“protective effect of ultra sonic bath assisted …

www.wjpr.net Vol 5, Issue 12, 2016.

205

Rao et al. World Journal of Pharmaceutical Research

“PROTECTIVE EFFECT OF ULTRA SONIC BATH ASSISTED

METHANOL EXTRACT OF LEPIDIUM SATIVUM LINN. SEEDS BY

USING CHEMOTHERAPY INDUCED NEUROPATHY IN RATS.”

*A.T. Chenna Kesava Rao, B. Priyanka, K. Maheswari, G. Rambabu, J. Vidya Sagar

Department of Pharmacy, Vision College of Pharmacy, Boduppal, Hyderabad. Telangana.

INTRODUCTION

Medicinal Plants & Their Importance

A medicinal plant is any plant which, in one or more of its organs,

contains substances that can be used for therapeutic purposes, or which

are precursors for chemo-pharmaceutical semi-synthesis. When a plant

is designated as „medicinal‟, it is implied that the said plant is useful as

a drug or therapeutic agent or an active ingredient of a medicinal

preparation. Medicinal plants may therefore be defined as a group of

plants that possess some special properties or virtues that qualify them

as articles of drugs and therapeutic agents, and are used for medicinal

purposes. [Bailey, C.J. and Day, C et al. (1989)].

History of Plant Based Traditional Medicine

Plants have formed the basis of sophisticated traditional medicine (TM) practices that have

been used for thousands of years by people in China, India and many other countries. Some

of the earliest records of the usage of plants as drugs are found in the Artharvaveda, which is

the basis for Ayurvedic medicine in India (dating back to 2000 BCE), the clay tablets in

Mesopotamia (1700 BCE) and the Eber Papyrus in Egypt (1550 BCE). Other famous

literature sources on medicinal plant include “De Materia Medica,” written by Dioscorides

between CE 60 and 78 and “Pen Ts‟ao Ching Classic of Materia Medica” (written around

200CE).

Nowadays plants are still important sources of medicines, especially in developing countries

that still use plant-based TM for their healthcare. In 1985, it was estimated in the Bulletin of

the World Health Organization (WHO) that around 80% of the world‟s population relied on

medicinal plants as their primary healthcare source. Even though a more recent figure is not

World Journal of Pharmaceutical Research SJIF Impact Factor 6.805

Volume 5, Issue 12, 205-233. Review Article ISSN 2277– 7105

*Corresponding Author

A.T. Chenna Kesava Rao

Department of Pharmacy,

Vision College of Pharmacy,

Boduppal, Hyderabad.

Telangana.

Article Received on

23 Sept. 2016,

Revised on 13 Oct. 2016,

Accepted on 03 Nov. 2016

DOI: 10.20959/wjpr201612-7318


206


available, the WHO has estimated that up to 80% of the population in Africa and the majority

of the populations in Asia and Latin America still use TM for their primary healthcare needs.

In industrialized countries, plant-based traditional medicines or phyto therapeutically are

often termed complementary or alternative medicine (CAM) and their use has increased

steadily over the last 10 years. In the USA alone, the total estimated “herbal” sales for 2005

was $4.4 billion, a significant increase from $2.5 billion in 1995. (Butlet MS.et al 2004)

However, such “botanical dietary supplements” are regulated as foods rather than drugs by

the United States Food and Drug administration(U.S FDA).

Role of Plants in Human History

Plants have also been used in the production of stimulant beverages (e.g. tea, coffee, cocoa,

and cola) and inebriants or intoxicants (e.g., wine, beer, kava) in many cultures since ancient

times and this trend continues till today. Tea (Camellia sinensis Kuntze) was first consumed

in ancient China (the earliest reference is around CE 350), while coffee (Coffea arabica L.)

was initially cultivated in Yemen for commercial purposes in the 9th century. The Aztec

nobility used to consume bitter beverages containing raw cocoa beans (Theobroma cacao L.),

red peppers and various herbs. Nowadays, tea, coffee and cocoa are important commodities

and their consumption has spread worldwide. The active components of these stimulants are

methylated xanthine derivatives, namely caffeine, theophylline and theobromine, which are

the main constituents of coffee, tea and cocoa, respectively. (K.G. Ramawat and J.M.

Mérillon et al, 2008).

Extraction methods from solid sample

Sample pretreatment

method Principles of the technique

Accelerated solvent Sample is placed in a sealed container and heated to a temperature

higher than its boiling point, causing pressure in the vessel to rise.

Automated Soxhlet A combination of hot solvent leaching and Soxhlet extraction; sample in thimble is first immersed in boiling solvent and then the

thimble is raised for Soxhlet extraction with solvent refluxing.

