes (Morazzoni and Bombardell, 1995, Houghton, 1999). GABA is the

major inhibitory neurotransmitter in the brain and the inhibition of its

neurotransmission has thought to be an underlying factor in epilepsy (Olsen, 1981).

Standard antiepileptic drugs, phenobarbitone and diazepam are thought to produce

their antiepileptic effects by enhancing GABA neurotransmission. This also may be

responsible for anticonvulsant and sedative action produced by V. wallichii.

Overproduction of NO has been linked to neurotoxicity during ischemia, some

forms of neurodegenerative brain diseases, and induction of seizures (Garthwaite,

1991). Nitric oxide (NO) has been linked to epileptic activity through the formation of

cyclic GMP (cGMP). Stimulation of the brain N-methyl-D-aspartate (NMDA)

receptors with glutamate or excitatory amino acids increases calcium influx, which

results in activation of cNOS and formation of NO. NO in turn activates guanylate

cyclase to synthesize cGMP, which is assumed to initiate seizures (Garthwaite, 1991).

NO modulates experimentally induced seizures in a complex manner. In addition to

stimulation of cGMP, NO has several other actions: it blocks NMDA receptors in a

negative feedback manner, thereby attenuating excitable activity (Manzoni et al.,

1992); promotes and suppresses glutamate release (Pelligrino et al., 1996) and reduces

the receptor activity of the inhibitory γ-aminobutyric acid (GABA) neurotransmitter

(Robello et al., 1996). PTZ induction of seizures may be related to the antagonistic

activity of the compound at the GABA-A receptor and to its activation of the NMDA

receptor (Kaputlu and Uzbay, 1997). Thus, the induced NO may augment the

capability of PTZ to induce seizures at both pathways. In case of valerian oils V-PA

produced a dose dependent effect and the results being significant at 80 mg/kg in

comparison to control group. V-MA produced an inverted U shaped curve in which

the effect produced at 40 mg/kg is significant in comparison to control group.

Valerian oil V-PA was found to be most effective in delaying onset of death in PTZ-

induced convulsions. Valerian oils may be effective because of terpenes present in

them as some researchers have reported anticonvulsant activity of monoterpenes

(Librowski et al., 2000). Therefore it seems that antiseizure profile of V. wallichii oils

may be related in part to terpenoids present in them like α-pinene, camphene and

terpineol (Arora and Arora, 1963) and the terpenes might be mediating anticonvulsant

effect by affecting nitric oxide pathway (Ahn et al., 2003). In another study done by

us we found that valerian oils produced an antidepressant effect mediated by NO

pathway and a similar inverted U-shaped activity curve or biphasic dose response

curve was observed with V-MA in producing antidepressant effect. Such biphasic

curve has been reported in other studies and it may be due to facilitatory and

inhibitory effect of NO on the NMDA receptor. In addition such mechanisms may

help explain why so many contradictory results have been found suggesting that NO

either facilitates or inhibits NMDA receptor mediated events such as seizures

(Rundfeldt et al., 1995), neurotoxicity (Weissman et al., 1992), anxiolysis and

nociception (Harkin et al., 1999).

The elevated plus maze (EPM) is a rodent model of anxiety that is used as a

screening test for putative anxiolytic compounds and as a general research tool in

neurobiological anxiety research. The test setting consists of a plus-shaped apparatus

with two open and two enclosed arms, each with an open roof, elevated 15 cm from

the floor. The model is based on rodents aversion for open spaces. This aversion leads

to the behavior termed thigmotaxis, which involves avoidance of open areas by

confining movements to enclosed spaces or to the edges of a bounded space (Pellow

et al., 1985; Treit et al., 1993; Rodgers, 1997; Carobrez and Bertoglio, 2005). So the

total time duration spent in closed arm was noted and compared with that of control

and standard drug treated group. Valerian extracts and oils were not found to have

neither anxiolytic nor muscle relaxant effect at the tested doses (10, 20 and 40 mg/kg

p.o).

CHAPTER-4

Screening of Valeriana wallichii chemotypes for Antidepressant effect

4.1. INTRODUCTION

Depression has attracted strong attention as a major public health problem

worldwide that’s why prevention and treatment of depression are urgent issues need

to be resolved. Exposure to chronic stress is one of the major factor that provoke

depressive illness (Sakakibara et al., 2008). The abnormalities of noradrenergic and

serotonergic transmission are known to cause mental depression. Subsequently a

dopamine hypothesis of depression was also put forward (Dhir and Kulkarni, 2007).

Biochemical theories of mood disorders have focused on the biogenic amines but also

the importance of gamma butyric acid (GABA) in the neurochemical pathophysiology

of depression has been demonstrated (Petty, 1995). Clinical data also indicate that a

decreased GABA function accompanies depression and there is accumulating

evidence which implicates a GABAergic dysfunction in depression (Kubacka et al.,

2006). On the other hand there are several evidences which suggest that deficiency of

behaviors in depressive illness is associated with an increase in presynaptic inhibitory

α2 receptors leading to a secondary decrease in norepinephrine (NE) release (David,

2006). Drugs that block α2 receptors and increase NE release have been shown to have

antidepressant effect in various animal models of depression.

Previous studies have demonstrated that antagonists at the NMDA receptor are

as efficacious as tricyclic antidepressants in pre-clinical antidepressant screening

procedures and in blocking or reversing the behavioral deficits associated with animal

analogs of major depressive symptomatology. The NMDA receptor complex gates

Ca2+ (Southam and Garthwaite, 1993), which interacts with calmodulin to

subsequently activate nitric oxide (NO) synthase. Since NMDA receptor antagonists

possess significant antidepressant-like properties (Trullas, 1997), it is hypothesized

that NO synthase inhibitors would possess antidepressant properties and there are

some reports supporting this hypothesis (Harkin et al., 1999). So L-arginine-nitric

oxide (NO)-cyclic guanosine monophosphate (cGMP) is an important signaling

pathway involved in depression (Dhir and Kulkarni, 2008).

So whatever is the cause there is a wide variety of antidepressant drugs

available to treat depression but most of the synthetic drugs are not without side

effects. On the other hand, drugs obtained from natural sources are perceived to have

the least risk and low side-effect profiles, while having the ability to cure psychiatric

disorders in much the same way as their synthetic counterparts. Ayurveda, the ancient

traditional system of medicine, mentions a number of single and compound drug

formulations of plant origin that are used for the treatment of psychiatric disorders.

Recently several clinical studies have presented some biological activities of valerian

root extracts and introduced the plant as anti-HIV (Murukami et al., 2002), sleep aid

(Francis and Dempster, 2002; Fernandez et al., 2004), tranquilizer (Houghton, 1999),

antidepressant (Andreatini et al., 2002; Miyasaka et al., 2006; Hattesohl et al., 2008)

agent. So the aim of the present study is to evaluate the effect of Valeriana wallichii

chemotypes on forced swim test and to explore the mechanism responsible for this

effect as there are few studies done on antidepressant-like effect of herbal

formulations like Trans-a1 (Shalam et al., 2007) and Sumind (Nanjappa et al., 2007)

containing Valeriana wallichii as one of the ingredient.

4.2. MATERIALS AND METHODS

4.2.1. Plant material and extraction

Same as in section 3.2.1. of chapter 3.

4.2.2. Animals

Same as in section 3.2.2. of chapter 3.

4.2.3. Drugs and treatment

Imipramine, L-NAME (N-omega-nitro-l-arginine-methyl-ester) a nitric oxide

synthase inhibitor were used. Extracts VVDR03, VVW02 and their oils V-PA and V-

MA were used at doses of 10, 20 and 40 mg/kg. Both the extracts and oils were

administered orally as an emulsion in 2% Tween-80 to the animals one hour prior to

forced swim test (FST). And then effect of extracts and oils in combination with L-

NAME (10 mg/kg, intraperitoneally) was studied in FST for mechanistic study.

4.2.4. Procedure

Locomotor activity

Six groups of animals were fed orally with VWDRO3 and VVWO2 at a dose

of 10, 20 and 40 mg/kg and another six groups were given 10, 20 and 40 mg/kg dose

of V-PA and V-MA. One group was given vehicle and acted as control and another

group received imipramine (10mg/kg i.p). The locomotor activity was monitored after

one hour using actophotometer (IMCORP, India). The animals were individually

placed in activity meter for three minutes before counting of actual locomotor activity

for the next 5 minutes. The locomotor activity was expressed in terms of total

photobeams counts, 5 min per animal (Reddy, 1998). Percentage increase or decrease

in locomotion was calculated.

Forced swim test

Briefly, a group of 64 mice was randomly divided into 14 groups and treated

as follows:

Group 1: Vehicle treated (control group)

Group 2-4: VVDR03 (10, 40 and 40 mg/kg p.o.)

Group 5-7: VVW02 (10, 20 and 40 mg/kg p.o.)

Group 8-10: V-PA (10, 20 and 40 mg/kg p.o.)

Group 11-13: V-MA (10, 20 and 40 mg/kg p.o)

Group 14: Impiramine (10 m/kg, p.o.)

Then one hour after the treatment mice were individually forced to swim

inside a rectangular glass jar (25x12x25 cm3) containing 15 cm of water maintained at

23–25°C. After the initial 2–3 min of vigorous activity the animals showed period of

immobility by floating with minimum movements. An animal is considered to be

immobile whenever it remained floating passively in the water in a slightly hunched

but upright position, its nose above the water surface (Fig. 4.1.). The total immobility

period for the period of 6 minutes was recorded with the help of stop-watch (Kulkarni

and Mehta, 1985).

Fig.4.1. Rectangular jar depicting the mobile (left) and immobile (right) postures of the animals

Extracts and oils in combination with L-NAME in forced swim test

In order to explore mechanism of action of valerian in decreasing immobility

period in FST, mice received following treatments:

Group 1: Vehicle treated (control group)

Group 2: L-NAME (10 mg/kg i.p.)

Group 3: VVDR03 (10 mg/kg, p.o.) + L-NAME (10 mg/kg i.p.)

Group 4: VVW02 (10 mg/kg, p.o.) + L-NAME (10 mg/kg i.p.)

Group 5: V-PA (10 mg/kg, p.o.) + L-NAME (10 mg/kg i.p.)

Group 6: V-MA (10 mg/kg, p.o.) + L-NAME (10 mg/kg i.p.)

All the test drugs were administered orally. L-NAME was given half an hour

after administration of test drugs and then mice were allowed to swim in Porsolt

apparatus 30 minutes after intraperitoneal injection of L-NAME.

4.2.5. Statistical analysis

The data were expressed as mean± SEM of 6 animals. Results were analysed

statistically by One-way ANOVA followed by Tukey’s multiple comparison using

sigma stat software. The difference was considered statistically significant if p<0.05.

4.3. RESULTS

4.3.1. Acute toxicity

Same as in section 3.3.1. of chapter 3.

4.3.2. Effect on locomotor activity

None of the dose of VVDR03 significantly decreased the locomotor activity

while VVW02 at 40 mg/kg produced significant decrease (45.8%) in locomotor

activity (Table 4.1.). V-MA did not alter the spontaneous locomotor activity except at

dose 40 mg/kg which significantly produced 44.9 % decrease in this parameter (Table

4.2.). While 40 mg/kg dose of V-PA produced a decrease in locomotor activity but the

effect was not significant. So the results depict that none of the doses of VVDR03 and

V-PA altered locomotor activity in mice. Imipramine the standard drug was also

devoid of any effect on locomotor activity.

4.3.3. Effect of acute dosing of extracts in FST

When single oral administration of different doses of VVDR03 and VVW02

were studied for effect in FST, 40 mg/kg dose of both the extracts (Fig. 4.2. & Fig.

4.3.) significantly decreased immobility period as compared to vehicle treated group.

The effect was found to be more pronounced with VVW02 (57% decrease in

immobility) than VVDR03 (32.7 % decrease in immobility) (statistical significant)

and group treated with standard drug imipramine (36.2 % decrease).

4.3.4. Effect of acute dosing of oils in FST

Different doses of V-PA produced a dose response curve i.e on increasing the

dose response was increased (Fig. 4.4.). The effect being significant at 40 mg/kg

(57.6% decrease) and 20 mg/kg (46.9% decrease). Whereas different doses of V-MA

produced an inverted U shaped curve, the effect being significant at 40 mg/kg and 10

mg/kg dose (Fig. 4.5.). 10 mg/kg produced 39.7% decrease while 40 mg/kg dose

produced 58% decrease in immobility as compared to control group. When compared

statistically both the oils were found equally effective in producing antidepressant

effect.

4.3.5. Effect of NOS inhibitor L-NAME on the action of VVDR03 and VVW02

(10 mg/kg) in FST

L-NAME was combined with 10 mg/kg dose of VVW02 and VVDR03 and

the modulation by L-NAME was then studied after half and hour of L-NAME

injection. L-NAME per se did not have any effect on the immobility period in FST

neither it potentiated the effect of VVDR03 and VVW02 (Fig 4.6.). The combination

did not change the locomotor activity (results not shown).

