biochemical studies on berberis lyceum royal and...
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BIOCHEMICAL STUDIES ON BERBERIS LYCEUM ROYAL AND ANALYSIS OF ITS EXTRACTS FOR BIOACTIVITY
ASIF AHMED (03-arid-748)
Department of Biochemistry Faculty of Sciences Pir Mehr Ali Shah
Arid Agriculture University Rawalpindi, Pakistan
2009
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BIOCHEMICAL STUDIES ON BERBERIS LYCEUM ROYAL AND ANALYSIS OF ITS EXTRACTS FOR BIOACTIVITY
by
ASIF AHMED (03-arid-748)
A thesis submitted in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
in
Biochemistry
Department of Biochemistry Faculty of Sciences Pir Mehr Ali Shah
Arid Agriculture University Rawalpindi, Pakistan
2009
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CERTIFICATION
I hereby undertake that this research is an original and no part of this thesis falls under
plagiarism. If found otherwise, at any stage, I will be responsible for the consequences.
Name: Asif Ahmed Signature: _______________________
Registration No. : 03-arid-748 Date:
Certified that the contents and form of thesis entitled “Biochemical studies on
Berberis lyceum Royal and analysis of its extracts for bioactivity” submitted by “Mr. Asif
Ahmed” has been found satisfactory for requirement of the degree.
Supervisor: __________________________ (Dr. Muhammad Gulfraz) Member: __________________________ (Dr. Ghazala Kaukab) Member: __________________________ (Dr. Muhammad Arshad) Chairman: ________________________ Dean: ____________________________ Director Advanced Studies: __________________________
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DEDICATED
TO
MY MOTHER &
WIFE
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CONTENTS
PAGES
LIST OF FIGURES viii
LIST OF TABLES ix
LIST OF PUBLICATIONS xi
LIST OF ABBREVIATIONS xii
ACKNOWLEDGEMENTS xiv
ABSTRACT xvi
1. INTRODUCTION 1
2. REVIEW OF LITERATURE 5
2.1 WORLD NATURAL MEDICINAL PLANT RESOURCES AND DEMAND
5
2.1.1 Therapeutic Potential of Medicinal Plants 5
2.1.2 Medicinal Plants in Pakistan and their Scope 6
2.2 BERBERICIDACEASE GENUS 7
2.2.1 Essential Minerals and Medicinal Plants: Therapeutic Role 8
2.2.2 Bioactive constituent of Berberis species 10
2.3 BERBERIS LYCEUM ROYAL 11
2.3.1 Systematic and Distribution of Berberis lyceum Royal 11
2.3.2 Berberis lyceum Royal in the traditional folk medicine 11
2.4 ISOLATION, PURIFICATION AND ANALYZING TECHNIQUES
12
2.4.1 Column Chromatography 13
2.4.2 Thin Layer Chromatography 14
2.4.3 Detection and Structure Elucidation Techniques 14
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2.5 BIOASSAY METHODS IN NATURAL PRODUCTS 15