Forced-flow leaching Sample is placed in a flow-through tube, and solvent is pumped or pushed through high pressure nitrogen gas, while the tube is heated

near the boiling point of solvent

Gas phase

After equilibrium, analytes partition themselves between a gas phase and the solid phase at a constant ratio with static headspace extraction, volatiles are sampled above the solid; with dynamic

headspace extraction, volatiles are sampled by continuously purging the headspace above a sample with inert gas, trapping them

on a solid medium, and then thermally desorbing them into a gas


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Ultrasonic bath

Introduction

Extraction techniques are widely accepted as a prerequisite for analytical determination of

both organic and inorganic analytes in a large variety of samples. As a part of an analytical

process, sample preparation is considered to be an essential step so that the entire process can

be simplified. In this case, the ability of many analytical systems to handle liquid samples has

brought about the development of separation methods which fulfill a main objective, i.e. to

obtain quantitative analyte leaching from the solid matrix using a suitable solvent, with little

or no matrix release, so that matrix effects can be kept to a minimum during the measurement

steps.

Fig 1.6 Ultrasonic Bath Sonicator

chromatograph.

Homogenization Sample is placed in a blender, solvent is added, and sample is homogenized to a finely divided state; solvent is removed for

further work-up.

Pervaporation

Volatile substances present in a heated donor phase placed inside a pervaporation module evaporate through a porous membrane and the vapour condenses on the surface of a cool acceptor stream on

the other side of the membrane.

Solid}liquid extraction Sample is shaken together with the appropriate solvent in a container and the liquid separated by filtration

Sonication

Finely divided sample in a container is immersed in ultrasonic bath

with solvent and subjected to ultrasonic irradiation; an ultrasonic probe or cell disrupter can also be used.

Soxhlet extraction

Sample is placed in a disposable, porous container (thimble);

constantly refluxing solvent flows through the thimble and leaches out analytes that are collected continuously.

Supercritical fluid

Sample is placed in flow-through container and a supercritical fluid (e.g. CO2) is passed through sample, after depressurization,

extracted analyte is collected in solvent or trapped on adsorbent and desorbed by rinsing with solvent.

Thermal A form of dynamic headspace analysis, but the sample is heated

(controlled) to much higher temperatures (as high as 3503oC).


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Sonication is usually recommended for pretreatment of solid environmental samples for the

extraction of nonvolatile and semi volatile organic compounds from solid, such as soils,

sludges and wastes. When comparing the different methods available for analyte extraction

from solid samples, sonication is considered as an effective method since unsophisticated

instrumentation is required and solid-liquid separations can usually be performed in a short

time using diluted reagents and low temperatures. To date, most of applications of ultrasonic

extraction have been carried out for organic compounds, but the usefulness of ultrasound for

element extraction is still to be explored. In general, the presence of an acidic liquid is an

important prerequisite for quantitative extraction to be achieved; nitric acid at low

concentration (e.g. 3-5% v/v) is usually chosen for extraction of elements from solid samples.

Quantitative extraction can be achieved for some analytes such as As, Cu, Pb, Cd, etc., from

plant and animal tissues. Nevertheless, incomplete extraction has been observed from

samples containing a typical inorganic matrix (e.g. sediment). It is believed that this finding

is related to the ability of ultrasound to penetrate the solid material. The extraction efficiency

obtained with ultrasound could be increased by addition of glass beads which promote

particle disruption by focusing the energy released by cavitation and by physical crushing.

Particle disruption could also be enhanced by increasing hydrostatic pressure and viscosity.

The use of a bubbling gas during sonication gives rise to an enhanced formation of H2O2 and

hydroxyl radicals (OH) thus aiding analyte extraction from oxidizable materials.

1.6.2 Principle

The high-frequency is generated electronically and the mechanical energy is transmitted to

the sample via a metal probe that oscillates with high frequency. The probe is placed into the

cell-containing sample and the high-frequency oscillation causes a localized low pressure

region resulting in cavitation and impaction, ultimately breaking open the cells. It uses sound

waves at frequencies above the range audible to humans to disrupt the plant cell wall, thereby

enhancing solvent penetration into the plant material and facilitating the release of extracts.

Fig1.7 Longitudinal sound waves


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1.6.3 Frequency

At high sonic frequencies, on the order of the MHz, the production of cavitation bubbles

becomes more difficult than at low sonic frequencies, of the order of the kHz. To achieve

cavitation, as the sonic frequency increases, so the intensity of the applied sound must be

increased, to ensure that the cohesive forces of the liquid media are over come and voids are

created. The broad classification of ultrasound as sound above 20 kHz and up to 100MHz can

be subdivided into two distinct regions Power and Diagnostic. The former is generally at

lower frequency end where greater acoustic energy can be generated to induce cavitation in

liquids, the origin of chemical effects. Sonochemistry normally uses frequencies between 20

and 40 kHz simply because this is the range employed in common laboratory equipment.