4.3.6. Effect of NOS inhibitor L-NAME on the action of V-PA and V-MA (10

mg/kg) in FST

To assess the modulatory effect of NOS inhibitor (L-NAME) on FST, V-PA,

V-MA (10 mg/kg p.o) was combined with L-NAME (10 mg/kg i.p). L-NAME per se

did not have any effect on the immobility period in FST. However, when L-NAME

was given 30 min after V-PA and V-MA it significantly potentiated the effect of V-

PA and V-MA (Fig. 4.7.). The combination did not increase the locomotor activity

(results not shown).

Table 4.1. Effect of different doses of extracts (VVDR03 & VVW02) of Valeriana wallichii in actophotometer apparatus in mice. (*) show significance at P<0.05 vs. vehicle group. Value in parenthesis is % decrease in locomotor counts. Treatment Dose (mg/kg. p.o) Mean ambulatory scores

Vehicle 240 ±32.4

VVDR03 10 235±64

VVDR03 20 139.6±65.4

VVDR03 40 210 ±58.5

VVW02 10 188.75±10

VVW02 20 105.3±62.7

VVW02 40 139±21 (45.8) *

Imipramine 10 200 ±6

Table 4.2. Effect of different doses of oils (V-MA & V-PA) of Valeriana wallichii in actophotometer apparatus in mice. (*) show significance at P<0.05 vs. vehicle group. Value in parenthesis is % decrease in locomotor counts.

Treatment Dose (mg/kg. p.o) Mean ambulatory scores

Vehicle 414 ±13.6

V-PA 10 383 ±45.4

V-PA 20 415 ±62.9

V-PA 40 319 ±36

V-MA 10 391±40

V-MA 20 324± 20.3

V-MA 40 228 ±48.7*(44.9)

Imipramine 10 300 ±40

Fig. 4.2. Effect of valerian extract VVW02 on the immobility period induced by FST. VVW02 at different doses were administered orally 60 min before FST challenge. *P<0.05 vs. vehicle group.

Fig. 4.3. Effect of valerian extract VVDR03 on the immobility period induced by FST. VVDR03 at different doses were administered orally 60 min before FST challenge. *P<0.05 vs. vehicle group.

Fig. 4.4. Effect of V-PA on the immobility period induced by FST. V-PA at different doses was administered orally 60 min before FST challenge. *P<0.05 vs. vehicle group.

Fig. 4.5. Effect of V-MA on the immobility period induced by FST. V-MA at different doses was administered orally 60 min before FST challenge. *P<0.05 vs. vehicle group.

Fig. 4.6. Effect of 10 mg/kg dose of VVDR03 & VVW02 in combination with L-NAME (10mg/kg i.p.).

Fig. 4.7. Effect of 10 mg/kg dose of V-PA & V-MA in combination with L-NAME (10mg/kg i.p.). a denotes significance at P < 0.05 in comparison to L-NAME per se, b denotes significance at P < 0.05 in comparison to V-PA per se and c denotes significance at P < 0.05 in comparison to V-MA per se. # denotes significance at P < 0.05 in comparison to vehicle treated group.

4.4. DISCUSSION

One of the purposes of this study was to evaluate the antidepressant-like effect

of valeriana by an animal model for depression. We employed the forced swimming

test (FST) on mice. The immobility displayed by rodents when subjected to an

unavoidable stress such as forced swimming is thought to reflect state of despair or

lowered mood, which reflects depressive illness in humans. Additionally, immobility

time has been shown to be reduced by treatment with antidepressant drugs (Porsolt,

1981). The FST was designed to detect potential antidepressant compounds based on

the abilities of clinically effective antidepressants to reduce the immobility in FST

(Porsolt et al., 1977). Though the FST does not adequately reflect the symptomalogy

of human depression, it appears to have a higher predictive validity compared to other

animal models Additionally, it is sufficiently specific, since it discriminates

antidepressants from neuroleptics and anxiolytics (Borsini and Meli, 1988; Willner,

1984). The present study has shown that valerian oils and extracts posses

antidepressant effect in FST.

In our study DCM extracts of both the chemotypes (VVW02 and VVDR03) at

40mg/kg dose significantly reduced the immobility time in FST in acute study.

VVW02 was found to produce 57 % decrease in immobility at 40 mg/kg as compared

to vehicle treated control group (Fig. 4.2.) while 40 mg/kg dose of VVDR03 produced

32.7% decrease in immobility which is comparable to the effect produced by standard

drug imipramine (Fig. 4.3.). In case of valerian oils V-MA produced significant

decrease in immobility at dosage 10 and 40 mg/kg while 20 mg/kg did not affect the

immobility period (Fig.4.5.) and produced an inverted U shaped curve. V-PA in single

dose study produced a dose dependent effect i.e. 20 and 40 mg/kg dose of V-PA

produced a significant decrease in immobility as compared to group receiving 10

mg/kg and control group (Fig.4.4.). Psychostimulants, which exert an indiscriminate

motor stimulating activity, have previously been shown to have efficacy at decreasing

immobility in FST, but no antidepressant activity (Porsolt, 1981). So in order to

exclude a false positive result, we employed an additional test to check motor

stimulating activity of extracts and oils. In the present study VVW02 and V-MA

lowered the locomotor activity at 40 mg/kg dose only whereas no alteration was seen

with VVDR03 and V-PA. Therefore the reduction in immobility by valerian extract

time can be attributed to an inherent antidepressant like effect.

A study depicted antidepressant effect of kessyl glycol diacetate, a valepotriate

possibly due to blockade of monoamine uptake (Miller and Murray, 1998). Methanol

extracts of Japanese valerian (Valeriana fauriei) also exhibited strong antidepressant

activity in the forced swimming test in mice (Oshima and Matsuoka, 1995). The

active fraction was identified as a sesquiterpenoid called a kessyl alcohol and its

activity compared favorably to that of imipramine. These studies support earlier

findings of a Japanese valerian root extract which significantly inhibited immobility

induced by a forced swimming test in rats (Sakomoto et al., 1992). Another recent

study showed anti-anxiety and anti-depressant benefits of valerian but failed to show

sedative or muscle relaxant benefits (Hattesohl et al., 2008). A study done on

Japanese valerian confirms that valerian extract and imipramine (a tricyclic anti-

depressant) significantly inhibited immobility in FST and reversed reserpine-induced

in mice, a test which suggests antidepressant actions (Sakamoto et al., 1992).

During the past few years, nitric oxide (NO), an inorganic free radical has

emerged as an important signal and effector molecule in several biological processes

including vasodilatation, inflammation and neurotransmission (Garthwaite, 1991;

Lowenstein, 1994; Moncada, 1991). NO is produced in neurons from the L-arginine

by the calmodium-dependent enzyme. NO synthase and NO fulfill some of the most

important criteria for a neurotransmitter (Knowles, 1994; Dawson, 1994). An

important target of NO is soluble guanlyl cyclase which is activated by NO binding to

heme part of the enzyme, causing elevation in cGMP. It appears that in different

systems, NO may be able to modulate vesicular release of neurotransmitter in either

direction or not all depending on the coincident level of presynaptic activity and NO

concentration (Garthwaite, 1995). In order to explore the involvement of nitric oxide

(NO) signaling pathway in the antidepressant-like effect of valerian extracts and oils,

10 mg/kg dose of VVDR03, VVW02, V-PA and V-MA were combined with L-

NAME (10 mg/kg) (a selective nitric oxide synthase inhibitor) (Babbedge et al., 1993;

Re es et al., 1990) and then tested for alteration in immobility in FST. No potentiation

was found in case of valerian extracts. It was found that L-NAME per se did not alter

the immobility period compared to control whereas it potentiated the action of V-PA

and V-MA significantly. The potentiation was even significant to group receiving L-

NAME only. The L-NAME was devoid of locomotor stimulatory property at 10

mg/kg dose which is in accordance to earlier study (Harkin et al., 1999). Moreover, a

study depicts that stimulation of NO synthase not only antagonized the behavioral

effect of NO synthase inhibitors but also of the prototypic antidepressant, imipramine.

So it may be possible that one or more component of essential oil (sesquiterpenes) of

Valeriana might be acting as NOS inhibitor as various studies report inhibition of NO

synthesis in lipopolysac-charide (LPS)-activated RAW 264.7 cells by sesquiterpenes

(Ahn et al., 2003; Motai and Kitanaka, 2005). In other studies, also sesquiterpenoids

have been found to have suppressive effect on NOS and COX-2 activity (Lee et al.,

2002; Yoon et al., 2008).

An inverted U-shaped activity curve or biphasic dose response curve observed

with V-MA has been reported in previous studies and it may be due to facilitatory and

inhibitory effect of NO on the NMDA receptor. NO regulates NMDA receptor

activity in a biphasic manner playing both a positive (via activation of guanylate

cyclase) and negative (via feedback effects on the NMDA receptor resulting in

decreased NMDA receptor and NO synthase activity) modulatory role in NMDA

receptor mediated events (Lei et al., 1992; Manzoni and Bockaert., 1993). In addition

such mechanisms may help explain why so many contradictory results have been

found suggesting that NO either facilitates or inhibits NMDA receptor mediated

events such as seizures (Rundfeldt et al., 1995), nociception, neurotoxicity (Weissman

et al., 1992) and anxiolysis (Harkin et al., 1999). Moreover, a study depicts that NO

has a dual role in the modulation of depression (Da Silva et al., 2000).

From our results it can be speculated that the level of NOS inhibition elicited

by valerian oils leads to a critical NO concentration which in turn, alters the vesicular

release of neurotransmitter like norepinephrine involved in depression. There are

other studies which also show that chronic blockade of nitric oxide synthesis elevates

plasma levels of catecholamines and their metabolites at rest and during stress in rats

(Richard et al., 1997). So we can say that the nitric oxide signaling pathway is

involved in the antidepressant-like effect of Valerian oils while valerian extracts

might be producing antidepressant by potentiating the action of GABA. As it has been

discussed that GABAergic dysfunction is also involved in depression (Kubacka et al.,

2006) and valepotriates and other constituents present in the extracts tends to enhance

the action of GABA (Riedel, 1982).

CHAPTER-5

Pharmacological and Neurobiochemical Evidence for Antidepressant like Effect of

Valeriana wallichii chemotypes

5.1. INTRODUCTION

Major depression has a lifetime prevalence rate of 17% (Andrews and

Nemeroff, 1994). Depression is more common in women and is seen throughout the

life. A major depressive disorder may occur as either a single or recurrent event.

Despite the advent of new molecules in the pharmacotherapy of depression, it is

unfortunate that this disorder goes undiagnosed and untreated. Although the currently

prescribed molecules provide some improvement in the clinical condition of the

patient, it is at the cost of having to bear the burden of their adverse effects (Stahl,

1998; Tripathi, 2005; Hardman et al., 2001). On the other hand, drugs obtained from

natural sources are perceived to have the least risk and low side-effect profiles, while

having the ability to cure psychiatric disorders in much the same way as their

synthetic counterparts. Ayurveda, the ancient traditional system of medicine,

mentions a number of single and compound drug formulations of plant origin that are

used for the treatment of psychiatric disorders. Patients with depression have

symptoms that generally reflect changes in the brain neurotransmitters serotonin,

norepinephrine, and dopamine. The "dysregulation hypothesis" proposes that

depression may be due to abnormalities in the pattern and responsiveness of

neurotransmitter systems that result from modifications in the receptor sensitivity and

function. Antidepressants are thought to increase levels of norepinephrine, serotonin,

or dopamine at the synapse to restore equilibrium to neurotransmitter activity.

The previous study revealed antidepressant effect of Valerian oils and extracts.

So this study aims at evaluating the alterations in neurotransmitter levels after 14 days

dosing of Valeriana wallichii extracts and oils.

5.2. MATERIALS AND METHODS

5.2.1. Plant material and extraction

Same as in section 3.2.1. of chapter 3.

5.2.2. Animals

Same as in section 3.2.2. of chapter 3.

5.2.3. Drugs and treatment

Extracts VVDR03, VVWO2 and their oils V-PA and V-MA were used at

doses of 10, 20 and 40 mg/kg. Both the extracts and oils were administered orally as

suspension in Tween-80 to the animals for 14 days and then the effect in forced swim

test (FST) was seen one hour after the drug administration on 14th day.

5.2.4. Procedure

Forced swim test

Briefly, a group of 64 mice was randomly divided into 14 groups, each group

containing six animals and treated as follows: one group was administered

imipramine (10mg/kg), three groups were administered VVDR03 (10, 20, 40 mg/kg,

p.o), three groups received VVW02 (10, 20, 40 mg/kg, p.o), three groups received V-

PA (10, 20, 40 mg/kg, p.o) and other three groups received V-MA (10, 20, 40 mg/kg.

p.o). One group receiving vehicle served as control. All the groups received the

treatment continuously for two week. Then one hour after the last day treatment mice

were individually forced to swim inside a rectangular glass jar (25×12×25 cm3)

containing 15 cm of water maintained at 23–25°C. After the initial 2–3 min of

vigorous activity the animals showed period of immobility by floating with minimum

movements. Depression produced in animal is characterized by increased immobility

period. An animal is considered to be immobile whenever it remained floating

passively in the water in a slightly hunched but upright position, its nose above the

water surface. The total immobility period for the period of 6 minutes was recorded

with the help of stop-watch (Kulkarni and Mehta, 1985).