2.5.1 Antimicrobial Bioassay 15
2.5.2 Wound Healing Bioassay 18
2.5.3 Anti diabetic Bioassay 21
3 MATERIALS AND METHODS 24
3.1 COLLECTION AND PREPARATION OF SAMPLES 24
3.2 BIOCHEMICAL ANALYSIS OF BERBERIS LYCEUM ROYAL 24
3.2.1 Wet and Dry Weight Analysis 24
3.2.2 Carbohydrate Analysis 25
3.2.3 Protein Analysis 26
3.2.4 Lipid Analysis 26
3.2.4.1 Analysis of fatty acid 26
3.2.4.2 Gas chromatography (GC) conditions 26
3.2.4.3 Gas chromatography –mass spectrometry (GC-MS) conditions 26
3.3 ESSENTIAL METAL ION ANALYSIS 27
3.4 QUANTIFICATION OF ALKALOIDS FROM ROOT OF BERBERIS LYCEUM ROYAL 27
3.4.1 Isolation of Alkaloid(s) by Column Chromatography 28
3.4.2 HPLC analysis of alkaloids 28
3.4.3 NMR analysis of alkaloids 29
3.4.3.1 Preparation of Samples 29
3.4.3.2 NMR spectra analysis 29
3.4.3.3 Recovery 29
3.5 BIOASSAYS 30
3.5.1 Antimicrobial Studies 30
3.5.1.1 Preparation of sample 30
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3.5.1.2 Microorganisms 30
3.5.1.3 Antimicrobial activity 30
3.5.1.4 Micro dilution assay 31
3.5.2 Wound Healing Activity of Berberis lyceum 32
3.5.2.1 Preparation of plant material 32
3.5.2.2 Drug formulation 32
3.5.2.3 Experimental animals 32
3.5.2.4.1 Wound healing activity: excision wounds 33
3.5.2.4.2 Wound healing activity: incision wounds 33
3.5.2.4.3 Wound healing activity: dead wound space 34
3.5.2.5 Acute toxicity and selection of dose 34
3.5.3 ANTI DIABETIC ACTIVITIES OF Berberis lyceum ROOTS EXTRACTS 34
3.5.3.1 Preparation of plant extracts 34
3.5.3.2 Experimental Animals 35
3.5.3.3 Acute toxicity and selection of doses 35
3.5.3.4 Effect of root extract on different animal models 35
3.5.3.4.1 Effect of the ethanol extracts of Berberis lyceum on serum glucose levels in normal fasted rats
35
3.5.3.4.2 Effect of the ethanol extracts of Berberis lyceum on serum glucose level in alloxan- induced diabetic rats
36
3.5.3.4.3 Effect of the ethanol extracts of Berberis lyceum on glucose tolerance test
36
3.5.3.4.4 Effect of aqueous extracts of Berberis lyceum on serum glucose level (mg/dl) in alloxan diabetic rats.
37
3.5.3.4.5 Effects of aqueous extracts of Berberis lyceum on serum glucose level (mg/dl) in oral glucose tolerance test
37
3.5.3.5 Determination of the serum glucose concentration 37
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3.5.3.6 Data and statistical analysis 37
4 RESULTS AND DISCUSSION 39
4.1 BIOCHEMICAL ANALYSIS OF BERBERIS LYCEUM ROYAL 39
4.1.1 Proximate Analysis 39
4.1.2 Determination of Oil from Root Samples 41
4.1.3 Metal ion analysis 46
4.2 EXTRACTION AND PURIFICATION OF ALKALOIDS 53
4.3 BIOASSAYS 60
4.3.1 Antimicrobial Bioassay 60
4.3.2 Wound Healing Activity 67
4.3.3 Anti Diabetic Activity 70
4.3.3.1 Effects of berberine and Berberis lyceum root extract on glucose tolerance, and glucose levels in normal and diabetic animals
70
4.3.3.2 Effects of berberine and Berberis lyceum root extracts on serum insulin and glycosylated Haemoglobin
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4.3.3.3 Effects of berberine and Berberis lyceum root extracts on lipid profiles
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4.3.3.4 Effects of berberine and Berberis lyceum root extracts on changes in body weight
74
5 SUMMARY 78
6 LITERATURE CITED 82
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LIST OF FIGURES
Fig. No. PAGES
4.1 Spectra of representative GC–MS for fatty acid analysis of Berberis lyceum Royal oil collected from different areas of Pakistan
42
4.2 Macro element analysis of Berberis lyceum Royal from different areas of Pakistan. (A): Magnesium and (B): Calcium
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4.3 Macro element analysis of Berberis lyceum Royal from different areas of Pakistan. (A): Sodium and (B): Pottasium
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4.4 Micro element analysis of Berberis lyceum Royal from different areas of Pakistan. (A): Manganese and (B): Iron
50
4.5 Micro element analysis of Berberis lyceum Royal from different areas of Pakistan. (A): Copper and (B): Zinc
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4.6 Peaks of HPLC (1) palmatine (2) berberine from root samples of B. lyceum
55
4.7 1H NMR spectroscopic analysis of berberine and palmatine 56
4.8 Structure of berberine 57
4.9 Structure of palmatine 57
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LIST OF TABLES
NO. TITILE PAGES
4.1 Proximate analysis of different Berberis lyceum Royal samples collected from different areas of Pakistan (%)
40
4.2a Percentage* (%) of saturated fatty acids of Berberis lyceum Royal oil analyzed by GC–MS and their retention times
45
4.2b Percentage* (%) of unsaturated fatty acids of Berberis lyceum Royal oil analyzed by GC–MS and their retention times
45
4.3 1H NMR chemical shifts (δ; in ppm) of berberine and palmatine (Solvent CDCl3)
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4.4 Antimicrobial activity of methanol extracts of Berberis lyceum (100 µg/disk tested against bacterial strains by using disk diffusion method. Zone of inhibition in diameter (mm) around test disk
61
4.5 Antimicrobial activity of aqueous extracts of Berberis lyceum (100 µg/disk tested against bacterial strains by using disk diffusion method. Zone of inhibition in diameter (mm) around test disk.
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4.6 Antimicrobial activity of methanol extracts of Berberis lyceum (100 µg/disk tested against yeast and fungi isolates by using disk diffusion method. Zone of inhibition in diameter (mm) around test disk
65
4.7 Antimicrobial activity of aqueous extracts of Berberis lyceum (100 µg/disk tested against yeast and fungi isolates by using disk diffusion method. Zone of inhibition in diameter (mm) around test disk
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4.8 The MIC (µg/ml) values of Berberis lyceum (methanol) tested against microorganisms in micro dilution assays.
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4.9 The MIC (µg/ml) values of Berberis lyceum (aqueous) tested against microorganisms in micro dilution assays
66
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4.10 Effect of topical application of the aqueous and methanol extracts of Berberis lyceum on epithelialisation (mm2) in the excision wound repair model
68
4.11 Effect of the aqueous and methanol extracts of Berberis lyceum on wound breaking strength in the incision model, and granulation in the dead space model
68
4.12 Blood glucose concentration on the oral glucose tolerance test after treatment with extract of Berberis lyceum or berberine in glucose loaded rats
71
4.13 Blood glucose concentration after treatment with extract of Berberis lyceum or berberine in normal rats
71
4.14 Blood glucose concentration after treatment with extract of Berberis lyceum or berberine in alloxan-induced diabetic rats
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4.15 Serum insulin and glycosylated haemoglobin in normal and alloxan induced diabetic rats after treatment with extract of Berberis lyceum or berberine
72
4.16 Serum lipid profiles in normal and alloxan induced diabetic rats after treatment with extract of Berberis lyceum or berberine
73
4.17 Body weight of alloxan induced diabetic rats after treatment with extract of Berberis lyceum or berberine
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LIST OF PUBLICATIONS
NO. TITILE
1 A. Asif, G. Kakub, S. Mehmood, R. Khunum and M. Gulfraz. 2007. Wound Healing Activity of Root Extracts of Berberis lyceum Royal in Rats. Phytother. Res. 21, 589–591
2 M. Gulfraz, S. Mehmood, A. Ahmad, N. Fatima, Z. Praveen and E. M. Williamson. 2008. Comparison of the Antidiabetic Activity of Berberis lyceum Root Extract and Berberine in Alloxan-induced Diabetic Rats. Phytother. Res. 22, 1208–1212
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LIST OF ABBREVIATIONS
(NH4)2SO4 Ammonium sulfate
AAS Atomic Absorption Spectrophotometer
ANOVA Analysis of Variance
Ca Calcium
CaCl2 Calcium chloride
CHCl3 Chloroform
CP Crude protein
Cu Copper
CuSO4 Copper sulfate
DM Dry Matter
DMRT Duncan’s Multiple Range Test
Fe Iron
FeSO4 Ferrous sulfate
FP Flame photometer
GC Gas Chromatography
GC-MS Gas Chromatography-Mass Spectrometry
H2S04 Sulfuric Acid
HCl Hydrochloric acid
HPLC High Performance Liquid Chromatography
K Potassium
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L Liter
M Molar
Mg Milli gram
MgCl2 Magnesium Chloride
Mgg-1 Milligram per gram
MgSO4 Magnesium sulfate
Min Minutes
MnCl2 Manganese chloride
MUFA Mono Unsaturated Fatty Acid
Na Sodium
NaCl Sodium Chloride
NaOH Sodium Hydroxide
oC Degree Centigrade
PUFA Poly Unsaturated Fatty Acid
Sec Seconds
SFA Saturated Fatty Acid
v/v Volume by volume
w/v Weight by volume
Wf Final Weight(Weight after drying)
Wi Initial Weight(Weight before drying)
Zn Zinc
ZnSO4 Zinc sulfate
μl Micro liter
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ACKNOWLEDGEMENTS
My all praise to Almighty Allah, The source of knowledge and wisdom, who
bestowed me loving parents, kind teachers and faithful friends that make me to prosper in life.