However since acoustic cavitation in liquids can be generated well above these frequencies,

recent researches into sonochemistry use a much broader range. High frequency ultrasound

from around 5MHz and above does not produce cavitation and this is the range used in

medical imaging.

Table 1.3 Frequency ranges of sound

Review of literature

Different genus with sativum species

Coriandrum sativum

Coriandrum sativum is a annual herb belonging to the family apiaceae. Its commonly known

as dhaniyalu in telugu, Dhania in oriya, kothmiri in gujarati. It is commonly used as a

digestant. Its fruits are considered carminative, diuretic, tonic, stomachic. and as a spice.

Oryza sativa

Rice is the seed of the monocot plants Oryza sativa (Asian rice) or Oryza glaberrima

(African rice). As a cereal grain, it is the most important staple food for a large part of the

world's human population, especially in Asia and the West Indies. Rice is an ingredient of

many soups and dishes. When combined with milk, sugar and honey, it is used to make

desserts. In some regions, bread is made using rice flour.

Human hearing 16Hz – 18 kHz

Conventional power sound 20 kHz – 100 kHz

Extended range of sonochemistry 20 MHz – 2 MHz

Diagnostic ultrasound 5 MHz – 10 MHz


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Pisum sativum

It is a annual herb belonging to the family fabaceae. The mature seeds are rich in protein and

can be cooked as a vegetable or added to soups etc The sprouted seeds are added to salads,

soups bread etc The roasted seed is a coffee substitute.

Cannabis sativa

Cannabis sativa is an annual herbaceous plant in the Cannabaceae family. It has many uses

and has been used for thousands of years for paper, fabric, food and medicinally it is used in

treating chronic Pain, Headache, Gastrointestinal (Nausea, Anorexia, Abdominal pain,

Irritable bowel syndrome, Crohns disease, Ulcerative colitis, Chemotherapy)Chronic

anxiety., etc.

Different species with Lepidium genus

Lepidium meyenii

It is known commonly as maca, is an herbaceous. The prominent product for export is maca

flour, which is a baking flour ground from the hard, dried roots, "harina de maca." Maca flour

(powder) is a relatively inexpensive bulk commodity, much like wheat flour or potato flour.

In Peru, maca flour is used in baking as a flour base and a flavoring. These are biennial plant

or annual plant.

Lepidium latifolium

It is known by several common names including Pepperweed, Pepperwort, Dittander.

Lepidium perfoliatum

It is a species of flowering plant in the mustard family known by the common name clasping

pepperweed. It is native to Europe and Asia and it can be found in other parts of the world as

an introduced species. This is an annual or biennial herb belongs to family Brassicaceae. The

plant is used as an antiscorbutic.

Lepidium fremontii

It is commonly called as desert pepper weed. Family: Brassicaceae .Parts used are seeds and

is distributed in U.S.A. It is used as flavouring agent.

Ethanomedical information of Lepidium sativum

Garden cress has been considered as an important medicinal plant since vedic era. In

ayurveda it is described as hot, bitter, galactogogue and aphrodisiac and claimed to destroy

http://en.wikipedia.org/wiki/Cannabaceae

http://en.wikipedia.org/wiki/Brassicaceae

http://en.wikipedia.org/wiki/Introduced_species


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vata (air) and kapha (phlegm). In unani system of medicine, seeds and leaves of this plant

have been reported to possess diuretics, aperients and aphrodisiac properties and are

recommended in inflammation, bronchitis, rheumatism and muscular pain. Lepidium sativum

is one of the most common plant species of genus Lepidium which is grown in Egypt for its

important uses in folk medicine for diabetes and antibacterial properties. A recent survey of

different regions of Saudi Arabia showed that the seeds are commonly used as febrifuge,

antirheumatic and he to enhance sexual desire (chopra rn). In china and other far eastern

countries, the seeds are dropsy. It is considered as the one of the better medicinal plants in

various African countries, where the seeds are chewed to cure throat disease, asthma and

headache and are useful for dieresis and rapid bone fracture healing. A tea spoon full of

garden cress seeds boiled in six ounces of water for ½ hour and the decoction with a table

spoon full of honey is given as an effective medicine to increase breast milk and sexual

disorders. Likewise, juice of the seed is also valued as medicine; one teaspoon obtained by

grinding with one ounce of water mixed with a glass full of tender coconut water given as a

folk medicine to cure diarrhea, dysentery, bleeding piles, scanty urination due to liver

disorders and irritation of the intestinal mucous membrane. Seeds roasted in ghee and mixed

with sugar are given as a tonic for a general weakness in young girls and after child birth to

increase breast milk and the oil extracted from seeds roasted in till oil is used as an analgesic

medicated oil in gout, rheumatism, glandular swelling, etc. It should be noted that this oil is

counter irritant and therefore, care must be taken to use a little at a time. (Pullaiah T,2006).