Measurement of biogenic amines

Biogenic amines (dopamine and norepinephrine) were estimated by high

performance liquid chromatography (HPLC) with electrochemical detector (ECD)

(Beyer et al., 2002). Waters® standard system consisting of a high pressure isocratic

pump, a 20µl sample injector valve, C18 reverse phase column and electrochemical

detector were used. Data were recorded and analyzed with the help of Empower

software. Mobile phase consisting of 0.15M NaH2PO4, 0.25mM EDTA, 1.75mM 1-

octane sulfonic acid, 2% isopropanol and 4% methanol (pH 4.8). Electrochemical

conditions for the experiment were +0.800 V, sensitivity ranges from 1 to 100 nA.

Separation was carried out at a flow rate of 1 ml/min. Samples (20µl) were injected

manually. Brain samples were homogenized in homogenizing solution containing

0.1M perchloric acid. After that samples were centrifuged at 24,000×g for 15 min.

The supernatant was further filtered through 0.25µm nylon filters before injecting in

the HPLC injection pump. Data were recorded and analyzed with the help of

Empower® software provided by Waters® (Beyer et al., 2002).

5.2.5. Statistical analysis

The data were expressed as mean± SEM of 6 animals. Results were analysed

statistically by One-way ANOVA followed by Tukey’s multiple comparison using

Jandel Sigma Stat Software. The difference was considered statistically significant at

p<0.05.

5.3. RESULTS

5.3.1. Acute toxicity

Same as in section 3.3.1 of chapter 3.

5.3.2. Effect of extracts in FST after 14 days of dosing

VVDR03 and VVW02 were found to produce a dose dependent response

curve in chronic study. 20 and 40 mg/kg dose of both the extracts decreased

immobility period significantly as compared to control group. The decrease being

30.8% and 55.9 % with VVW02 (Fig. 5.1) while VVDR03 (Fig. 5.2) produced 17.2

% and 45 % decrease in immobility in FST.

5.3.3. Effect of oils in FST after 14 days of dosing

Only 20 mg/kg dose of V-PA produced a significant decrease in immobility in

FST (Fig. 5.3.) as compared to vehicle treated group. V-PA (20 mg/kg) produced 69.6

% decrease which is much more marked than the effect produced by 40 mg/kg dose of

V-PA. 40 mg/kg dose of V-MA produced a significant decrease (70.9%) in

immobility while 20 mg/kg V-MA has no significant effect in FST (Fig. 5.4.).

5.3.4. Effect of extracts and oils on the neurotransmitter levels in mice brain

When checked for the alterations in the neurotransmitter levels after 14 days

of dosing 20 and 40 mg/kg dose of VVW02 increased norepinephrine levels dose

dependently and significantly. No alteration in dopamine levels is seen with any dose

of VVW02 (Fig. 5.5.). In case of VVDR03 both 20 and 40 mg/kg increased the

norepinephrine levels in whole brain significantly. The alterations being dose

dependent. None of the dose altered dopamine levels as compared to control except

20 mg/kg (Fig. 5.6.). 40 mg/kg and 20 mg/kg of V-PA increased norepinehrine levels

by 48.5 % and 84.96% respectively but the effect is significant at 20mg/kg dose (Fig.

5.7.). Similarly V-MA at 40 mg/kg produced a marked increase in norepinehrine

levels (Fig. 5.8.) while 20 mg/kg did not affect norepinehrine levels. None of the dose

of V-PA and V-MA altered dopamine levels in brain.

0

50

10 0

150

2 0 0

2 50

3 0 0

V ehicle V V W 0 2( 10 )

V V W 0 2( 2 0 )

V V W 0 2( 4 0 )

Mea

n im

mob

ility

per

iod

(sec

)

*

*

Fig. 5.1. Effect of VVW02 on the immobility period induced by FST. VVW02 at different doses was administered orally repeatedly for 14 days before FST challenge. *P<0.05 vs. vehicle group.

0

50

10 0

150

2 0 0

2 50

3 0 0

V ehicle V V D R 0 3( 10 )

V V D R 0 3( 2 0 )

V V D R 0 3( 4 0 )

Mea

n im

mob

ility

per

iod

(sec

)

*

*

Fig. 5.2. Effect of VVDR03 on the immobility period induced by FST. VVDR03 at different doses was administered orally repeatedly for 14 days before FST challenge. *P<0.05 vs. vehicle group.

0

50

10 0

150

2 0 0

2 50

V ehicle V - PA ( 10 ) V - PA ( 2 0 ) V - PA ( 4 0 )

Mea

n im

mob

ility

per

iod

(sec

)

* *

Fig. 5.3. Effect of V-PA on the immobility period induced by FST. V-PA at different doses was administered orally repeatedly for 14 days before FST challenge. *P<0.05 vs. vehicle group.

0

50

10 0

150

2 0 0

2 50

V ehicle V - M A ( 10 ) V - M A ( 2 0 ) V - M A ( 4 0 )Mea

n im

mob

ility

per

iod

(sec

)

*

Fig.5.4. Effect of V-MA on the immobility period induced by FST. V-MA at different doses was administered orally repeatedly for 14 days before FST challenge. *P<0.05 vs. vehicle group.

Fig. 5.5. Effect of different doses of VVW02 on the alteration in neurotransmitter levels in the mouse whole brain. VVW02 was administered for 14 days before sacrificing the animals. (*) P<0.05 as compared to vehicle treated group.

Fig. 5.6. Effect of different doses of VVDR03 on the alteration in neurotransmitter levels in the mouse whole brain. VVDR03 was administered for 14 days before sacrificing the animals. (*) P<0.05 as compared to vehicle treated group.

Fig. 5.7. Effect of different doses of V-PA on the alteration in neurotransmitter levels in the mouse whole brain. V-PA was administered for 14 days before sacrificing the animals. (*) P<0.05 as compared to vehicle treated group.

Fig. 5.8. Effect of different doses of V-MA on the alteration in neurotransmitter levels in the mouse whole brain. V-MA was administered for 14 days before sacrificing the animals. (*) P<0.05 as compared to vehicle treated group

5.4. DISCUSSION

In this chronic study repeated administration of VVW02 and VVDR03 for 14

days produced a dose dependent response curve (Fig. 5.1 & 5.2). The effect being

significant at 20 and 40 mg/kg for both the extracts and out of them VVW02 (Maaliol

chemotype) was found to more effective than VVDR03 (Patchouli alcohol

chemotype).

In case of oils, V-PA produced a U shaped curve and the effect was found to

be significant at 20mg/kg in comparison to control (Fig. 5.3). Such U-shaped activity

curve may be due to multiple receptor action and has also been reported for some

herbal medicines (Butterweck et al., 2000, Butterweck et al., 2001 & Sakakibara et

al., 2006). V-MA produced a dose dependent response curve and the effect being

significant at 40 mg/kg (Fig. 5.4).

When checked for the alterations in the neurotransmitter levels after 14 days

of dosing, 20 and 40 mg/kg dose of VVW02 increased norepinephrine levels dose

dependently and significantly. No alteration in dopamine levels was seen with any

dose of VVW02 (Fig. 5.5). Similarly 20 and 40 mg/kg dose of VVDR03 increased the

norepinephrine levels in whole brain significantly and dose dependently (Fig. 5.6).

None of the dose altered dopamine levels as compared to control except 20 mg/kg of

VVDR03. 20 mg/kg of V-PA increased norepinehrine levels significantly and V-MA

at 40 mg/kg produced a marked increase in norepinehrine levels (Fig. 5.7 & 5.8).

From the previous study done by us we found that a NOS inhibitor (L-NAME)

potentiated the antidepressant effect of valerian oils in FST depicting the alteration of

NO level in mice brain by valerian oils leading to antidepressant effect. So it appears

that in different systems, NO may be able to modulate vesicular release of

neurotransmitter in either direction or not all depending on the coincident level of

presynaptic activity and NO concentration (Garthwaite, 1995). There are other studies

which also show that blockade of nitric oxide synthesis elevates plasma levels of

catecholamines and their metabolites at rest and during stress in rats (Richard et al,

1997). So from our results it can be speculated that the level of nitric oxide synthase

(NOS) inhibition elicited by valerian oil leads to a critical NO concentration which in

turn, alters the vesicular release of neurotransmitter like norepinephrine involved in

depression and we can say that the nitric oxide signaling pathway is involved in the

antidepressant-like effect of V. wallichii oils. While in case of valerian extracts no

potentiation was seen with L-NAME which suggests that some other pathway is

involved in antidepressant effect of extracts (VVDR03 and VVW02).

Biochemical theories of mood disorders have focused on the biogenic amines

but the importance of gamma butyric acid (GABA) in the neurochemical

pathophysiology of depression has also been demonstrated (Petty, 1995). Clinical data

also indicate that a decreased GABA function accompanies depression and there are

accumulating evidences which implicates a GABAergic dysfunction in depression

(Kubacka et al., 2006). There are various studies showing that the constituents present

in valeriana enhance GABA levels (Morazzoni and Bombardell, 1995, Houghton,

1999). So we can hypothesize that valerian extracts might be altering norpeinephrine

levels in brain by enhancing GABA neurotransmission.

CHAPTER-6

Studies on Analgesic Activity of Valeriana wallichii chemotypes

6.1. INTRODUCTION

Pain is a sensorial modality which is universally understood as a signal of

disease and it is the most common symptom requiring treatment with analgesic

agents. So pain often has a protective function. Throughout history, man has used

many different forms of therapy for the relief of pain, among them; medicinal herbs

are highlighted due to their wide popular use. In the relief of pain, opiates are

generally considered to act on the central nervous system exercising their effects

through three opioid receptors (µ, κ and δ) and such drugs are especially important for

the treatment of chronic pain. Although morphine has reigned for centuries as the king

of painkillers, its rule hasn’t been totally benign. There are concerns about its

addictive properties and side effects, which include respiratory depression,

drowsiness, decreased gastrointestinal motility, nausea and several alterations of the

endocrine and autonomic nervous system. Natural products in general and medicinal

plants in particular, are believed to be an important source of new chemical

substances with potential therapeutic efficacy. Taking into account the most important

analgesic, prototypes (e.g. salicylic acid and morphine) were originally derived from

the plant sources. The study of plant species traditionally used as pain killers should

still be seen as a fruitful research strategy in the search of new analgesic and anti-

inflammatory drugs.

Valeriana wallichii or Indian Valerian is a plant that steeped in history. Its

original use in herbal medicine today is as sedative and calming agent. Valepotriates

are responsible for the sedative action of the plant. It is used as a carminative and

antispasmodic in hysteria and similar nervous manifestations. Spasmolytic effect of

the V. wallichii has been used in different gastrointestinal disorders such as diarrhea

and abdominal spasm (Holmes, 1989). The studies depict that mild myorelaxant

action of Valeriana is attributed to the valepotriates component of the herb (Dunaev et

al., 1987). The valepotriates isovaltrate and valtrate, and the essential oil compound

valeranone were observed to suppress the rhythmic contractions in a closed part of the

guinea-pig ileum in vivo (Hazelhoff et al., 1982). The same compounds and

didrovaltrate relaxed potassium stimulated contractures and inhibited BaCl2

contractions in guinea-pig ileum preparations in vitro. The inhibition of muscle

contractions by the valium chemicals valeranone and didrovaltrate were as potent as

papaverine. There is a report showing analgesic effect of dried leaves of V. jatamansi

at a dose of 2 mg (Shrivastava and Sisodia, 1970). It has potential ethnomedicinal

uses like V. officinalis but till date there is no evidence-based clinical report. In this

context, we aimed to investigate the medicinal plant, V. wallichii for its analgesic

effect.

6.2. MATERIAL AND METHODS

6.2.1. Plant material and its extraction

Same as in section 3.2.1. of chapter 3.

6.2.2. Animals

Same as in section 3.2.2. of chapter 3.

6.2.3. Drugs

Aspirin was procured from Panacea Biotech, Lalru, India. To the test drugs

(VVW02, VVDR03, V-PA and V-MA) one drop of Tween-80 was added and then

volume was made up with distilled water. Solution of aspirin was also prepared using

Tween-80.

6.2.4. Procedure

Effect of valerian extracts and oils in Acetic acid-induced writhing

Acetic acid (1%) was injected i.p. in mice and the writhing response

characterized by abdominal constriction and hind limb stretching was counted for 10

min (Kulkarni, 1999). V-PA, V-MA, VVDR03 and VVW02 at doses of 20, 40 and 80

mg/kg were administered orally to mice 1 hr before the test. Aspirin 100mg/kg, p.o.

was used as a standard for comparison while vehicle treated group was kept as

control. The number of writhings in these groups were recorded and compared with

control and standard groups.