Glory and praise to our Last Prophet Hazrat Muhammad , who is forever a
symbol of direction and knowledge for whole humanity. I pay my regards to my parents that
brought me up and chalked my way to seek knowledge.
I pay my thanks and respect to my Supervisors, Dr. Muhammad Gulfraz, Associate
Professor, Pir Mehr Ali Shah Arid Agriculture University Rawalpindi (PMAS AAUR) who
guided me at every step of research and provided me a lot of opportunities to build up my
research carrier. I am also thankful to the Dean, Faculty of Sciences and Founder Chairperson,
Department of Biochemistry, Professor Dr. Azra Khanum and Chairman, Department of
Biochemistry, Professor Dr. S.M. Saqlain Naqvi for their sympathy and guidance regarding
every aspect of the student life in the department.
I am thankful to all my teachers especially Dr. Ghazala Kaukab, Associate Professor,
Department of Biochemistry and Prof. Dr. Muhammad Arshad, Department of Botany who
supported me by sharing their valuable suggestions during the preparation of this manuscript.
My special thanks and gratitude to my friends Dr. Sajid Mehmood and Mr. Muhammad
Zeeshan Hyder, Ph.D. Scholar, for their help during Ph.D studies. I am also thankful to all
my research fellows and friends, especially Khalid Khan, Nosheen Fatima Rana, Zahida
Parveen, Safoora Shaukat, Mushtaq Ahmad, Ahmad Waseem Shahid and Zohaib
Imtiaz.
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I am also thankful to the staff of National Agriculture Research Council (NARC,
Islamabad, especially Dr. Nasim Akhtar), Poultry Research Institute (PRI, Rawalpindi),
and Gazi University, Turkey, for their help and without their co-operation I would have not
reach at the stage of thesis completion. I am also thankful to the Departmental and other
departmental employees, especially Yasin Shah, Amir Shahzad and Aurangzeb for their co
operation during my research.
May Allah almighty bless all of them.
Asif Ahmed
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ABSTRACT
Medicinal plants are major source of drugs used for the treatment of various health
disorders. Berberis lyceum Royal, an indigenous plant of the North-East of Pakistan was selected
to explore its medicinal value during this study. This plant has many therapeutic values and is
being used against many diseases / infections by local population since centuries. B.lyceum
remedies provided against swollen and sore eyes, broken bones, wounds, gonorrhea, curative
piles, unhealthy ulcers, acute conjunctive, and in chronic ophthalmia. Therefore, thorough
investigation was conducted for proximate analysis, fatty acid contents, metal ion analysis,
isolation and purification of alkaloids. Bioactivity of crude extract for antimicrobial, antidiabetic
and wound healing have been investigated in this study. Biochemical analysis of root samples of
B. lyceum Royal showed the variation among different parameters, which include protein contents
(4.4 – 6.24 %), crude fiber (14.96 – 16.40 %) and crude ash (3.79 – 6.99 %) on dry weight basis.
No variation regarding crude fats (0.5 %) was found in any samples analyzed. The oil contents
were determined by Soxhlet method and results revealed that the principal saturated and
unsaturated fatty acid components of B. lyceum Royal root were Palmitic (16:0), Oleic (18:1)
and Linoleic (18:2) acids. Palmitic acid (11.73 – 32.04 %), stearic acid (1.09 – 2.66 %), oleic
acid (12.01 – 39.67 %), Linoleic acid (42.59 – 47.43 %) and linolenic acid (1.70 – 5.71) were
found when oil was analyzed by gas chromatography-mass spectrometry. In all cases
polyunsaturated fatty acids (PUFAs) were greater than monounsaturated fatty acids (MUFAs).
The micro and macro elements of different samples were analyzed by atomic absorption
spectrometry and flame photometer. The results showed that the higher mineral ion contents
under investigation were found in Mansehra sample i.e. 599.12 μg /g, whereas Abbotabad had
the lowest content, 242.63 μg/g. The total mineral ion contents was in the sequence of
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Mansehra> Kotlisattian> Bagh> Abbotabad. Calcium (Ca2+) was the highest, ranging from
456 to 187.33 μg/g and copper (Cu2+) was the lowest, ranging from 0.37 to 0.013 μg/g.
Two alkaloids, berberine and palmatine were analyzed and quantified by TLC and
HPLC and proton and carbon signals were detected in 1H and 13C -NMR spectra. The analysis
of the NMR spectra of berberine and Palmatine revealed that the proton H-13 resonating as a
singlet (H-13 of 1: δ 8.72; H-13 of 2: δ 8.81) could be used for quantification. The 1H NMR
method used in this study was found to be simple, rapid and specific for the analysis of
protoberberine alkaloids and required reference compound, apart from the internal standard,
and an overall profile of the preparation was obtained directly. Using this method the content
of protoberberine alkaloids can be determined in Berberis lyceum and other plant extracts in a
shorter time than conventional method of HPLC.