Pharmacological activities proved from Lepidium sativum

Lepidium sativum is reported to exhibit antihypertensive (Maghrani M, Zeggwagh NA,2005),

diuretic (Patel U, Kulkarni,2009), anti-inflammatory, analgesic, anticoagulant (Al-Yahya

MA, Mossa JS,1994), antirheumatic (Ahsan SK, Tariq M, Ageel M,1989), hypoglycemic

(Patole AP,1998), laxative, prokinetic (Rehman N, Journal of Ethnopharmacology. 2011),

antidiarrheal and antispasmodic (Rehman N, Mehmood MH et.al.,) properties. It has been

shown to possess antiasthmatic (Paranjape AN, Mehta AA, 2006) and bronchodilatory (Mali

RG, Mahajan SG, 2008) activity.

Active constituents isolated from Lepidium sativum

O

OH

O

sinapin

N

phenyl acetonitrile


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N

lepidine

N C S

benzyl isothiocyanate

O

OH

OO

HO

sinapic acid

2.6: Aim & Plan Of Work

• The plan of work involved collection and authentication of plant material, size reduction

after shade dried, extraction of powdered plant material using sonicator in methanol

(95%), phytochemical investigation of the extract and estimation of protective effect on

neuropathic pain.

AIM OF THE INVESTIGATION

Neuropathic pain is defined as the pain condition that results from damage affecting

peripheral nerves, posterior roots and spinal cord or certain regions of brain. Increased

neuronal excitability is thought to be the underlying mechanism involved in all forms of

neuropathic pain. Tricyclic antidepressants, often the first choice causes sedation and

cardiovascular issues and are only partially effective. Opioids prescribed for moderate to

severe pain sometimes avoided because of the potential dependence and tolerance as their

side effects. To overcome the side effects we made an attempt to develop newer drug.

Literature supports the involvement of adenosine receptors in pain. We framed the work to

study the adenosine based treatment for chemotherapy induced neuropathic pain. Adenosine

was found to be significant as it is involved in nociception.

Bearing the above points in mind Lepidium sativum is selected which is used for various

ailments like oxidative stress (anti oxidant), diabetics, cancer, aging, fatigue, pain. Hence to

identify a new drug an attempt is made to investigating the activity of Lepidium sativum to


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treat neuropathic pain by carrying out pharmacological studies. The present study was aimed

to carry out the in vivo chemotherapy induced neuropathic pain screening models.

Lepidium sativum Description

Taxonomical Classification (George H.M Lawerence,1959.)

Kingdom = Plantae

Division = Angiospermae

Class = Dicotyledonae

Sub Class = Polypetalae

Series = Thalamiflorae

Order = Parietales

Family = Cruciferae ( Brassicaceae)

Genus = Lepidium Linn

Species = Lepidium sativum Linn sp.

Description

It is an erect branched, glabrous herb with 60 cm height. Leaves are entire or pinnately

dissected, variously lobed often with linear segments; upto 6 -5 cm long and lobes are 0.7 –

1.2 to 0.3 – 0.6 cm size, upper leaves usually entire and 2 to 3 cm long, oblanceolate, sessile.

Recemes are 7 to 15 cm long axillary and terminal; flowers are pale pink; pedicels are 3 to

5mm long. Pods are obovate are broadly elliptical, roundate, emarginated slightly but thickly

winged above.

Fig: 3.1 Lepidium sativum plants

Fig: 3.2 Lepidium sativum Flowers Fig: 3.3 Lepidium sativum Leaf


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Fig: 3.4 Lepidium sativum Germinated seeds Fig: 3.5 Lepidium sativum Flex seeds

Seeds are small oval shaped pointed and triangular at one end, smooth about 2 to 3 mm long

1 to 1.5 mm wide, reddish brown and arrow present on both surfaces, extending upto two

thirds down wards, a slight wing like extension present on both the edges of seed when

soaked in water seedcoat swell and gets covered with a transparent, colourlessmucilage.

Distribution

Plant is a native of Mediterranean region. Now, it is being cultivated through out India very

likely indigenous in West Asia. In India it is mainly cultivated in U.P., Rajasthan, Gujarath,

Maharastra and Madhya Pradesh. (Indian medicinal plants Kirtikar et al.).