Effect of valerian extracts and oils on tail-flick latency

Analgesia was assessed with analgesiometer (Techno Electronics, India).

Basal reaction time of animals to radiant heat was recorded by placing the tip (last 1-2

cm) of the tail on the radiant heat source. The tail withdrawal from the heat (flicking

response) was taken as the end point. The animals, which showed flicking response

within 3-5 secs, were selected for the study. A cut off period of 12 secs was observed

to avoid damage to the tail (Kulkarni, 1999). The measurement of tail flick latency

was recorded at 15, 30, 60, 120 and 180 min after administration of V-PA, V-MA,

VVDR03 and VVW02 (20, 40 and 80 mg/kg).

Effect of valerian extracts and oils in combination with aspirin in acetic acid

writhing

For mechanistic study sub-effective dose i.e 20 mg/kg dose of VVDR03,

VVW02, V-PA and V-MA was combined with 5 mg/kg dose of aspirin and was then

studied in acetic acid induced writhing. Aspirin was given intraperitoneally 30

minutes after the administration of test drugs and acetic acid was injected 30 minutes

after aspirin. Per se effect of 5 mg/kg dose of aspirin was also studied in acetic acid

writhing.

6.2.5. Statistical analysis

The data were expressed as mean± SEM of 6 animals. Results were analysed

statistically by One-way ANOVA followed by Tukey’s multiple comparison using

sigma stat software. The difference was considered significant at p<0.05.

6.3. RESULTS

Acute toxicity

Same as in section 3.3.1 of chapter 3.

Effect of valerian extracts and oils in Acetic acid-induced writhing

Different doses of VVDR03 and VVW02 dose dependently inhibited the

number of writhings. VVDR03 produced significant inhibition of writhing at 80mg/kg

(54.3%) and 40 mg/kg (29.8%) (Fig. 6.1.). While VVW02 produced significant effect

only at 80 mg/kg and the percentage inhibition with it was found to be 29.8% (Fig

6.2.). The standard drug aspirin at 100mg/kg dose significantly inhibited the writhing

movements (64.9%) and the results were found to be highly significant (p<0.05) in

comparison to control group.

Similarly V-MA and V-PA at doses 20, 40 and 80 mg/kg, p.o. produced dose

dependent inhibition. V-PA at doses 80 and 40 mg/kg produced significant inhibition

of writhings i.e. 50% and 31.1% respectively (Fig. 6.3.). V-MA produced significant

inhibition in number of writhings at 80 mg/kg (63.6 %) and 40 mg/kg (27.2 %) as

compared to control group (Fig. 6.4.).

Effect of valerian extracts and oils on tail flick latency

In tail flick model different doses of VVDR03 (Table 6.1.) and V-PA (Table

6.3.) showed no significant increase in tail flick latency at different time interval when

compared to the predrug reaction time. But both VVW02 (Table 6.2.) and V-MA

(Table 6.4.) at 80 mg/kg dose produced significant increase in tail flick latency after

two hours of drug administration in comparison to predrug reaction. Similarly the

standard drug aspirin produced significant increase in tail flick latency after one and

two hours of drug administration.

Effect of valerian extracts in combination with aspirin in acetic acid writhing

Aspirin (5 mg/kg i.p) produced significant inhibition of writhings as compared

to vehicle treated group. But when 20 mg/kg dose of VVDR03 and VVW02 was

given in combination with aspirin (5 mg/kg) no potentiation was seen.

Effect of valerian oils in combination with aspirin in acetic acid writhing

V-PA (20 mg/kg) per se was found to have no effect on acetic acid writhing.

Aspirin per se produced 35% inhibition of writhing as compared to vehicle treated

group while when aspirin was given in combination with V-PA (20 mg/kg)

percentage inhibition was found to be 61.7% (Fig. 6.7.). Similarly V-MA in

combination with aspirin produced 52.4 % decrease in number of writhings (Fig.

6.8.). Thus the results show that aspirin potentiated the action of V-MA and V-PA in

acetic acid induced writhing.

0

5

10

15

2 0

2 5

3 0

3 5

4 0

4 5

V V D R 0 3( 2 0 )

V V D R 0 3( 4 0 )

V V D R 0 3( 8 0 )

V ehicle A sp ir in

Num

ber o

f writ

hing

s

*

*

*

Fig. 6.1. Effect of different doses of VVDR03 in acetic acid induced writhing in mice. Aspirin (100 mg/kg p.o) was used as standard. (*) denotes significance at P<0.05 versus vehicle treated group

0

5

1015

2 0

2 5

3 0

3 54 0

4 5

50

V V W 0 2( 2 0 )

V V W 0 2( 4 0 )

V V W 0 2( 8 0 )

V ehicle A sp ir in

Num

ber o

f writ

hing

s

*

*

Fig. 6.2. Effect of different doses of VVW02 in acetic acid induced writhing in mice. Aspirin (100 mg/kg p.o) was used as standard. (*) denotes significance at P<0.05 versus vehicle treated group

0

5

10

15

2 0

2 5

3 0

3 5

4 0

4 5

V - PA( 2 0 )

V - PA( 4 0 )

V - PA( 8 0 )

V ehicle A sp ir in

Num

ber o

f writ

hing

s

* *

*

Fig. 6.3. Effect of different doses of V-PA in acetic acid induced writhing in mice. Aspirin (100 mg/kg p.o) was used as standard. (*) denotes significance at P<0.05 versus vehicle treated group.

0

5

10

15

2 0

2 5

3 0

3 5

4 0

4 5

V - M A( 2 0 )

V - M A( 4 0 )

V - M A( 8 0 )

V ehicle A sp ir in

Num

ber o

f writ

hing

s

*

*

*

Fig. 6.4. Effect of different doses of V-MA in acetic acid induced writhing in mice. Aspirin (100 mg/kg p.o) was used as standard. (*) denotes significance at P<0.05 versus vehicle treated group. Table 6.1. Effect of different doses of VVDR03 in tail flick latency in mice. (*) denotes significance at P<0.05 versus predrug reaction Tail flick latency in seconds

Group (mg/kg) Pre-drug reaction

0.25 h 0.5 h 1 h 2 h 3 h

VVDR03 (20) 2.04±0.17 1.75±0.6 2.11±0.2 2.4±0.01 2.5±0.4 2.15±0.4

VVDR03 (40) 2.08±0.4 2.44±0.3 2.46±0.27 3.48±0.6 3.88±0.8 3.57±0.7

VVDR03 (80) 2.22±0.3 1.89±0.18 2.18±0.5 3±0.5 2.7±0.3 2.28±0.6

Aspirin (100) 3.79±0.2 3.94±0.8 4.5±0.5 7.17±0.07* 5.77±0.8* 3.88±0.19

Table 6.2. Effect of different doses of VVW02 in tail flick latency in mice. (*) denotes significance at P<0.05 versus predrug reaction

Tail flick latency in seconds

Group (mg/kg)

Pre-drug reaction

0.25 h 0.5 h 1 h 2 h 3 h

VVW02 (20) 2.00±0.17 2.75±0.6 2.13±0.2 2.6±0.01 2.5±0.4 2.15±0.4

VVW02 (40) 2.32±0.3 2.47±0.3 2.6±0.5 2.9±0.3 3.48±0.6 3.57±0.7

VVW02 (80) 1.18±.07 1.37±0.01 1.69±0.2 2±0.4 3.14±0.8* 2.3±0.8

Aspirin (100) 3.79±0.2 3.94±0.8 4.5±0.5 7.17±0.07* 5.77±0.8* 3.88±0.19

Table 6.3. Effect of different doses of V-PA in tail flick latency in mice. (*) denotes significance at P<0.05 versus predrug reaction.

Tail flick latency in seconds

Group (mg/kg)

Pre-drug reaction

0.25 h 0.5 h 1 h 2 h 3 h

V-PA (20) 2.89±0.13 3.31±0.15 2.79±0.41 2.99±0.36 3.32±0.3 3.0±0.31

V-PA (40) 2.23±0.27 2.49±0.18 3.15±0.3 3.29±0.4 3.2±0.4 2.3±0.4

V-PA (80) 2.74±0.19 2.8±0.07 3.24±0.3 2.9±0.29 3.6±0.4 3.3±0.195

Aspirin (100) 3.79±0.2 3.94±0.8 4.5±0.5 7.17±0.07* 5.77±0.8* 3.88±0.19

Table 6.4. Effect of different doses of V-MA in tail flick latency in mice. (*) denotes significance at P<0.05 versus predrug reaction

Tail flick latency in seconds

Group (mg/kg)

Pre-drug reaction

0.25 h 0.5 h 1 h 2 h 3 h

V-MA (20) 3.5±0.17 2.14±0.19 2.88±0.17 2.5±0.15 3.29±0.5 2.97±0.2

V-MA (40) 2.88±0.3 3.3±0.12 6.17±1.9 3.8±0.9 3.97±0.6 3.14±0.7

V-MA (80) 2.57±0.2 2.9±0.3 3.32±0.52 3.4±0.95 4±0.4* 3.4±0.175

Aspirin (100) 3.79±0.2 3.94±0.8 4.5±0.5 7.17±0.07* 5.77±0.8* 3.88±0.19

0

5

10

15

2 02 5

3 0

3 5

4 0

4 5

V V D R 0 3( 2 0 )

V V D R 0 3( 2 0 ) +A sp

( 5)

A sp ( 5) V ehicle

Num

ber o

f writ

hing

s

*, a *

Fig. 6.5. Effect of aspirin (5 mg/kg, i.p) in combination with VVDR03 (20 mg/kg, p.o) in acetic acid induced writhing in mice. (*) denotes significance at P<0.05 as compared to vehicle treated group. (a) denotes significance at P<0.05 as compared to VVDR03 per se.

05

1015

2 02 53 03 54 04 550

V V W 0 2( 2 0 )

V V W 0 2( 2 0 ) +A sp

( 5)

A sp ( 5) V ehicle

Num

ber o

f writ

hing

s

*, a *

Fig. 6.6. Effect of aspirin (5 mg/kg, i.p) in combination with VVW02 (20 mg/kg, p.o) in acetic acid induced writhing in mice. (*) denotes significance at P<0.05 as compared to vehicle treated group. (a) denotes significance at P<0.05 as compared to VVW02 per se.

0

5

10

15

2 0

2 5

3 0

3 5

4 0

4 5

V - PA ( 2 0 ) V - PA( 2 0 ) +A sp

( 5)

A sp ( 5) V ehicle

Num

ber o

f writ

hing

s

*, a, b

*, a

Fig. 6.7. Effect of aspirin (5 mg/kg, i.p) in combination with V-PA (20 mg/kg, p.o) in acetic acid induced writhing in mice. (*) denotes significance at P<0.05 as compared to vehicle treated group. (a) denotes significance at P<0.05 as compared to V-PA per se and (b) denotes significance at P<0.05 as compared to Aspirin per se.

0

5

10

15

2 0

2 5

3 0

3 5

4 0

4 5

V - M A( 2 0 ) )

V - M A( 2 0 ) +A sp

( 5)

A sp ( 5) V ehicle

Num

ber o

f writ

hing

s

*, a, b *, a

.Fig. 6.8. Effect of aspirin (5 mg/kg, i.p) in combination with V-MA (20 mg/kg, p.o) on acetic acid induced writhing in mice. (*) denotes significance at P<0.05 as compared to vehicle treated group. (a) denotes significance at P<0.05 as compared to V-MA per se and (b) denotes significance at P<0.05 as compared to Aspirin per se.

6.4. DISCUSSION

In this study, analgesic activity of valerian extract and oils were investigated

in both acetic acid-induced writhing and tail flick models in mice. The acetic acid-

induced writhing reaction in mice, described as a typical model for inflammatory pain,

has long been used as a screening tool for the assessment of analgesic or anti-

inflammatory properties of new agents (Collier et al., 1968; Khandelwal, 2007). This

method presents a good sensitivity; however, it shows poor specificity, leaving scope

for the misinterpretation of results. This can be avoided by complementing the test

with other models of nociception and by a performance motor test. Acetic acid causes

inflammatory pain by inducing capillary permeability (Amico-Roxas et al, 1984) and

liberating endogenous substances that excite pain nerve endings (Raj, 1996). The

abdominal constriction is related to sensitization of nociceptive receptors to

prostaglandins, bradykinin and substance-P (Huo et al, 2007, Singh and Majumdar,

1995). The nociceptive response caused by acetic acid is also dependent on the release

of TNF-α, interleukin-8 via modulation of macrophages and mast cells localized in

the peritoneal cavity (Ikeda et al., 2001). Because the writhing test usually lasts for

less than an hour, it is too short to cover central prostaglandin-dependent sensitization,

which requires spinal COX-2 induction (Hanns, 2007) however it primarily involves

COX-1 derived prostanoids and thus non-selective COX inhibitor offers a better

antinociceptive effect as compared to selective COX-2 inhibitors (Kulkarni and Jain,

2005).