Bioactivity of crude extract and Berberine of B. lyceum Royal was evaluated for
antimicrobial, antidiabetic and wound healing. For antimicrobial bioassay, root extracts of B.
lyceum prepared in three different solvents, methanol, ethanol and aqueous and tested against
different bacteria, fungi and yeast strains. Antimicrobial activities were assessed by using
Disc diffusion method and Micro dilution assays. It was observed that all root extracts of
Berberis lyceum were highly effective against different bacteria and fungi. The methanolic
and ethanolic extracts have inhibited growth of microorganisms more effectively as compared
to aqueous extract. The results obtained in present study indicates that root of B. lyceum
contained some phytochemicals having antimicrobial activity and could be used for
pharmaceutical industries for the development of new drugs required for human and animal
health. The wound healing activities of the aqueous and methanol extracts of the root of B.
lyceum were assessed using incision, excision and dead wound space models of wound repair
in rats. After application of both extracts it was observed that the area of epithelialization
xx
increased, followed by an increase in wound contraction, skin breaking strength, tissue
granulation, dry weight and hydroxyproline content. Histopathological studies of the
granulation tissue also indicated that there was an increase in collagen formation in those rats
treated with the methanol extract, compared with the control group animals. The methanol
extract was more effective than the aqueous extract, but both showed significant results as
compared to the control.
The antidiabetic activity of the ethanol root extract of Berberis lyceum was compared
with pure berberine in normal and alloxan-diabetic rats using similar doses of each. The
purpose of the study was to investigate the effects of berberine and a whole extract of B.
lyceum on blood glucose and other parameters associated with diabetes, to compare the
effects of the crude extract with those of pure berberine and thus validate its use as a
therapeutic agent, and finally to identify any contribution of the other components of the
extract to these effects. Oral administration of 50 mg/kg of Berberis extract and berberine to
normal and experimental diabetic rats produced a significant (p < 0.05) reduction in blood
glucose levels from days 3 –7 days of treatment. Significant effects were also observed on the
glucose tolerance, glycosylated haemoglobin, serum lipid profiles and body weight of
experimental animals. Berberis extract and berberine demonstrated similar effects on all
parameters measured, and although the extract was comparable in efficacy to berberine, it did
not produce any effects additional to those shown by pure berberine. The results support the
use of the extract in traditional medicine, and demonstrate that apart from being a highly cost-
effective means of treating with berberine, as compared to root extract which is cheaper,
easily available to rural community and also show no or very less adverse effects as compared
to pure compound (berberine).
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Chapter 1
INTRODUCTION
Medicinal plants consist of components of therapeutic values and have been used
as remedies for human diseases since long. Recently, due to the pathogens resistance
against the available antibiotics and the recognition of traditional medicine as an
alternative form of health care has reopened the research domain for the biological
activities of medicinal plants (Arias et al., 2004). Medicinal plants being as an important
natural resource and potentially safe drugs can play an important role in assuaging human
health by contributing herbal medicines. In the rural and remote areas of Pakistan, more
than 70 percent of population depends on folk and traditional system of medicines
obtained from plants. The high cost of allopathic medicine and their potential side effects,
encouraged the people to use the traditional medicine (Zaidi, 1998). The increasing
demand of plant extracts to be use in the cosmetic, food and pharmaceutical industries
suggests that systematic studies of medicinal plants are very important in order to find
active compounds and their use as a medicine for curing various diseases (Nostro et al.,
2001).
It has been widely observed in developing countries that, the use of traditional
medicines are common to the maintenance of the health (UNESCO, 1996). In the
developing countries, for the treatment of minor ailments, and cost for personal health
maintenance, herbal medicines have become more popular (Cowan, 1999). In addition,
the use of medicinal plants in the developed societies have been recognized which can be
seen by the extraction and development of several drugs and chemotherapeutics from
plants and traditionally used herbal remedies (UNESCO, 1998). Today about 1500
species of medicinal plants are being used in many countries including Albania, Bulgaria,
2
Croatia, France, Germany, Hungary, Spain, Turkey and the United Kingdom (Hoareau,
1999).
It has been reported that there are 8000 species of known medicinal values in
South Asia are considered an essential part of traditional health care systems. More than
80 percent of Asian population is dependent on these cheap and effective traditional
medicines used against many diseases and infections. Although in Pakistan, 2000
medicinal plant species are reported however, very few of them are being exploited and
90 percent of the country’s medicinal herb requirements are imported from other
countries (Aslam, 2002). A survey of the natural plant wealth of Pakistan showed that
there is profusion growth of medicinal plants in Murree Hills, Kotli sattian, Malakand
Kurram Agency, Hazara, Azad Kashmir, Northern areas and Baluschistan. Further more
some medicinal plants are also cultivated on farmlands in Punjab, Sindh, NWFP and
Kashmir areas (Choudhary et al., 2003).
Berberis lyceum Royal is considered to be important medicinal plant in practice of
herbal medicine. The main therapeutic features described for this plant includes , its
roots that are used as remedy for swollen and sore eyes, healing of broken bones internal
injuries, gonorrhea, curative piles, unhealthy ulcers, acute conjunctive and in chronic
ophthalmic. It is also used as bitter tonic astringent, diaphoretic and febrifuge
traditionally. Berberis lyceum Royal belongs to family Berberidaceae. It is a semi
deciduous shrub, 2 to 4 meter high, leaves are lanceolate or narrowly obovate-oblong,
entire or with a few large spinous teeth, arranged alternately on stem. Inflorescence a
racemes, flowers yellow born in auxiliary clusters longer than the leaves. Fruit is black in
colour and called berries (Aslam, 2002). It is found in Pakistan, India, Kashmir, Bhutan,
3
China, Japan, Nepal, United Kingdom, Ireland and Turkey and distributed in temperate
and sub tropic parts of Asia, Europe and America (Hoareau, 1999). In Pakistan its habitat
is Murree, Gilgit, Nomal, Nalter, Hunza, Nagar, Chilas, Puniyal, Ghupis, Mansehra and
Baagh. Many alkaloids have been extracted from Berberis species that includes
berberine, baluchistanamine, chenabine, gilgitine, jehlumine, palmatine, punjabine and
sindamine. Other alkaloids identified are umbellatine, oxyberberine, and berbamine.
Three more alkaloids berbenine, berbericine hydrochloride and berbericinine hydro
iodide has been isolated from its roots. Work has been done on the identification of
alkaloids from roots of many species of Berberidaceae family (Miana, 1973).