Cultivation & Harvesting

It is a cool season annual plant, cultivated throughout India. It has long leaves at the bottom

of the stem and small bright green feather like once arrange on opposite side of its stalk at the

top. There are plain broad leaf and curly leaf that differ in texture but not taste. Garden cress

can be harvested throughout the year whether in door or out door and is cut when the sprouts

are 5 to 10cm tall. Cress grows in well worked soil with good drainage. It flourishes in shade

or semi shade and can tolerate a wide range of temperature. while preparing the soil, it should

be dug and mixed with a well balanced fertilizer. The seed sowed thickly a 5 – 6 cm deep in

wide rows, 45 – 60 cm apart to have a continuous crop. The leaves should not remain wet

more than the requirement since the soil that lodges there when water splashes is impossible

to washout damaging the leaf. Cress has no serious pest problem. Its growth is rapid and

harvesting can begin in the same month as sowing with yields reaching as high as 6 tonnes

per hectare. The plant prefers light (sandy), medium (loamy) and heavy (clay) soils. It can

grow in semi shade (light woodland) or even without any shade. It requires moist soil and

also some shade during the summer to prevent heat from running straight to seed. It can be

grown at all elevations, the whole around. However, the best crop is obtained in the winter

season. Seeds are sown in the plains from September to February and on the hills, from


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March to September. They are sown thick and covered until germination begins. In few days

sowing the plants are ready for cutting. To get a continuous supply of leaves, sowing is done

in succession at intervals of 8 days. Garden cress leaves are consumed raw in salads; they are

also cooked with vegetable curries and used as fodder for horses, camels, etc. firm and evenly

colored rich green specimen. Its stem should be stuck in a water filled glass, then should be

selected for storage. Cress with any signs of slime, wilting or discolouration should be

avoided. The cress can be stored under refrigeration in plastic for up to five days. Its stem

should be stuck in a water filled glass, the glass bagged and refrigerated to prolong life. Until

they are needed for use, the leaves should be left on the stem. (The wealth of india new delhi

vol 6, 1962).

Chapter 4: Extraction and qualitative analysis of extracts

MATERIALS AND METHODS

Plant material

The Plant material was collected from the distributor SHRI-SHAIL MEDI-FARM, Nagpur,

Maharashtra, India. It was identified by Prof. Dr. B.Pratibha Devi Department of Botany,

Osmania University and Hyderabad. A voucher no.032 of the plant was deposited in the

Department of Botany, Osmania University, and Hyderabad. Air-dried under the shed at

room temperature. Dried seed material was pulverized and the powder kept in polyethylene

bags.

Preparation of extract

Accurately weighed plant material was soaked in the conical flask by using methanol solvent.

Extraction was done by using ultra sonic bath sonicator. Solvent recovery done by using

simple distillation method. Extract was collected and stored in refrigerator.

Preliminary phytochemical screening

Tests for Alkaloids (Kokate, 2006b)

A small portion of the solvent free petroleum ether, alcohol extracts were stirred separately

with few drops of dilute hydrochloric acid and filtered. The filtrate was tested with various

reagents for the presence of alkaloids.

Mayer‟s Reagent – Cream colored precipitate.

Dragendroff‟s Reagent – Orange - brown colored precipitate.

Hager‟s Reagent – Yellow Colored precipitate.


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Wagner‟s Reagent – Reddish brown precipitate

Test for Saponins

The extract was diluted with 20ml of distilled water and it was agitated in a graduated

cylinder for 15mins. The formation of 1cm. layer of foam shows the presence of saponins.

Test for Phenolic Compounds and Tannins

Small quantities of the extracts were taken separately in water and test for the presence of

phenolic compounds and tannins was carried out with the following reagents.

Dilute ferric chloride solution (5%) – Violet color.

1% solution of gelatin containing 10% sodium chloride – White precipitate.

10% lead acetate solution – White precipitate.

Test for Flavonoids

Extracts were taken with aqueous sodium hydroxide solution – blue to violet color

(anthocyanins) yellow color (flavones), yellow to orange (flavonones).

With concentrated sulfuric acid – yellow to orange color (anthocyanins) yellow to orange

(flavones), orange to crimson (flavonones).

Shinoda‟s Test: Small quantities of the extracts were individually dissolved in alcohol, to

them piece of magnesium followed by concentrated hydrochloric acid drop – wise added

and heated. Appearance of magenta color shows the presence of flavonoids.

Test for Proteins (Khandelwal, 2006c)

Small quantities of the extract was dissolved in water and treated with the following

reagents.

Biuret test: An equal volume of 5% sodium hydroxide and 1% copper sulfate solution

was added – appearance of pink or purple color shows the presence of free amino acids or

proteins.

Millon‟s Reagent: Appearance of red color shows the presence of protein and free amino

acid.

Test For Terpenoids

Noller’s test

A pinch of powder was taken in a dried test tube. A bit of tin foil and 0.5ml of thinly chloride

was added and heated gently. Formation of pink colour indicates the presence of terpenoids.


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Test For Carbohydrates (Kokate et al.,2006a)

Molisch’s test

The substance was treated with α-napthal and concentrated sulphuric acid. Formation of

violet colour indicates the presence of carbohydrates.