The results suggest that in acetic acid writhing model VVDR03 produced

significant effect at 40 and 80 mg/kg while VVW02 was found to produce significant

effect at only 80 mg/kg. Both V-MA and V-PA produced significant effect in acetic

acid induced writhing at 40 and 80 mg/kg dose. The highest dose of V-MA tested (80

mg/kg) produced 63.6 % inhibition whereas that of V-PA produced 50 % inhibition.

Statistical comparison shows that both the oils are equally effective in producing

antinociceptive in acetic acid writhing. Aspirin also inhibited the number of writhings

significantly and the effect produced by 80 mg/kg V-MA is comparable to that of 100

mg/kg aspirin (64.9%).

Tail flick is an acute spinally mediated reflex to noxious thermal stimuli. The

fast rising pain in the tail flick gives rise to rapid tail withdrawal at the lowest possible

threshold for pain before it reaches to higher level. In the tail flick model, none of the

doses of V-PA and VVDR03 were found to have effect on tail flick latency when

compared to the predrug reaction time while 80 mg/kg dose of V-MA and VVW02

produced significant increase in tail flick latency after two hours. This indicates that

higher center is also involved in antinociceptive effect of the maaliol chemotype but

at higher doses.

The present study demonstrated that both valerian extracts and oils were found

to be more effective as peripheral analgesics. When studied for mechanism of action

in acetic acid writhing model aspirin potentiated the action of valerian oils while no

potentiation was seen with extracts. NSAIDs can inhibit COX in peripheral tissues

and, therefore, interfere with the mechanism of transduction of primary afferent

nociceptors (Fields, 1987). So the mechanism of analgesic effect of valerian oil could

probably be due to blockade of the effect or the release of endogenous substances that

excite pain nerve endings similar to that of indomethacin and other NSAIDs.

There are few reports on analgesic activity of Valeriana wallichii (Cao and

Hong, 1994; Schultz and Eckstein, 1962; Shrivastava and Sisodia, 1970). There is

another report which demonstrates analgesic activity of the volatile oil of Valeriana

amurensis in acetic acid writhing (Wu et al., 2007). There are many studies reporting

analgesic activity of essential oils and sesquiterpenes (Golshani et al, 2004; Santos et

al, 1997; Sayyah et al, 2002; Sayyah et al, 2003; Koudou et al, 2005; Ahmed et al,

1997). A recent report presents natural terpene 1,8-cineole for its analgesic effect

(Santos and Rao, 2000). In a study β-caryophyllene inhibited in vitro formation of

PGE1 (Burstein et al, 1975) and sesquiterpenoids have been found to have suppressive

effect on iNOS and COX-2 activity (Lee et al., 2002; Yoon et al., 2008). Anti-

inflammatory effect has been reported for limonene and terpineol (Duke and

Beckstrom, 1996). The limonene has been tested in the lipopolysaccharide (LPS)-

induced pleurisy model and when administered orally was able to inhibit the LPS-

induced inflammation including cell migration and inhibiting the production of nitric

oxide, gamma-interferon and IL-4 (Souza, 2003). The inhibitory effect of terpineol, a

volatile terpenoid alcohol on the compound action potential of rat sciatic nerve has

been reported (Moreira, 2001).

Few studies on Indian valerian have demonstrated that it contain flavonoid

compounds (Marder et al., 2003; Tang and Liu, 2003). Flavonoids are known to target

prostaglandins which are involved in the late phase of acute inflammation and pain

perception (Rajnarayana et al., 2001). Iridoids found in many medicinal plants exhibit

a wide range of bioactivities including cardiovascular, antiphepatotoxic, chlorectic,

hypoglycemic, anti-inflammatory, antispasmodic, antitumor, antiviral, immuno-

modulator and purgative activities (Didna et al., 2007). In the present study, GC-MS

screening of both the plant showed that their essential oil is dominated by

sesquiterpenes so we can speculate that the inhibitory effect of extracts and essential

oils in acetic acid writhing and hot plate test is due to the presence of terpenes,

sesquiterpenes (Cabo et al, 1986; Ozbek et al., 2006) and iridoids. Natural terpenoids

are reported to be natural inhibitors of NF-κB signaling (Salminen et al., 2008) just

like aspirin and other NSAIDs (Muller et al., 2001; Tegeder et al., 2001). Valerian

oils inhibited number of writhings by acting in a way similar to aspirin while effect of

extracts in acetic acid writhing may be attributed to some other mechanism possibly

to KATP channel activation which can then led to antispasmodic effect (Gilani et al,

2005). Analgesic effect of DCM extract of maaliol chemotype on tail flick model may

be attributed to mechanism other than that of COX-inhibiton. Hence the presence of

flavonoids, terpenes and iridoids may be contributory to the analgesic activity of

Valeriana in acetic acid induced writhing and tail flick model.

CHAPTER-7

Evaluation of in vitro Antioxidant Profile of Valeriana wallichii chemotypes

7.1. INTRODUCTION

Free radicals have aroused significant interest among scientists in the past

decade. Their broad ranges of effects in the biological systems have drawn on the

attention of many experimental works. Highly reactive free radicals, especially

oxygen-derived radicals which are formed by exogenous chemicals or endogenous

metabolic processes in the human body, are capable of oxidizing biomolecules,

resulting in cell death and tissue damage. Oxidative stress, defined as an imbalance

between oxidants and antioxidant in favour of the former, leads to many biochemical

changes due to overproduction of free radicals. DNA is probably the most

biologically significant target of oxidative attack, and it is widely thought that

continuous oxidative damage to DNA is a significant contributor to the age-related

development of the major cancers (Ames et al., 1993). Not only this, oxidative

damage has been implicated in the pathology of many other diseases and conditions

including diabetes, cardiovascular disease, inflammatory conditions and aging.

Almost all organisms possess antioxidant defenses and numerous damage removal

and repair enzymes to remove or repair damaged molecules. However, the natural

antioxidant mechanisms can be inefficient, hence dietary intake of antioxidant

compounds become important. Several authors demonstrated that antioxidant intake is

inversely related to mortality from coronary heart disease and to the incidence of heart

attacks (Hertog et al., 1993; Anderson et al., 1996). Antioxidants like vitamin C,

vitamin E (tocopherol), carotenoids and several polyphenolic compounds including

flavonoids that are mainly supplied as dietary consumptions can also impede

carcinogenesis by scavenging oxygen radical or interfering with the binding of

carcinogens to DNA (Stoner and Mukhtar, 1995). Synthetic antioxidants are widely

used because they are effective and cheaper than natural types. However, the safety

and toxicity of synthetic antioxidants have been important concerns (Imaida et al.,

1983).

Many herbal plants contain antioxidant compounds and these compounds

protect cells against the damaging effects of reactive oxygen species, such as singlet

oxygen, superoxide, peroxyl radicals, hydroxyl radicals and peroxynitrite. The food

industries also use natural antioxidants as a replacement of conventional synthetic

antioxidants in food by natural products that are considered to be promising and a safe

source. As a result of which, much attention has been directed towards the

characterization of antioxidant properties of plant extracts/their fractions and

identification of the constituents responsible for those activities. Considering the

pathological implications of reactive oxygen species (ROS), it is important to identify

antioxidants, which can scavenge several free radicals and prevent multiple diseases.

This can be achieved by simple in vitro test systems. Subsequent to this preliminary

screening, the results can be confirmed with ex vivo and in vivo systems.

V. wallichii is used as carminative and forms an ingredient of a number of

ayurvedic recepies. The drug contains a group of iridoid or monoterpene derivatives

known as valepotriates, which are useful as tranquilizers. It is also used for its

antispasmodic properties. V. officinalis a related species is reported to posses

neuroprotective activity and in another study its efficiency as antioxidant was tested

by the influence of its extract on the yield of chemiluminesence of Gly-Trp solution

(Bol’shakova et al., 1997). V. wallichii and V. officinalis share a group of active

constituents and both of them are characterized by an unusual fragnance, derived from

the essential oil component. There are reports depicting the presence of lignans,

terepenes, iridoids and flavonoids in Indian valerian. Besides this V. wallichii is being

used as a substitute for V. officinalis. We thus evaluated the antioxidant properties of

V. wallichii in different in vitro models. The antioxidant activity of the tested

compounds was compared with the standard antioxidant vitamin C.

7.2. MATERIAL AND METHODS

7.2.1. Chemicals

Potassium ferricyanide, sodium nitroprusside, ferric chloride and

trichloroacetic acid. Sulfanilamide, napthylethylene diamine hydrochloride,

orthophosphoric acid and 1, 1,-diphenyl 2-picryl hydrazyl (DPPH) were obtained

from Sigma Chemicals, USA. Ascorbic acid was procured from Ranbaxy, Gurgaon.

All other unlabeled chemicals and reagents were of analytical grade and were used

without further purification.

7.2.2. Plant material and its extraction

Same as in section 3.2.1. of chapter 3.

7.3. IN VITRO ANTIOXIDANT ASSAYS

In vitro antioxidant activity was determined by using DPPH radical

scavenging, nitric oxide scavenging and reductive ability. Different concentrations of

both the extracts (VVDR03 and VVW02) and oils (V-PA and V-MA) were prepared

in dimethyl sulfoxide (DMSO) ranging from 1-10mg/ml.

7.3.1. DPPH Radical Scavenging assay

DPPH (1,1,-diphenyl 2-picryl hydrazyl) scavenging activity was measured by

the spectrophotometric method (Sreejayan and Rao, 1996). DPPH, is a stable free

radical having a purple colour. When free radical scavengers are added, DPPH is

reduced and its colour is changed to yellow, based on the efficacy of antioxidants

(Fig. 1.). To a methanolic solution of DPPH (100 µM) 2.95 ml, 0.05 ml of test

compounds (extracts and oils of both the chemotypes) dissolved in DMSO were

added at different concentrations (1-10 mg/ml). Equal amount of DMSO was added to

the control. Absorbance was recorded at 517 nm after 20 min. Ascorbic acid was used

as a standard. Radical scavenging activity was expressed as percent inhibition and

was calculated using the following formula.

% DPPH radical scavenging activity= (1- Sample OD) x100 Control OD

NO2

NO2

NN NO2[ H ]

NO2N N

NO2

NO2

H

DPPH Reduced form of DPPH

Fig. 1. Reduction of 1,1, Diphenyl -2-Picryl hydrazyl (DPPH) free radical.

7.3.2. Nitric Oxide Scavenging Effect

Nitric oxide is generated from sodium nitroprusside, which at physiological

pH (7.4) liberates nitric acid. This nitric acid gets converted to nitrous acid and further

forms nitrite ions on contact with air. The nitrite ions diazotise with sulphanilic acid

and couple with naphthylethylenediamine (Griess reagent), producing pink colour

which can be measured spectrophotometrically (Sreejayan and Rao, 1997) (Fig. 2.).

Sodium nitroprusside (5 mM) in phosphate buffer saline was mixed with different

concentrations of extracts and oils (1-10mg/ml) dissolved in DMSO and incubated at

25º C for 30 min. After 30 min 1.5 ml of incubated solution were removed and diluted

with 1.5 ml of Griess reagent (1% sulphanilamide, 2% phosphoric acid and 0.1%

napthylethylene diamine dihydrochloride). The absorbance of the chromophore

formed during diazotisation of the nitrite with sulphanilamide and subsequent

coupling with napthylethylene diamine was measured at 546 nm along with a control.

Ascorbic acid was used as a standard. The percentage inhibition of nitric oxide

generated was measured by comparing the absorbance values of control and test.

% nitric oxide scavenging activity= (1- Sample OD) X 100 Control OD

H 2 NO 2 S N = N NH NH 2

+

NH NH 2

+N

N

SO 2 NH 2

H 2+

SO 2 NH 2

N

NO

O 2

+NO

NH 2

SO 2 NH 2

NO

NO + O 2

SO 2 NH 2

NH 2

Fig. 2. Generation of nitric oxide and its reaction with Griess reagent.

7.3.3. Reducing Power

Reductive ability of the extracts and oils was measured according to the

method of Oyaizu (Oyaizu, 1986). Different concentrations (1-10mg/ml) of extract

were mixed with 2.5 ml of sodium phosphate buffer (pH 6.6) and 2.5 ml of potassium

ferricyanide (1%). The mixture was incubated at 50ºC for 20 min. Trichloroacetic

acid (2.5 ml of 10%) was added to it, the mixture was mixed and centrifuged at 650

rpm for 10 min. The upper layer (5 ml) was mixed with 5 ml of deionised water and 1

ml of ferric chloride (1%) and absorbance was measured at 700 nm. Higher

absorbance indicated higher reducing power.