Berberis lyceum is the versatile matrix, producing a broad range of secondary
metabolites, having different functional groups and polarity. The metabolites having
chemically diverse compounds often specific to a particular species. There are number of
techniques which employed in natural product isolation, purification and characterization
from these diverse groups of plants. These techniques include column chromatography,
thin layer chromatography (TLC), High performance liquid chromatography (HPLC)
Mass spectrometry (MS), Nuclear magnetic resonance (NMR) and Gas chromatography
(GC). Highly specific in vitro and in vivo bioassay techniques, chromatographic methods
and spectroscopic techniques especially NMR have made much easier to screen, isolate
and identify potential bioactive compounds quickly and precisely.
Chemical, biological and physical assays are necessary to identify the target
compound(s) from a complex natural product extract. In order to discover bioactive
substances, different bioassays are used for detection purposes, guidance for the chemical
analysis, isolation and preliminary biological characterization of new compounds. Now
days, this research area is more focused on isolating target compounds rather than
isolating all compounds in the extract. These compounds may belong to certain chemical
4
classes, having certain physical and biological activities. Different bioassays are available
to check the biological significance of the compound which includes antimicrobial, anti
fungal and wound healing and anti diabetic assay etc.
Biochemical studies are very helpful in exploring the beneficial features of
medicinal plants. These may range from complete chemical compositional analysis to
detailed biochemical analysis of bioactive compounds. The family members of
Berberidaceae have been characterized in details but there is a little work on this species.
It was expected that Berberis lyceum Royal may also contains many valuable attributes
(phytochemicals). Keeping in view the importance of this important medicinal plant
species the present study is was undertaken with the following aim and objectives:
1. Composition analysis of roots of Berberis lyceum protein, carbohydrates, lipids
and essential metal ions.
2. In vivo assessment of anti diabetic, wound healing and in vitro antimicrobial
activities of various root extracts berberine and palmatine against selective micro-
organisms.
3. Isolation and Purification of alkaloid(s) from roots of Berberis lyceum.
5
Chapter 2
REVIEW OF LITERATURE
2.1 WORLD NATURAL MEDICINAL PLANT RESOURCES AND DEMAND
A significant revival of interest in natural products as a potential source
for new medicine has been observed recently both among the academia as well as in
pharmaceutical companies. There are around 250,000 species of higher plants that exist
on earth, but merely only 5 to 10 per cent of these have been investigated so far (Gragg
and Newman, 2001). In USA, the botanical market, including herbs and medicinal plants
is estimated around US$1.6 billion per annum. The dominating countries are China with
exports of over 120,000 tonnes annually and India with some 32,000 tonnes annually. It
is estimated that Europe imports medicinal plant from Asia and Africa is about 400,000
tonnes annually which cost approximately US$ 1 billion. With the growing awareness
about this new commodity towards the foreign-exchange reserves, a number of national
economies are beginning to emerge. Surveys are being conducted to unearth new plant
sources of herbal remedies and medicines to satisfy this growing demands.
2.1.1 Therapeutic Potential of Medicinal Plants
Nature has been a source of therapeutic agents for thousands of years and an
impressive number of modern drugs have been derived from natural sources. Various
active compounds (or their semi-synthetic derivatives) derived from medicinal plants
have been assessed for their efficacy and tolerability in the treatment of different
diseases. Beside the therapeutic potential of medical plant/drugs, therapeutic index also
indicates the drug safety. The therapeutic index is a measure of the drug’s beneficial
6
affects at a low dose versus its harmful effects a high dose. A high therapeutic index is an
indication of large safety margin between beneficial and toxic dose.
2.1.2 Medicinal Plants in Pakistan and their Scope
There are numbers of medicinal and aromatic plants which are being used in
natural health care system of Pakistan. Medicinal Plants are well grown in different areas
according to their habitate and main areas are Murree Hills, Abbotabad, Mansehra,
Northern Areas, Murree Hills, Azad Kashmir, Sindh and Baluchistan, and/or cultivated
on farmlands in Sindh, Punjab, Baluchistan and North West Frontier Province of
Pakistan. It has been estimated that there are 6000 species of wild plants are present in
Pakistan and among them; 3200 and 1000 medicinal plants species are distributed in
upper and lower part respectively. 75 crude herbal drugs are extensively exported while
200 species are treaded within Pakistan. 500 tons of medicinal plants are produced in
Hazara and Malakand, 38 tons in Azad Kashmir, 24 tons in Northern Areas and about 16
tons in Murree Hills according to the surveys carried out by Pakistan Forest Institute
(1989). Pakistan obtains more than 80 % of its medicaments from higher plants.
The actual demand/supply of herbs and medicinal plants are in the range of
20,000 tonnes per annum which cannot be fulfilled by indigenous production. During
1999, national and multinational companies imported medicinal plants worth of US$ 31.0
million. Beside this Pakistan only exported medicinal plants of only US$ 6 million (EPB,
1999). This point to a massive imbalance between import and export regarding medicinal
plants. To overcome this problem, during 2001, the Central Board of Revenue (CBR) has
allowed duty free temporary importation of medicinal herbs for subsequent exportation.
7
2.2 BERBERICIDACEASE GENUS
Berbericidacease is a large genus of medicinal plants of five hundred
species (Bhattacharjee, 2001). These are shrubs or small trees distributed in the
temperature and sub tropical parts of Asia, Europe and America. Among many species of
berberis growing wild in the Himalayan subtropical belt at altitudes ranging from 1000-
2200 meters, the most commonly reported ones are B. asiatica, B. aristata, B. chitria, B.
osmastonii, B. insignis, B. vulgaris, B. wallichinana, B. coriaria, B. floribunda, B.
himalaica, B. lambertii, B. tinctoria, B. virescens, B. nepalensis, B. petiolaris and B.
umbellate (Chopra et al., 1998). It is commonly known as berberry, several species are
grown in gardens for their ornamental leaves and bunches of succulent, acidic and edible
berries. They are also planted in hedges due to their strangling habit. Barberry flowers
bloom from February to June attracting bees for the pollen and nectar. The honey
obtained is dark and has a strong flavor. The bright yellow wood of various species
cannot be distinguished with certainty. It is used for making picture frames, scales,
pattern work, carvings and toys.