Chemicals

Chemicals were obtained from the sources mentioned as follows. Pregabalin, Ketamine was

purchased from NIMS medicals Hyderabad. Vincristine was purchased from NIMS medicals

Hyderabad The Plant material was collected from the distributor SHRI-SHAIL MEDI-

FARM, Nagpur, Maharashtra, India.

All chemicals and reagents used were of analytical grade.

Experimental animals

Adult male Wistar rats weighing 200-250gm were used in the pharmacological studies. The

inbred animals were taken from the animal house in Peerzadiguda, uppal, Hyderabad 500008.

The animals were housed in groups of 6 per cage. They were maintained in well-ventilated

room at a temperature of 22-24ºc with relative humidity at 45-55% and natural 12h: 12h day-

night cycle in propylene cages. All the experiments were carried out between 10:00 am to

5:00 pm. The animals were housed one week prior to experiments to acclimatize laboratory

temperature. Food was withdrawn 3hrs before and during experiment.

Preparation of drug solution

Drug was dissolved in saline and administered to animals through intravenous and intra

peritoneal routes.

Assessment of effectiveness of Lepidium sativum seeds extract for neuropathic pain

using chemotherapy induced neuropathic pain model

Chemotherapy induced neuropathic pain was performed following the method described by

Joseph et al., 2003. Rats were first treated with vincristine sulphate (100 µg/kg, i.v). A single

intravenous dose of vincristine causes painful peripheral neuropathy. Baseline readings for

mechanical hyperalgesia and thermal hyperalgesia are noted. After 5days of administration of

vincristine test drug and standard drug are administered for 5days. The total period of study

lasts for 10 days. Mechanical hyperalgesia and thermal hyperalgesia of all groups were

measured during test drug administration in order to confirm the development of neuropathic


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pain. hyperalgesia of test drug treated group were compared with vincristine treated group in

order to confirm the effectiveness of drug in neuropathic pain.

Experimental groups

The rats were divided into four groups consisting of 6 per each group

Group I -Vincristine (100 µg/kg i.v.)

Group II - Vincristine (100 µg/kg i.v.) + test extract (50 mg/kg i.p.)

Group III- Vincristine (100 µg/kg i.v.) + test extract (100 mg/kg i.v.)

Group IV- Vincristine (100 µg/kg i.v.) + Pregabalin (80 mg/kg i.p.)

Behavioural tests

Mechanical hyperalgesia (Decosterd et al., 1998).

Mechanical hyperalgesia was tested by using the pin prick test. Animals were placed on the

elevated grid; a pin prick test was performed using a safety pin. The lateral plantar surface of

the right hind paw was briefly stimulated at intensity sufficient to indent but not penetrate the

skin (pin prick test). The duration of paw withdrawal was recorded, with an arbitrary minimal

time of 0.5 (sec) and a maximal cut off 15 (sec).

Thermal Hyperalgesia (Hot Plate Test) (Jain V et al., 2009).

The thermal nociceptive threshold, as an index of thermal hyperalgesia, was assessed by the

Eddy‟s hot plate, which is an instrument designed by Eddy and co-workers to assess thermal

sensitivity. The plate was preheated and maintained at a temperature of 40 ± 2.0°C. The rat

was placed on the hot plate and nociceptive threshold, with respect to licking of the hind paw,

was recorded in seconds. The cut-off time of 20 s was maintained.

Statistical analysis

The data are represented as mean±S.E.M and statistical significance between groups were

analyzed by means of student paired t-test, one way ANOVA followed by Dunnet‟s t-test, for

behavioral parameters as applicable and two way ANOVA for motor coordination. P<0.05

implies significance. All the statistical analysis was carried out using graph pad prism 5.0

version software.


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Table no. 5.1 Preliminary phytochemical screening of Lepidium sativum seed extract

Table5.2: Base line readings of Eddy’s hot plate for category 1:

Paw licking

Animal 1 ( head) 8

Animal 2 (body) 7

Animal 3 (tail) 3

Animal 4 (head +body) 2

Animal 5 (body + tail) 5

Animal 6(head + body +tail) 6

Fig 5.2 Base line readings of Eddy’s hot plate for category 1

Extract Alkaloids Carbo

hydrates Glycosides Tannins

Amino

acids Flavanoids Saponins Inulin

Tri

terpenoids

Methanol + + _ + + + _ + +

‘+’ indicates-presence of constituents ‘-’ indicates-absence of constituents


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Table 5.3: Base line readings of Eddy’s hot plate for category 2:

Paw licking

Animal 1 (head) 7

Animal 2 (body) 6

Animal 3 (tail) 5



Animal 6 (head + body +tail) 5

Fig 5.3 Base line readings of Eddy’s hot plate for category 2:

Table 5.4: Base line readings of Eddy’s hot plate for category 3

Paw licking

Animal 1 ( head) 7

Animal 2 (body) 6

Animal 3 (tail) 7




Fig 5.4 Base line readings of Eddy’s hot plate for category 2:


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Table 5.5: Base line readings of Eddy’s hot plate for category 4

Paw licking

Animal 1 ( head) 6

Animal 2 (body) 4

Animal 3 (tail) 6




Fig 5.5: Base line readings of Eddy’s hot plate for category 4:

Table 5.6: Base line readings of actophotometer for category 1:

Readings \ count for 5 mins

Animal 1 ( head) 148

Animal 2 (body) 455

Animal 3 (tail) 343




Fig 5.6: Base line readings of actophotometer for category 1


222




Animal 1 (head) 220

Animal 2 (body) 342

Animal 3 (tail) 227




Fig 5.7 Base line readings of actophotometer for category 2:



Animal 1 (head) 341

Animal 2 (body) 421

Animal 3 (tail) 356






223




Animal 1 (head) 456

Animal 2 (body) 236

Animal 3 (tail) 274





Table 5.10: Actophotomer readings when vincristine induced (Category1):

Day1 Day2 Day3 Day4 Day5 Day6

Animal 1 (head) 145 138 128 118 98 85

Animal 2 (body) 425 389 357 298 276 232

Animal 3 (tail) 324 318 297 257 232 198

Animal 4 (head +body) 324 298 276 243 208 187

Animal 5 (body + tail) 328 317 298 253 231 187

Animal 6 (head + body +tail) 317 298 254 221 197 167

Fig 5.10 Actophotomer Readings When Vincristine Induced (Category1):


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Table 5.11 Actophotometer readings when vincristine is induced: (Category 2)

Day 1 Day 2 Day 3 Day 4 Day 5 Day 6

Animal 1 (Head) 162 158 144 136 128 119

Animal 2 (Body) 200 191 188 178 162 156

Animal 3 (Tail) 200 196 178 160 142 128

Animal 4 (Head + Body) 196 172 164 158 144 122

Animal 5 (Body + Tail) 245 218 185 172 165 148

Animal 6 (Head + Body + Tail) 233 218 189 171 155 132

Fig 5.11 Actophotomer Readings When Vincristine Induced:(Ct-2).

Table 5.12 Actophotometer readings when vincristine is induced: (Category 3)


Animal 1 (Head) 160 152 132 122 111 98

Animal 2 (Body) 222 198 188 172 152 123

Animal 3 (Tail) 218 192 179 162 151 132

Animal 4 (Head + Body) 216 201 189 165 148 125

Animal 5 (Body + Tail) 252 232 199 187 165 148


Fig 5.12 Actophotomer Readings When Vincristine Induced:(Ct-3)


225


5.13 Actophotometer readings when vincristine is induced: (Category 4)


Animal 1 (Head) 158 132 111 96 82 72

Animal 2 (Body) 260 243 221 188 162 126

Animal 3 (Tail) 210 196 178 165 132 108

Animal 4 (Head + Body) 192 172 144 128 94 81

Animal 5 (Body + Tail) 236 222 190 172 152 128


Fig 5.13 Actophotomer Readings When Vincristine Induced:(Ct-4)

Table 5.14: Actophotomer readings when test extract(50µg\ml) induced


Animal 1 (head) 87 101 123 135 142 156

Animal 2 (body) 243 287 308 376 398 432

Animal 3 (tail) 198 243 265 294 308 334

Animal 4 (head +body) 194 221 254 294 308 334

Animal 5 (body + tail) 195 241 267 302 327 335

Animal 6(head + body +tail) 175 208 234 276 308 324

fig 5.14: Actophotomer readings when test extract(50µg\ml) induced


226


Table 5.15: Actophotomer readings when test extract(100µg\ml) induced


Animal 1 ( head) 91 111 126 141 149 160

Animal 2 (body) 261 294 317 384 401 446

Animal 3 (tail) 200 249 272 301 318 346

Animal 4 (head +body) 201 234 267 303 321 354

Animal 5 ( body + tail ) 200 254 273 318 330 345

Animal 6(head + body +tail) 184 215 245 286 318 348

Fig 5.15: Actophotomer readings when test extract(100µg\ml) induced

Table 5.16: Eddy’s hot plate readings when vincristine induced

Day 1 Day 2 Day 3 Day 4 Day 5

Animal 1 (head) 7 7 4 3 4

Animal 2 (body) 5 5 4 2 1

Animal 3 (tail) 4 4 3 2 1

Animal 4 (head +body) 6 5 4 2 1

Animal 5 (body + tail) 4 4 3 2 1

Animal 6(head + body +tail) 5 4 3 2 1

fig 5.16: Eddy’s hot plate readings when vincristine induced


227


Table: 5.17 Eddy’s hot plate readings when vincristine is induced: (category 2)