7.3.4. Statistical Analysis

For all the drugs and in all the test systems, percentage inhibition curve was

plotted by taking concentration on x-axis and percentage scavenging on the y-axis and

IC50 values (amount of drug required to scavenge 50% of free radicals) were obtained

from the linear plots (r value ranging from 0.92-0.98 for all the test systems). The data

was statistically analyzed by one-way ANOVA followed by Tukey’s test for multiple

pair wise comparison in all the test systems.

7.4. RESULTS

7.4.1. DPPH Radical Scavenging Activity

This activity has been used by various researchers as quick and reliable

parameters to assess the in vitro antioxidant activity of crude plant extracts (Navarro

et al., 1992; Thabrew et al., 1998). In this test DPPH radicals react with suitable

reducing agents and then electrons become paired off and the solution loses its colour

and becomes yellow. The ability of a compound to act as donor for hydrogen atoms or

electrons is measured spectrophotometrically. The DPPH scavenging activity exerted

by VVDR03 and VVW02 as well as ascorbic acid is summarized in Fig. 7.1. and

exhibited a concentration dependent inhibition. VVDR03 at 10 mg/kg exhibited

63.8% inhibition, VVW02 and ascorbic acid at the same concentration exhibited 34.5

% and 96% inhibition. In this study VVDR03 at different concentration’s

demonstrated significant DPPH scavenging activity as compared to VVW02. Free

radical scavenging activity of both the extracts is moderate as compared to ascorbic

acid. IC50 values were found to be 7.028 mg/ml for VVDR03 and 13.653 mg/ml for

VVW02 (Fig. 7.7.). While the IC50 of standard drug Ascorbic acid is 0.022 mg/ml. So

the study demonstrated that both have abilities to act as radical scavengers. Valerian

oils also produced concentration dependent inhibition but were found to have weak

scavenging action on DPPH as compared to extracts. Both V-PA and V-MA exhibited

almost similar scavenging effect in DPPH assay i.e 23.2 and 26.2 % at 10mg/ml (Fig.

7.4.).The IC50 was found to be 26.707 mg/ml for V-PA and 21.06 for V-MA which is

much greater than that of ascorbic acid (Fig. 7.7.).

7.4.2. Nitric Oxide Scavenging Effect

Nitric oxide (NO) is an important chemical mediator generated by endothelial

cells, macrophages, neurons etc. and is involved in the regulation of various

physiological processes. Excessive nitric oxide is associated with several diseases.

Nitric oxide reacts with superoxide and forms peroxynitrite radicals and is responsible

for the inflammatory response by the release of prostaglandin. Some scientists believe

that repeated infections throughout life, cause an excessive production of NO, which,

over time, causes toxic damage to the body resulting in age-related diseases such as

heart disease, Alzheimer’s disease and Diabetes. Peroxynitrite, produced in vivo from

nitric oxide and superoxide, leads to oxidation of low-density lipoprotein.

Peroxynitrite is a strong nitrating species with oxidizing effect. It may oxidize all

major types of biomolecules like DNA, proteins and lipids. Peroxynitrite is thought to

be involved in neurodegenerative diseases like Alzheimer’s disease, Parkinson’s

disease etc. Thus nitric oxide scavenging may prevent the diseases caused by it.

Fig. 7.2. illustrates the percentage inhibition of nitric oxide generation by

VVDR03 and VVW02. Ascorbic acid was used as a standard compound and its IC50

is 0.02243 mg/ml. IC50 of VVW02 was found to be 8.3 mg/ml whereas that of

VVDR03 is 0.6 mg/ml (Fig. 7.8.). This shows that nitric oxide scavenging activity of

VVDR03 is more than VVW02.

Similarly valerian oils were tested for nitric oxide scavenging effect and

showed a concentration dependent scavenging effect. The IC50 of V-MA was found to

be 12.7 mg/ml while that of V-PA is 7.09 mg/ml (Fig. 7.8.).

7.4.3. Reductive Ability

The antioxidant activity has been reported to be concomitant with the

development of reducing power. The reductive ability of VVDR03 and VVW02 has

been shown in Fig. 7.3. The reducing power increased as the extract concentration

increased, indicating that some compound in extract is both electron donor and could

react with free radicals to convert them in to more stable products and to terminate

radical chain reaction. All the concentrations of both the extracts showed higher

activity than the control and out of them VVDR03 was found to be having more

reducing power than VVW02. Both the oils when tested for reducing power were

found to have almost similar reducing power at tested concentrations (Fig. 7.6.). The

reducing power was found to be low for oils than the extracts. Ascorbic acid at all the

concentrations gave absorbance as 3.9 nm while control gave an absorbance of 0.054

nm. This shows that ascorbic acid has very high reducing property.

0

20

40

60

80

100

120

0 1 2 4 6 8 10

Conc (mg/ml)

% in

hibi

tion

V V D R 0 3 V V W 0 2 A sco rb ic acid

Fig.7.1. Percentage inhibition of DPPH radical by different concentrations of VVDR03, VVW02 and ascorbic acid

0

20

40

60

80

100

120

0 1 2 4 6 8 10

Conc (mg/ml)

% in

hibi

tion

V V D R 0 3 V V W 0 2 A sco rb ic acid

Fig. 7.2. Percentage inhibition of nitric oxide by different concentrations of VVDR03, VVW02 and Ascorbic acid

0

1

2

3

4

5

0 1 2 4 6 8 10

Conc (mg/ml)

Abs

orba

nce

(nm

)

V V D R 0 3 V V W 0 2 A sco rb ic acid

Fig. 7.3. Reductive ability of different concentrations of VVDR03, VVW02 and Ascorbic acid

0

20

40

60

80

100

120

0 1 2 4 6 8 10

Conc (mg/ml)

% in

hibi

tion

V - PA V =M A A sco rb ic acid

Fig. 7.4. Percentage inhibition of DPPH radical by different concentrations of V-PA, V-MA and ascorbic acid

0

20

40

60

80

100

120

0 1 2 4 6 8 10

Conc (mg/ml)

% in

hibi

tion

V - PA V =M A A sco rb ic acid

Fig. 7.5. Percentage inhibition of nitric oxide by different concentrations of V-PA, V-MA and Ascorbic acid

0

1

2

3

4

5

0 1 2 4 6 8 10

Conc (mg/ml)

Abs

orba

nce

(nm

)

V - PA V =M A A scorb ic acid

Fig. 7.6. Reductive ability of different concentrations of V-PA, V-MA and Ascorbic acid

7.0 2 8

13 .6 53

2 6 .70 7

2 1.0 6

0 .0 2 2 20

5

10

15

2 0

2 5

3 0

Conc

entra

tion

(mg/

ml)

V V D R 0 3 V V W 0 2 V - PA V - M A A scorb icacid

Fig. 7.7. Histogram showing IC50 values of V. wallichii extracts and oils in DPPH

assay

0 .6

8 .3

7.0 9

12 .7

0 .0 2 2 4 30

2

4

6

8

10

12

14

Conc

entra

tion

(mg/

ml)

V V D R 0 3 V V W 0 2 V - PA V - M A A scorb icacid

Fig. 7.8. Histogram showing IC50 values of V. wallichii extracts and oils in nitric oxide scavenging assay 7.4. DISCUSSION

Free radical oxidative stress has been implicated in the pathogenesis of a

variety of clinical disorders, resulting usually from deficient natural antioxidant

defenses. Potential antioxidant therapy therefore should include either natural free

radical scavenging enzymes or agents which are capable of augmenting the activity of

these enzymes. If human disease is believed to be due to the imbalance between

oxidative stress and antioxidative defense, it is possible to limit oxidative tissue

damage and hence prevent disease progression by antioxidant defense supplements.

Medicinal herbs are known to contain a variety of antioxidants. It has been revealed

that various phenolic antioxidants such as flavonoids, tannins, coumarins, xanthones

and more recently procyanidins present in plants scavenge radicals dose dependently,

thus they are viewed as promising therapeutic drugs for free radical pathologies. The

antioxidant activity of putative antioxidants have been attributed to various

mechanisms, among which are prevention of chain initiation, binding of transition

metal ion catalysts, decomposition of peroxides, prevention of continued hydrogen

abstraction, reductive capacity and radical scavenging (Diplock, 1997; Oktay et al.,

2003). Numerous antioxidant methods and modifications have been proposed to

evaluate antioxidant activity and to explain how antioxidants function. Of these, total

antioxidant activity, reducing power, DPPH assay, metal chelating, active oxygen

species such as H2O2, O2•− and OH• quenching assays are most commonly used for the

evaluation of antioxidant activities of extracts (Duh et al., 1999; Amarowicz et al.,

2000; Chang et al., 2002).

DPPH radicals react with suitable reducing agents losing colour

stoichometrically with the number of electrons consumed which is measured

spectrophotometrically at 517 nm. The DPPH method is widely used to determine

antiradical/antioxidant activity of purified phenolic compounds as well as natural

plant extracts (Brand-Williams et al., 1995; Sripriya et al., 1996; Bondet et al., 1997;

Mahinda and Shahidi, 2000; Peyrat-Mallard et al., 2000). The method also has good

repeatability and is used frequently however color interference of DPPH with samples

that contain anthocyanins leads to underestimation of antioxidant activity (Arnao,

2000). Used as a reagent, DPPH evidently offers a convenient and accurate method

for titrating the oxidizable groups of natural or synthetic antioxidants. Lower

absorbance of the reaction mixture indicated higher free radical scavenging activity

(Cao et al., 1997). All the tests were performed in triplicate and the results averaged.

From the present results it may be postulated that DCM extracts, VVDR03 and

VWR02 reduces the radical to the corresponding hydrazine when it reacts with the

hydrogen donors in the antioxidant principles. The free radical scavenging effect was

found to be dose dependent. Standard ascorbic produced 96% scavenging of DPPH at

10mg/ml and 72.3% at 1 mg/ml. The IC50 of VVDR03 (7.028 mg/ml) and VVW02

(13.653 mg/ml) demonstrated that VVDR03 is more potent than VVW02 in

scavenging free radicals, however the scavenging potential is moderate in comparison

to ascorbic acid.

Nitric oxide is generated from the amino acid L-arginine by vascular

endothelial cells, phagocytes and certain cells in the brain. Some scientists believe

that repeated infections throughout life cause an excessive production of NO, which

over time, causes toxic damage to the body resulting in age related diseases such as

heart disease, Alzheimer’s disease and diabetes. NO reacts with superoxide and forms

peroxynitrite radicals and is responsible for the inflammatory response by the release

of prostaglandin. The extracts moderately inhibited nitric oxide in dose dependent

manner, the effect being more prominent in case of VVDR03 than VVW02. Whereas

the oils V-PA and V-MA showed a week nitric oxide scavenging activity. Thus the

present study offers scientific evidence for the use of the plant in the indigenous

system in inflammatory conditions.

Reducing power of extracts and oils was good and increased with increasing

concentration. On a comparative basis, the extracts were found to have more

antioxidant property than the oils, which posses only weak antioxidant activity. Out of

the two extracts VVDR03 was found to have more antioxidant potential than the

VVW02. The antioxidant property of extracts can be attributed to the presence of

iridoids (Gamboa and Castro, 2004; Raju et al., 2004), lignans (Yamauchi et al., 2007;

Piccinelli et al., 2004) and flavonoids. There are studies showing that extract of

valerian root are endowed with flavonoids (Wasowski et al., 2002; Marder et al.,

2003) making this popular and very well tolerated plant attractive for the search of a

daily-used neuroprotective product for human consumption (Tang et al., 2003). The

weak antioxidant effect of valerian oils could be due to their chemical composition, as

the essential oil contains mainly monoterpene hydrocarbons such as sabinene,

terpinene-4-ol, α-pinene, p-cymene, terpinene, limonene, β-pinene and α-terpineol.

These compounds are known to possess a weak antioxidant activity (Tepe et al.,

2005).

In vitro scavenging is only a preliminary test for selecting potent antioxidants.

These compounds should be tested in vivo for their activity in different disease states

so that their potential uses will be explored. Their bioavailability and interaction with

biomembranes can also be determined. If these parameters are established properly,

the scope for developing phytochemicals into pharmaceuticals will be practical.

CHAPTER-8

Modulation of Antioxidant Defense System in Mice Brain by Valeriana wallichii

chemotypes

8.1. INTRODUCTION

Oxygen is essential for the survival of all on this earth. During the process of

oxygen utilization in normal physiological and metabolic processes approximately 5%

of oxygen gets univalently reduced to oxygen-derived free radicals. Free radicals are

partially reduced metabolites of oxygen and are highly toxic, mutagenic and reactive.

A free radical has one or more unpaired electrons in its outer orbital and they react

rapidly with adjacent molecules, donating, abstracting and even sharing its outer

orbital electron(s). Sometimes free radicals generate a second free radical or other

reactive oxygen species (ROS). Oxidative damage by oxygen free radicals are known

to be one of the mechanisms of chronic disorders such as atherosclerosis or cancer

(Halliwell and Gutteridge, 1989). Oxygen free radicals are created through aerobic

metabolism and are mostly removed by antioxidants in vivo (Diplock, 1994). An

antioxidant is a substance, which at low concentration relative to oxidisable substrate

significantly delays or reduces oxidation of the substrate by itself becoming oxidized.