The roots and stem of barberry yield a yellow dye useful for tanning and coloring
leather and cloth. The stems are used in Tibet to stir boiling butter to impart a fresh
golden color to ghee. Root bark extracts of various Berberis species are being used as
folk remedy worldwide as a remedy for various inflammatory ailments including
lumbago, rheumatism and to reduce fever (Yesilada and Kupeli, 2002).
There are many uses of Berberis species. Berberis vulgaris, a plant native to
Britain is a perennial shrub and grows up to height of 3 meters. Its fruit is rich in vitamin
C. A wine is prepared from barberry fruits and used in malignant, choleric ad pestilential
fever and diarrhea. Squeezed blooms are applied to clean old ulcer. The concentrated
8
juice of berries is used for gums and teeth trouble. The root which is bitter in taste is used
as a purgative tonic. The bark has been used to treat dysentery and indigestion. The plant
extract is aloss used for jaundice and dropsy and congestion of liver and spleen
(Bhattacharjee, 2001).
According to Yesilada and Kupeli, 2002 Berberis aristata is one of the chief
sources of rasaut sold in the Indian market. Alcoholic extract of bark yielded berberine,
berberine chloride and palmatine chloride. Total alkaloid content was four percent. The
plant is emmenagogue and is useful in the treatment of jaundice and enlargement of
spleen. The berries are edible and are laxative, antiscrobutic and are useful in piles, sores
and eye diseases, particularly conjunctivitis.
2.2.1 Essential Minerals and Medicinal Plants: Therapeutic Role
It has been recognized that the possible influences of herbal medicines have not
yet been thoroughly investigated compared to its potential use in health care. In many
Asian countries the use of natural products from different plants and animals sources is
likely to be in parallel with the use of conventional medicines (Zhu, 1999). These
resources contain minerals, which could be additionally used as medicine therapy.
Human and animal tissue contains about 65 to 75 metals and the role of minerals
depends upon the certain tissues/cell that how it uses it after the ingestion and absorption.
To learn the role of microelement in the metabolic state of organisms has become
interesting field for research now days. Microelements play an important role due to their
requirements as co factor for a large number of enzymes in the cellular metabolism.
(Gadd, 1992). For example Copper plays essential role as trace mineral being involved in
many important functions of human body, including build up of bones and blood and it is
also important in forming elastin and collagen; the connective tissues of muscles, blood
9
vessels skin, heart, and lungs. Copper is also involved in the healing process, energy
production, hair and skin coloring, and taste sensitivity (Tenaud, 2001).
Iron mainly functions in the hemoglobin in our red blood cells, which transports
oxygen from the lungs to the body's tissues, including the muscles and the brain. Calcium
and magnesium are also very important as Calcium plays an important role in the
development and maintenance of the bones, tooth formation and cell signaling. Ca2+ has
also chemopreventive activity against colon cancer (Chakrabarty, 2003). Magnesium is
an extremely important and valuable mineral and act as a critical co-factor in more than
300 enzymatic reactions in the human body. Magnesium and potassium are the most
abundant cat ions found within the cells of the body. Potassium plays important role in
normal cell respiration and its deficiency can cause the deficiency of oxygen resulting in
the decrease in the efficiency of normal cell function. An ample amount of potassium is
also required normal functioning of heart beat and to regulate normal muscle contraction
and transference of nutrients to cells.
Mertz and Swartz (1957) identified Chromium as the active component of the
"glucose tolerance factor (GTF)" and its deficiency causes the Type II Diabetes
(Anderson, 2000). It has been reported recently that chromium helps to raise HDL
cholesterol and involves in muscle-enhancing and fat-reducing effects (Balk, 2007).
Manganese is needed for bone development and maintenance of strong bones. It is also
important in the utilization of thiamine, helps to activate enzymes that are necessary for
the body's proper use of biotin, B1 and vitamin C.
10
2.2.2 Bioactive constituent of Berberis species
A number of alkaloids have been extracted from Berberis species that includes
berberine, baluchistanamine, chenabine, gilgitine, jehlumine, palmatine, punjabine and
sindamine. Other alkaloids identified are umbellatine, oxyberberine, and berbamine.
Three more alkaloids berbenine, berbericine hydrochloride and berbericinine hydro
iodide has been isolated from its roots. Work has been done on the identification of
alkaloids from roots (Miana, 1973).
The presence of alkaloids oxyacanthine, berbebamine, columbine, jatrorhizine,
isotetrandine, palmatine and berberine in B. aemulans, B. cnadidula, b. dasystachya, B.
poiretti and B. pruinosa was determined using RP-HPLC (Lu et al., 1995). Berbernine
showed depressant action on isolated rabbit heart and produced acute fall in blood
pressure in dogs (Rastogi et al., 1993).
Berberine is a plant alkaloid with a long history of medicinal use concerning its
significant antimicrobial activity against a variety of organisms including bacteria,
viruses, fungi, protozoans, helminthes and Chlamydia (Huang et al., 2002). Berberine
produced sedation in mice and conscious cats and potentiated phenobarbitone sleeping
time. In isolated guinea pig ileum smaller doses of berberine sulphate potentiated
spasmogenic actions of prostaglandins (Rastogi et al., 1993).
Berberine inhibited by 70 percent the secretary responses of the heat labile
enterotoxins of Vibrio cholera and E. coli in experimental animals. It produced long
lasting, dose related fall in blood pressure, when administered into an ear vein of
anaesthetized rabbits. Berberine hydrochloride and sulphate help in the diagnosis of latent
11
malaria by releasing the parasites into the blood stream. It also inhibit HIV-1 reverse
transcriptase (Gudima et al., 1994).
2.3 Berberis lyceum Royal 2.3.1 Systematic and Distribution of Berberis lyceum Royal
Berberis lyceum Royal is a semi deciduous shrub which is about 2 to 4
meter high having lanceolate or narrowly obovate-oblong leaves, with few large spinous
teeth arranged alternately on stem. Inflorescence pattern is raceme and flowers are yellow
which born in axillary clusters longer than the leaves.