Animal 1 (Head) 3 2 2 1 1 0

Animal 2 (Body) 2 2 1 1 1 0

Animal 3 (Tail) 2 2 1 1 0 0

Animal 4 (Head + Body) 2 1 1 1 0 0

Animal 5 (Body + Tail) 3 2 2 1 1 0


Fig: 5.17 Eddy’s hot plate readings when vincristine is induced: (category 2)

Table 5.18: Eddy’s hot plate readings when vincristine is induced: (category 3)


Animal 1 (Head) 2 1 1 0 0 0

Animal 2 (Body) 3 2 2 1 1 0

Animal 3 (Tail) 3 2 1 0 0 0




Fig 5.18: Eddy’s hot plate readings when vincristine is induced: (category 3


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Table 5.19 Eddy’s hot plate readings when vincristine is induced: (category 4)


Animal 1 (Head) 3 2 2 1 0 0

Animal 2 (Body) 3 2 2 1 1 0

Animal 3 (Tail) 2 1 1 0 0 0




Fig: 5.19 Eddy’s hot plate readings when vincristine is induced: (category 4)

Table 5.20: Eddy’s hot plate readings when test extract(50µg\ml) induced


Animal 1 ( head) 5 6 6 7 8

Animal 2 (body) 2 3 4.5 6 7

Animal 3 (tail) 1.5 2 3.5 4 6

Animal 4 (head +body) 2 3.5 5 6 8

Animal 5 (body + tail) 1.5 2 3.5 5 6

Animal 6 (head + body +tail) 2 2.5 4 5.5 7

Fig: 5.20 Eddy’s hot plate readings when test extract(50µg\ml) induced


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Table 5.21: Eddy’s hot plate readings when test extract(100µg\ml) induced


Animal 1( head) 6 6.5 7 8 9

Animal 2 (body) 3 3.5 5 7 8.5

Animal 3 (tail) 2 3 4 6 7

Animal 4 (head +body) 3 5 6 7 9

Animal 5 (body + tail) 2 3 4 6 7

Animal 6 (head + body +tail) 3 4 5 6 8

Fig 5.21 Eddy’s hot plate readings when test extract(100µg\ml) induced

Table 5.22: Eddy’s hot plate readings when standard(pregabalin) drug induced


Animal 1 (head) 10 10 11 12 12

Animal 2 (body) 10 11 11 11 12

Animal 3 (tail) 10 10 12 12 12

Animal 4 (head +body) 10 10 11 11 12

Animal 5 (body + tail) 10 11 11 12 12


fig: 5.22: eddy’s hot plate readings when standard(pregabalin) drug induced


230


Table 5.23: Actophotometer readings when standard(pregabalin) drug induced

Day1 Day2 Day3 Day4 Day5

Animal 1 (head) 100 142 162 190 220

Animal 2 (body) 104 158 172 185 210

Animal 3 (tail) 110 154 184 216 255

Animal 4 (head +body) 118 162 194 216 250

Animal 5 (body + tail) 140 180 240 290 311


Fig 5.23: Actophotometer readings when standard(pregabalin) drug induced

CONCLUSION

The Plant material was collected from the distributor Shri-Shall Medi-Farm, Nagpur and

Maharashtra, India. It was identified by prof. Dr. Mrs. B. Prathibha Devi. Department of

Botany, Osmania University and Hyderabad. A Voucher No. 032 of the plant was deposited

in the Department of Botany, osmania University and Hyderabad. Air-dried under the shed at

room temperature. Dried seed material was pulverized and the powder kept in polyethylene

bags.

Extraction was done by using ultra sonic bath sonicator. Solvent recovery done by using

simple distillation method. Extract was collected and stored in refrigerator. Preliminary

phytochemical screening results shown that, Alkaloids carbohydrates, tannins, aminoacids,

flavonoids are present so we concluded that the plant material contains the activity towards

neuropathic pain, thus we undergone animal studies. To identify pharmacological properties

against neuropathy of our seed extract we undergone Behavioral tests. In that we performed

thermal hyperalgesia tests and locomotor activity studies. By conducting Thermal


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Hyperalgesia test on animals by using Eddy‟s hot plate and by observing the results we

concluded that plant extract has shown some activity towards neuropathy by comparing with

standard. By conducting Locomotor Activity on animals by using Actophotometer and by

oberserving the results we concluded that plant extract has shown some activity towards

neuropathy by comparing with standard. Finally, we concluded that Lepidium sativum seeds

extract having anti-neuropathic activity.

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“protective effect of ultra sonic bath assisted …

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