The antioxidant defense systems in the body include superoxide dismutase (in

mitochondria and cytosol), catalase (in peroxisomes), glutathione peroxidase, α-

tocopherol (in membranes and lipoproteins). As with the chemical antioxidants, cells

are protected against oxidative stress by an interacting network of antioxidant

enzymes (Davies, 1995; Sies, 1997). Here, the superoxide released by processes such

as oxidative phosphorylation is first converted to hydrogen peroxide and then further

reduced to give water. This detoxification pathway is the result of multiple enzymes,

with superoxide dismutases catalysing the first step and then catalases and various

peroxidases removing hydrogen peroxide. The glutathione system includes

glutathione, glutathione reductase, glutathione peroxidases and glutathione S-

transferases (Meister and Anderson,1983). This system is found in animals, plants and

microorganisms (Meister and Anderson, 1983; Creissen et al., 1996). Glutathione

peroxidase is an enzyme containing four selenium-cofactors that catalyzes the

breakdown of hydrogen peroxide and organic hydroperoxides. But when generation

of ROS overtakes the antioxidant defense of the cells, the free radicals start attacking

the cell proteins, lipids and carbohydrates leading to a number of physiological

disorders. They have also been implicated in pathogenesis of a number of disorders

like diabetes, liver damage, nephrotoxicity, inflammation, cancer, cardiovascular

disorders, and neurological disorders and in the process of aging. Antioxidants can

cancel out the cell-damaging effects of free radicals (Sies, 1997). Furthermore, people

who eat fruits and vegetables, which are good sources of antioxidants, have a lower

risk of heart disease and some neurological diseases (Stanner, 2004) and there is

evidence that some types of vegetables, and fruits in general, probably protect against

a number of cancers. People taking vitamin E supplements had a lower risk of

developing heart disease (Rimm et al., 1993). There is also some evidence that

antioxidants might help prevent diseases such as macular degeneration (Bartlett and

Eperjesi, 2003), suppressed immunity due to poor nutrition (Wintergerst et al., 2006),

and neurodegeneration (Wang, 2006). These observations suggested that antioxidants

might help prevent these conditions.

Therefore there is a constant need to replenish antioxidant resources, either

endogenously or through supplementation. For this, there is a need to identify

antioxidants, which can scavenge several free radicals and prevent multiple diseases.

Although synthetic antioxidants such as butylated hydroxytoluene (BHT), butylated

hydroxyanisole (BHA) and tertiary butylhydroquinone are widely used as

antioxidants in various industries, they are suspected of being responsible for liver

damage and carcinogenesis in laboratory animals (Grice, 1986). Therefore, the

development and utilization of more effective antioxidants of natural origin is desired

(Gulcin et al., 2002). Therefore search into the determination of natural antioxidant

sources is much important. The present study has been directed to investigate the

modulatory effect of Valeriana wallichii on antioxidant defense system in mice.

8.2. MATERIAL AND METHODS

8.2.1. Plant material and its extraction

Same as in section 3.2.1. of chapter 3.

8.2.2. Animals

Same as in section 3.2.2. of chapter 3.

8.2.3. Experimental protocol and procedure

Both the extracts VVW02 and VVDR03 and oils V-PA and V-MA were

studied for modulatory effect on antioxidant defense system of mice brain. Mice were

divided into thirteen groups, each group consisting of six animals. Group I, II and III

received 10, 20 and 40 mg/kg of VVDR03. Group IV, V and VI received 10, 20 and

40 mg/kg dose of VVW02. Group VII, VIII and IX received 10, 20 and 40 mg/kg

dose of V-PA. Group X, XI and XII received 10, 20 and 40 mg/kg dose of V-MA.

Group XIII received vehicle only i.e a drop of Tween-80 dissolved in distilled water

and served as control. The drugs were dissolved in one drop of Tween 80 and solution

was made up with sterile water. All the drugs were given orally to mice continuously

for 14 days. On 14th day all the animals were sacrificed, blood was collected and

whole brains were removed and weighed. Brains were processed to get 10%

homogenate in cold buffer using glass Teflon homogenizer. The homogenates were

used to estimate brain malondialdehyde (MDA), catalase and reduced glutathione

(GSH). Brain homogenates were centrifuged at 800g for 5 min at 4ºC to get the post

mitochondrial supernatant (PMS) which was used to assay superoxide dismutase

(SOD) activity.

Estimation of lipid peroxidation

The quantitative measurement of lipid peroxidation in terms of

malondialdehyde levels in the whole brain was performed according to the method of

Wills (Wills, 1966). The malondialdehyde (MDA) content, a measure of lipid

peroxiadtion, was assayed in the form of thiobarbituric acid reacting substances

(TBARS). Briefly, the reaction mixture consisted of 0.5 ml homogenate and 0.5 ml

Tris HCl pH 7.4 incubated at 37ºC for 2 hrs. Then 1ml of 10% Trichloroacetic acid

was added to the above mixture and centrifuged at 1000 rpm for 10 min. 1ml of

supernatant was removed, to which is added 1 ml of 0.067% thiobarbitric acid. The

tubes containing reaction mixture were then kept in boiling water for 10 min. They

were then cooled 1ml of distilled water was added. The absorbance of the colour

developed in the tubes was measured at 532 nm.

Estimation of reduced glutathione

Content of reduced glutathione in the brain was assayed by the method of

Jollow (Jollow et al., 1974). 1ml of tissue homogenate (10%) was precipitated with

1ml of sulfosalicylic acid (4%). The samples were kept at 4ºC for at least 1 hr and

then subjected to centrifugation at 1200g for 15 min at 4ºC. The assay mixture

contained 0.1 ml aliquot and 2.7 ml phosphate buffer (0.1 M pH 7.4) in a total volume

of 3 ml. The yellow colour developed was read immediately at 412 nm on a

spectrophotometer.

Estimation of superoxide dismutase (SOD)

Assay of SOD was done in post mitochondrial supernatant (Kono, 1978). The

assay system consisted of EDTA 0.1mM, sodium carbonate 50mM and 96mM of

nitroblue tetrazolium (NBT). In the cuvette, 2 ml of above mixture, 0.05 ml

hydroxylamine and 0.05 ml of PMS were taken and the auto-oxidation of

hydroxylamine was observed by measuring the absorbance at 560 nm.

Estimation of catalase

Catalase activity was assayed by the method of Luck (Luck, 1963). Briefly the

assay mixture consisted of 3 ml of H2O2 phosphate buffer (1.25x10–2 M H2O2) and

0.05 ml of supernatant of the brain homogenate (10%) and the change in absorbance

was read at 240 nm using the spectrophotometer. Enzyme activity was calculated

using the millimolar extinction coefficient of H2O2 (0.07). The results were expressed

as micromoles of H2O2 decomposed per min, per mg of protein.

8.2.4 Statistical analysis

All the values are expressed as mean±SEM. The data were analysed by

analysis of variance (ANOVA) followed by Tukey test. The criterion for statistical

significance was at p<0.05.

8.3. RESULTS

Effect on TBARS levels

Peroxidation of lipids causes severe damage to cell membrane because lipids

are the main component of the cell membrane. Its initiation occurs when a radical

species attacks and removes allylic hydrogen from an unsaturated fatty acid, resulting

in a radical chain reaction. Once lipid peroxidation begins, iron may participate in

driving the process. The possible sources of iron in body are iron-containing proteins.

There are a number of methods available to measure the markers of lipid

peroxidation, either by measuring the formation of products, or the degradation of

reactants. The most commonly used method is the thiobarbituric acid reactive

substances (TBARS) test. The results of the effect of VVW02 and VVDR03 on the

lipid peroxidation of mice brain homogenates are shown in Fig. 8.1 & 8.3. VVW02

did not alter the lipid peroxiadtion in brain as compared to control group. Similarly

none of the dose of VVDR03 except 40 mg/kg attenuated the level of lipid

peroxidation significantly. In case of oils none of the dose of V-PA and V-MA altered

the TBARS levels in mice brain as compared to control group (Fig. 8.5 & 8.7).

Effect on glutathione levels

Glutathione is a cysteine-containing peptide found in most forms of aerobic

life (Meister and Anderson, 1983). It is not required in the diet and is instead

synthesized in cells from its constituent amino acids (Meister, 1988). Glutathione has

antioxidant properties since the thiol group in its cysteine moiety is a reducing agent

and can be reversibly oxidized and reduced. In cells, glutathione is maintained in the

reduced form by the enzyme glutathione reductase and in turn reduces other

metabolites and enzyme systems as well as react directly with oxidants (Linster and

Van, 2007). Due to its high concentration and its central role in maintaining the cell's

redox state, glutathione is one of the most important cellular antioxidants.

In the present study control group was found to have 0.0121micromoles/mg pr

as level of reduced glutathione. VVW02 (10, 20 & 40 mg/kg) did not result in a

significant alteration in reduced glutathione levels (Fig. 8.1.) While 40 mg/kg of

VVDR03 significantly increased the GSH levels (Fig. 8.3.) as compared to control

group. No effect was found on brain glutathione levels with V-PA (Fig. 8.5.) and V-

MA (Fig. 8.7.) (10, 20 and 40 mg/kg).

Effect on catalase

Catalases are enzymes that catalyse the conversion of hydrogen peroxide to

water and oxygen, using either an iron or manganese cofactor (Chelikani, 2004;

Zámocký and Koller, 1999). This protein is localized to peroxisomes in most

eukaryotic cells (Del Río, 1992). Hydrogen peroxide is generated by a number of

intracellular flavin oxidases like xanthine oxidase and also from the autooxidation of

numerous xenobiotics (Hochstein and Atallah, 1988). Hydrogen peroxide can directly

affect the DNA and sulfhydryl groups apart from its participation in hydroxyl radical

generation. H2O2 is reduced by three general mechanisms. 1) It is the substrate for two

enzymes, catalase and glutathione (GSH) peroxidase that catalyze the conversion of

H2O2 to H2O + O2; this presumably is a detoxification mechanism (Maddipati and

Marnett, 1987). 2) H2O2 is converted by myeloperoxidase (MPO) in neutrophils to

hypochlorous acid (HOCl). This appears to be a mechanism for a physiological toxic

agent, since HOCl is a strong oxidant that acts as a bactericidal agent in phagocytic

cells. Reaction of HOCl with H2O2 yields singlet oxygen (1O2) and water. 3) H2O2 is

converted in a spontaneous reaction catalyzed by Fe2+ (Fenton reaction) to the highly

reactive hydroxyl radical (OH.). The hydroxyl radical reacts instantaneously with any

biological molecule (RH) from which it can abstract a hydrogen atom. The resulting

free radical is more stable and hence longer-lived than the hydroxyl radical Thus

removal of hydrogen peroxide significantly lowers the toxic effects of hydroxyl

radicals. VVW02 at 10, 20 and 40 mg/kg failed to improve the levels of catalase (Fig.

8.2.) in mice brain but VVDR03 significantly increased the level of catalase at all the

doses (Fig. 8.4.). No alteration was seen on catalase level with different doses of V-

PA and V-MA (Fig. 8.6. & 8.8.).

Effect on SOD

Superoxide anion is generated from mitochondria during respiratory chain,

enzymatic glycosylation of proteins and also from the activation of phagocytes (Beyer

et al., 1991). Superoxide anions undergo dismutation which causes the production of

hydrogen peroxide (Hochstein and Atallah, 1988; Riley and Behrman, 1991).

Superoxide anion is normally involved in inflammatory conditions. Formation of

superoxide anion radical leads to a cascade of other ROS (Grisham, 1992).

Superoxide dismutates to hydrogen peroxide (H2O2) and oxygen. It is less reactive

when compared to the other free radicals. But it leads to the production of hydroxyl

and peroxynitrite radicals by combination with hydrogen peroxide and nitric oxide

radical respectively. The pathological effects of superoxide anion are indirect in the

sense that it is the subsequently formed hydroxyl and peroxynitrite radicals which are

involved in different pathological conditions like cancer, cardiac, renal ischemia,

atherosclerosis, diabetes etc. Thus if, superoxide anion production is controlled or

scavenged, the formation of hydroxyl and peroxynitrite radicals can be limited, such

that, the diseases caused by them can sufficiently be reduced.

In our study none of the dose of VVW02 (Fig. 8.2.) altered brain SOD levels.

While VVDR03 at 40 mg/kg produced significant increase in SOD levels as

compared to control group (Fig. 8.4.). Similarly when valerian oils V-PA and V-MA

were studied for modulation of SOD levels in brain no effect was found when

compared with control group (Fig. 8.6. & 8.8.).