2.3.2 Berberis lyceum Royal in the traditional folk medicine
Berberis lyceum is locally known as sumbloo. Powdered roots are taken
orally with mild to treat rheumatic and muscular pains in the northern area. The root
which is bitter with an unpleasant taste is used in diabetes, spleen troubles, intestinal
astringent, good for cough, chest and throat troubles, eye sores and itching of eye, piles
and menorrhagia, useful in chronic diarrhea and healing of broken bones (Kirtikar and
Basu, 1999).
The roots of Berberis lyceum with that of Acacia modesta are boiled in water and
the decoction is used for toothache and septic gums (Humayun et al., 2003). Berberis
lyceum roots are dried and crushed to a powder to cure a mouth disease call chall and to
heal bone fractures (Pitman, 2000). The leaves are administered in Baluchistan as a cure
for jaundice. In Indo China the fruit is given as a tonic in kidney troubles (Kirtikar and
Basu, 1999). In Mansehra the root is used for the relief of intestinal colic and treatment of
pharyngitis. The bark is used as an astringent and improvement of internal wounds throat
pains and chall. The root is used as a febrifuge and also in menorrhagia, chronic diarrhea
and piles whereas leaves are used against jaundice (Imtiaz and Manzoor, 2003).
12
Many Indian practitioners use its root extract for the treatment of malarial fever. It
is claimed to be administered at the height of febrile paroxysm and never produces any
effects that follow the administration of quinine. Its tincture is considered valuable in the
enlargement of liver and spleen (Brahmananda, 2000). The herb is administered as a
febrifuge against bilious fevers also promotes digestion and acts as an aperient. Tincture
is valuable in periodic neuralgia, in enlargement of liver and spleen and recommended in
fevers accompanied with bilious symptoms and diarrhea. Root extract is directly used for
relieving stomach heat, skin diseases (absess/pimples) and diabetes before breakfast early
in the morning. It is also used for intestinal trauma. Bark’s paste is used for articulation of
bones (Majid et al., 2004). Berberis lyceum is successfully propagated in the Potohar
region where it constitutes an important flora (Bahadur et al., 2001). In Azad Khasmir, its
infusion is also used to treat intestinal colic, pharyngitis, urine burning, jaundice, eye
disease, piles, diarrhea and opthlmia and is also used as an astringent (Dastagir, 2001).
2.4 ISOLATION, PURIFICATION AND ANALYZING TECHNIQUES
Plants are the multifaceted matrix, producing a wide range of secondary
metabolites, having different functional groups and polarity. The metabolites having
chemically diverse compounds often specific to a particular species. These products have
to be extracted (released) from the biomass before isolation and purification work. As
diversity is present in natural products due to their distinct physiochemical properties, for
example solubility so there is a need of efficient extraction system to release the
compounds from biomass. Several approaches can be used to extract the plant material.
Although water is used as an extractant in many protocols but different organic extraction
is also developed for the various solubilites of plant constituents. Solvent extraction
procedures applied to plant for compound(s) extraction are maceration, Soxhlet
13
extraction, pressurized solvent extraction, ultrasound assisted solvent extraction,
extraction under reflux and steam distillation.
The ideal extraction procedure should be exhaustive, fast, simple and
reproducible. This process can use water-miscible or water-immiscible solvents which
should have a low potential for artifact formation, a low toxicity, a low flammability and
a low risk of explosion. The extraction may be for selective or total compound isolation
depending upon the choice of interest. For selective extraction, a specific solvent system
could be used for specific type of compound(s). For example non polar solvent are used
for alkanes, fatty acids, pigments, waxes, sterols, some terpenoids, alkaloids and
coumarins, medium polar solvents are used for some alkaloids and flavonoids and more
polar solvents are used for flavonoid glycosides, tannins and some other alkaloids. For
the total compound extraction solvents are used sequentially from non polar to polar with
n-hexane, chloroform, ethyl acetate, acetone, butanol, ethanol and water. The next step
after extraction is to isolate and to purify the different components from total compound
extraction. For this purpose Thin layer chromatography (TLC) is used which is easy,
cheap and rapid method.
2.4.1 Column Chromatography
Open-column chromatography is often used as a first fractionation step for
crude extract, which provides a partial separation of the different groups of the
constituents. One of the major problems in column chromatography is the length of time
required to perform the separations with large numbers of fractions to be analyzed. In
column chromatography technique, the stationary phase i.e. a solid adsorbent, is placed in
a vertical glass (usually) column and the mobile phase i.e. a liquid, is added to the top
which flows down through the column by either gravitional force or by external pressure.
14
Column chromatography is generally used as a purification technique: it isolates desired
compounds from a mixture.
2.4.2 Thin Layer Chromatography
Thin Layer Chromatography (TLC) is a cheap, easy, rapid and solid-liquid
technique which is widely used for the analysis and isolation of synthetic and natural
product(s). The two phases used in TLC are a solid (stationary phase) and a liquid
(mobile phase). Silica gel (SiO2 x H2O) and alumina (Al2O3 x H2O) are the two most
commonly used solids. Silica is polar and acidic in nature while alumina is available in
neutral, basic or acidic forms. Sensitivity of TLC micro gram level and as little as 10-9g
of material can be detected by this technique.
Information on semi-purified samples for example, column fractions can also be
invaluable. One must record the 1H NMR spectra to get the information about the classes
of compounds present prior to TLC purification. This will give the information about the
solvent system used for complete purification. The solvent system may be run
isocractically or as using a step gradient. In step gradient, isolation could be made by
initially using a non polar solvent and then increase the polarity after each development.
After development, effective visualization or detection is crucial to obtain pure
compounds while the poor detection resulted in a low recovery of the product from the
sorbent.