00 .1

0 .20 .3

0 .40 .50 .6

0 .70 .8

0 .91

V V W 0 2( 10 )

V V W 0 2( 2 0 )

V V W 0 2( 4 0 )

V ehicle

nmol

es/m

g pr

0

0 .0 0 2

0 .0 0 4

0 .0 0 6

0 .0 0 8

0 .0 1

0 .0 12

0 .0 14

0 .0 16

0 .0 18

V V W 0 2( 10 )

V V W 0 2( 2 0 )

V V W 0 2( 4 0 )

V ehicle

mic

rom

oles

/mg

pr

(a) (b)

Fig 8.1. Effect of different doses of VVW02 on a) Lipid peroxidation in mice brain b) Glutathione content in mice brain

0

2

4

6

8

10

12

14

16

18

V V W 0 2( 10 )

V V W 0 2( 2 0 )

V V W 0 2( 4 0 )

V ehicle

mic

rom

oles

of

H2O

2 de

com

pose

d/m

in/m

g p

r

0

0 .5

1

1.5

2

2 .5

3

V V W 0 2( 10 )

V V W 0 2( 2 0 )

V V W 0 2( 4 0 )

V ehicle

un

its/

mg

pr

(a) (b)

Fig. 8.2. Effect of different doses of VVW02 on a) Catalase level in mice brain b) Superoxide dismutase level in mice brain

0

0 .1

0 .2

0 .3

0 .4

0 .5

0 .6

0 .7

0 .8

0 .9

V V D R 0 3( 10 )

V V D R 0 3( 2 0 )

V V D R 0 3( 4 0 )

V ehicle

nmol

es/m

g pr * a

0

0 .0 0 5

0 .0 1

0 .0 15

0 .0 2

0 .0 2 5

0 .0 3

V V D R 0 3( 10 )

V V D R 0 3( 2 0 )

V V D R 0 3( 4 0 )

V ehicle

mic

rom

oles

/mg

pr * a b

(a) (b)

Fig. 8.3. Effect of different doses of VVDR03 on a) Lipid peroxidation in mice brain b) Glutathione content in mice brain. Results are significant at P<0.05. (*) versus vehicle group, a versus VVDR03 (20 mg/kg), b versus VVDR03 (10 mg/kg)

02

46

810

1214

1618

2 0

V V D R 0 3( 10 )

V V D R 0 3( 2 0 )

V V D R 0 3( 4 0 )

V ehicle

mic

rom

oles

of H

2O2

deco

mpo

sed/

mim

/mg

pr

**

*

00 .5

11.5

22 .5

3

3 .54

4 .55

V V D R 0 3( 10 )

V V D R 0 3( 2 0 )

V V D R 0 3( 4 0 )

V ehicle

units

/mg

pr

* a b

(a) (b)

Fig. 8.4. Effect of different doses of VVDR03 on a) Catalase level in mice brain b) Superoxide dismutase level in mice brain. Results are significant at P<0.05. * versus vehicle group, a versus VVDR03 (20 mg/kg) b versus VVDR03 (10 mg/kg)

2 . 8

2 . 9

3

3 . 1

3 . 2

3 . 3

3 . 4

3 . 5

3 . 6

V- P A ( 10 ) V- P A ( 2 0 ) V- P A ( 4 0 ) Ve hi c l e

nmol

es/m

g pr

0 .0 12

0 .0 12 5

0 .0 13

0 .0 13 5

0 .0 14

0 .0 14 5

0 .0 15

V- P A ( 10 ) V- P A ( 2 0 ) V- P A ( 4 0 ) Ve hi c l e

mic

rom

oles

/mg

pr

(a) (b)

Fig. 8.5. Effect of different doses of V-PA on a) Lipid peroxidation in mice brain b) Glutathione content in mice brain

2 .6

2 .7

2 .8

2 .9

3

3 .1

3 .2

3 .3

3 .4

3 .5

V- P A ( 10 ) V- P A ( 2 0 ) V- P A ( 4 0 ) Ve hi c l e

mic

rom

oles

of H

2O2

deco

mpo

sed/

min

/mg

pr

0

0 .5

1

1.5

2

2 .5

3

3 .5

4

4 .5

5

V- P A ( 10 ) V- P A ( 2 0 ) V- P A ( 4 0 ) Ve hi c l e

units

/mg

pr

(a) (b)

Fig. 8.6. Effect of different doses of V-PA on a) Catalase level in mice brain b) Superoxide dismutase level in mice

0

0 .5

1

1.5

2

2 .5

V- M A ( 10 ) V- M A ( 2 0 ) V- M A ( 4 0 ) Ve hi c l e

nm

ole

s/m

g p

r

0

0 .0 0 1

0 .0 0 2

0 .0 0 3

0 .0 0 4

0 .0 0 5

0 .0 0 6

0 .0 0 7

0 .0 0 8

V- M A ( 10 ) V- M A ( 2 0 ) V- M A ( 4 0 ) Ve hi c l e

mic

rom

oles

/mg

pr

(a) (b)

Fig. 8.7. Effect of different doses of V-MA on a) Lipid peroxidation in mice brain b) Glutathione content in mice brain

0

0 . 5

1

1. 5

2

2 . 5

V- M A ( 10 ) V- M A ( 2 0 ) V- M A ( 4 0 ) Ve hi c l e

mic

rom

oles

of H

2O2

deco

mpo

sed/

min

/mg

pr

0

0 .5

1

1.5

2

2 .5

3

3 .5

4

4 .5

V- M A ( 10 ) V- M A ( 2 0 ) V- M A ( 4 0 ) Ve hi c l e

units

/mg

pr

(a) (b)

Fig. 8.8. Effect of different doses of V-MA on a) Catalase level in mice brain b) Superoxide dismutase level in mice

8.4. DISCUSSION

Antioxidants are radical scavengers which protect the human body against free

radicals that may cause pathological conditions such as ischemia, anaemia, asthma,

arthritis, inflammation, neuro-degenertion, Parkinson's diseases, mongolism, ageing

process and perhaps dementias (Polterait, 1997). Plants are potent biochemical

factories and have been components of phytomedicine since times immemorial and

man is able to obtain from them a wondrous assortment of industrial chemicals. Plant

based natural constituents can be derived from any part of plant like bark, leaves,

flowers, roots, fruits, seeds. The beneficial medicinal effects of plant materials

typically result from the combinations of secondary products present in the plant.

Flavonoids and flavones are widely distributed secondary metabolites with

antioxidant and antiradical properties (Nakayoma and Yamada, 1995). Antioxidant-

based drugs/formulations for the prevention and treatment of complex diseases like

atherosclerosis, stroke, diabetes, Alzheimer's disease, and cancer have appeared

during the last 3 decades (Devasagayam et al., 2004). This has attracted a great deal of

research interest in natural antioxidants. Subsequently, a worldwide trend towards the

use of natural phytochemicals present in berry crops, tea, herbs, oilseeds, beans, fruits,

and vegetables has increased.

In the present study different doses (10, 20 and 40 mg/kg) of valerian extracts

VVDR03, VVW02 and valerian oils V-PA and V-MA were administered orally to

mice daily for 14 days. And then on 14th day mice were sacrificed and brain

homogenate was prepared. Brain homogenates were then evaluated for modulation of

antioxidant defense system. It was found that none of the dose of VVW02 produced

any alteration in TBARS levels, glutathione content, SOD and catalase levels in brain

as compared to control group. VVDR03 however exerted antioxidant effect at 40

mg/kg and significantly increased the glutathione levels and SOD levels. All the doses

(10, 20 and 40 mg/kg) of VVDR03 increased catalase levels significantly. Similarly

40 mg/kg dose of VVDR03 significantly decreased the TBARS levels as compared to

control group. When the effect of valerian oils on antioxidant system was evaluated

then none of the dose of V-PA and V-MA altered TBARS levels, glutathione content,

catalase and SOD levels in mice brain as compared to control group.

From the in vitro study it was demonstrated that valerian extracts VVDR03

and VVW02 exhibited antioxidant activity out of which VVDR03 was more effective.

In the present study however only VVDR03 at 40 mg/kg was found to have

modulated antioxidant defense system in mice brain after two weeks dosing. VVW02,

V-PA and V-MA were not found to be effective in this study and the results correlate

well with the results of in vitro study. Iridoids, present in roots of Valeriana wallichii

might be responsible for modulatory effect on antioxidant defense system of mice

brain (Gamboa and Castro, 2004; Raju et al., 2004; Ahmad et al., 2008).

CONCLUSION

Chemical Screening

• The major constituents of the oils from roots and rhizomes of Valeriana

wallichii collected from different geographical region were separated and

characterized by GC, GC/MS and NMR. The chemical compositions of the

oils show two chemotypes within Valeriana wallichii. Patchouli alcohol and

8-Acetoxyl patchouli alcohol were isolated from the plant VW-II and since

Patchouli alcohol was the major compound it was characterised as V. wallichii

patchouli alcohol chemotype. From the plant VW-I, Maaliol and Patchouli

alcohol were isolated. Since Maaliol was one the major component of the

essential oil it was characterised as V. wallichii maaliol chemotype.

OH

H

H

OH

H

H

OAc

H

OH

H

H Patchouli alcohol 8-Acetoxyl patchouli alcohol Maaliol

• The extracts from the roots and rhizomes of both the chemotypes were

subjected to HPLC screening. The residue obtained after extraction was

fractionated on column chromatography to yield valepotriates. Didrovaltrate,

Isovaleroxyhydroxy didrovaltrate and 1-α Acevaltrate were isolated from V.

wallichii maaliol chemotype while Isovaleroxy didrovaltrate, 1-α Acevaltrate

and 1-Homoisovaltrate from patchouli alcohol chemotype.

O

O

O

O H3C

CH3

H

O

O

CH3

CH2OCOCH2CH(CH3)2

O

O

O

H

O

O

CH3CH3

O

O

O

O

Didrovaltrate Acevaltrate

O

O

O

H

O

CH3

O

CH3

O

O

CH3

O

O

O

O H3C

CH3

HO

O

O

CH3

O

CH3CH3

O

O

O

1-Homoisovaltrate Isovaleroxyhydroxy didrovaltrate (IVHD)

Biological screening

• When evaluated for psychopharmacological effect, none of the chemotypes

was found to have anxiolytic and muscle relaxing effect at tested doses (10, 20

& 40 mg/kg p.o).

• Extracts and oils of both the chemotypes were found effective in delaying

onset of death when tested in PTZ-induced convulsions. However they didn’t

afford any effect on percentage mortality as compared to control group. The

standard drug diazepam produced 100 % protection from mortality. Essential

oil of patchouli alcohol chemotype (V-PA) was more effective than maaliol

chemotype (V-MA) while both the extracts were equally effective in delaying

onset of death. V-PA was found to be most effective. Thus we can say that

both chemotypes possess very weak anticonvulsant effect which can be

attributed to the presence of valepotriates and sesquiterpenes.

• In acute study DCM extracts and essential oils of both the chemotypes

produced antiimmobilty effect without producing any motor stimulating

effect, however extract and oil of maaliol chemotype significantly decreased

locomotor activity as compared to vehicle treated group. Essential oils of both

the chemotypes were equally effective as antidepressant while extract of

maaliol type was found to be more effective than patchouli alcohol type. Out

of all, extract of patchouli alcohol type was found to be least effective in

producing antimmobilty effect. Since nitric oxide synthase inhibitor, L-

NAME, potentiated the effect of essential oils they are postulated to produce

antidepressant effect by modulating nitric-oxide signaling pathway while

GABA potentiating effect of extracts might be responsible for antidepressant

effect.

• In chronic study, both the oils were found to be equally effective as

antidepressants as in acute study. Extract of maaliol type was more effective

than patchouli alcohol type and this correlates well with acute study. The

results are further supported by neurobiochemical studies used to check

alterations in the neurotransmitter levels and both oils and extracts of both the

chemotypes significantly increased norepinephrine levels in mice brain after

14 days of treatment. Thus we can conclude that DCM extract of maaliol type

was more effective than patchouli alcohol type. Both the oils and DCM extract

of maaliol type were equally effective and the effect produced by them is

slightly more than the standard antidepressant, imipramine.

• While evaluating for analgesic effect, extract of chemotype patchouli alcohol

was found to be more effective than maaliol type in acetic acid writhing test

while both the oils produced comparative results in inhibiting acetic acid

writhings. From the present results we can conclude that analgesic effect

produced by both the oils in acetic acid writhing is equal to standard drug,

Aspirin. In tail flick test, only maaliol chemotype (both extract and oil) was

found to be effective but only at higher dose. Essential oils exerted action in

acetic acid writhing through COX pathway as they potentiated the effect of

aspirin while DCM extracts acted through pathway other than COX pathway.

• From the present study we found that DCM extracts of both the chemotypes

possess moderate in vitro antioxidant activity. Out of which, patchouli alcohol

type was more effective than maaliol type and this correlates well with in vivo

study also, done on mice. While in case of oils, both possess very weak in

vitro antioxidant activity while none was effective in modulating antioxidant

defense system in mice brain at the tested doses. Thus we can say that the

mixture of iridoids and essential oil present in DCM extracts of both the

chemotypes may be responsible for antioxidant effect.

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