2.4.3 Detection and Structure Elucidation Techniques
Mass Spectrometry or nuclear magnetic resonance (NMR) used as
detection method having higher throughput because these techniques are capable of
selective and simultaneous detection of multiple components. NMR is the most powerful
one-dimensional technique for the structure elucidation chemistry. Solid state NMR
15
spectroscopy is used to determine the molecular structure of solids. Two-dimensional
techniques are also used to determine the structure of more complicated molecules. High
resolution Fourier transform–ion cyclotron resonance (FT–ICR)–MS is also used for
structure elucidation now days which may couple with any other one-dimensional
technique.
2.5 BIOASSAY METHODS IN NATURAL PRODUCTS 2.5.1 Antimicrobial Bioassay
Medicinal plants are potential sources of new compounds of therapeutics value
and are sources of lead compounds in the drug development (Kumar et al, 2006). These
compounds show antimicrobial activity against a wide range of microbes.
Long before mankind discovered the existence of microbes, the idea that certain
plants had healing potential has been established well (Rojas et al., 2006). Since
antiquity, man has used plants to treat common infectious diseases, Some of these
medicines are still the part of treatment of various maladies (Black et al., 2008). For
example, bearberry (Arctostaphylos uvaursi) and cranberry juice (Vaccinium
macrocarpon) were reported in different phytotherapy manuals to treat urinary tract
infections, while lemon balm (Melissa officinalis), garlic (Allium sativum) and tee tree
(Melaleuca alternifolia) are reported as broad-spectrum antimicrobial agents (Heinrich et
al., 2004). In recent years, the indiscriminate use of commercial antimicrobial
drugs/chemical to treat infectious diseases, has resulted in the development of multiple
drug resistance in both human and plant pathogens. (Kumar et al., 2006).
One alternative approach to prevent antibiotic resistance of pathogenic species is
by using new compounds that are not based on existing synthetic antimicrobial agents.
This situation has forced scientists to search new antimicrobial substances in various
16
sources like medicinal plants (Edeoga et al., 2005). In traditional medication many plants
been claimed for their effective or superior properties over synthetic drugs, like medicinal
plants such as bixa spp. and bidens spp. have been claimed more efficient to treat
infectious diseases than synthetic antibiotics by traditional healers (Rojas, 2006). So it
becomes necessary to evaluate the scientific base for the potential use of folk medicine
for the treatment of infectious diseases produced by common pathogens. Medicinal plants
might represent an alternative treatment in non-severe cases of infectious diseases (Shah,
2005). They can also be a possible source for new potent antibiotics to which pathogen
strains are not resistant.
Many medicinal plants have been evaluated for their antimicrobial activites for
example Bidens pilosa L. (Asteraceae), Bixa orellana L. (Bixaceae), Cecropia peltata L.
(Moraceae), Cochlearia officinalis L. (Rubiaceae), Jacaranda mimosifolia D.Don
(Bignoniaceae), Justicia. secunda Vahl. (Acanthaceae), Piper pulchrum C.DC
(Piperaceae), Peltogyne paniculata L. (Polygalaceae), and Spilanthes americana Hieron
(Asteraceae) (Rojas et al., 2005).
Several studies revealed that phenolics are the predominant active chemical in
these plants, especially against gram positive bacteria which are most sensitive against
these compounds. Many focused on determining the antimicrobial activity of plant
extracts found in folk medicine (Ngwendson et al., 2003), alkaloids (Klausmeyer et al.,
2004), essential oils (Alma et al., 2003), flavonoids (Sohn et al., 2004), sesquiterpene
lactones (Lin et al., 2003), triterpenes (Katerere et al., 2003), diterpenes (El-Seedi et al.,
2002), or naphtoquinones (Machado et al., 2003), among others. These papers comprise
about 65% of all the articles on microbial activity and medicinal plants.
17
It has been reported that hydroalcoholic extracts of root and stem of the four
Berberis spp. were effective against Bacillus cereus, Escherichia coli, Staphylococcus
aureus, Aspergillus flavus, Bacillus cereus and Streptococcus pneumonia bacteria. B.
lycium, B. aristata and B. asiatica root extract showed significant antifungal activity
against Aspergillus terreus and A. flavus. (Singh et al., 2007). Musumeci et al., (2003)
stated that berberine-containing Berberis species synthesise the substances 5′-
methoxyhydnocarpin-D (5′-MHC-D) and pheophorbide having no antimicrobial activity
but inhibit the expression of multidrug resistant efflux pumps (MDRs) in Staphylococcus
aureus and potentiate the action of berberine. (Li et al., 2007) also reported the
antibacterial activity of some medicinal plants including Berberis thunbergii specie
against gram positive bacteria. Anti bacterial and antifungal activity was seen against
Bacillus subtllis NCIM-2349, Bacillus coagulans NCIM-2323 Staphylococcus aureus
NCIM-2492, Escherichla coil NCIM-2345 and Candida albicans (different strains of
Candida albicans), Aspergillus flavus, Aspergillus niger, Aspergillus xyllnum and
Aspergillus fumigates of chloroform and methanol extracts of Berberis tinctoria Lesch
(Berberidaceae) root and root bark (Duraiswamy et al., 2002).
The solvent and the extraction system may both modify the final results. The most
appropriate method would be that in which the extract were the same as that used in folk
medicine or phytotherapy, although in the lab the use of methanol or ethanol extract is
much more common. (R´ıos and Recio, 2005). The methodology employed is another
point, which needs to be considered in more depth. For non-polar extracts, the use of
diffusion techniques seems to be inadequate, although many reports with these kinds of
techniques have been published. R´ıos and Recio, (2005) study also shows the use of
solid dilution techniques for studying plant extracts or nonpolar compounds. The use of
diffusion techniques possibly more appropriate when a small amount of sample is
18
available. The medium’s composition could also influence the growth and activity of the
tested extracts or compounds. Thus, Ross et al. (2001) studied the effects of garlic
powder and garlic oil, the antimicrobial activity of garlic oil was found to be greater, in
media lacking tryptone or cysteine, which led to the hypothesis that the effects may
involve sulfhydryl reactivity.
Summing up, it is believed that for gaining insight the study of medicinal plants
as therapeutic agents the standard methods for investigation are essential. Moreover,
research in this area should be carried out to the level of determination of active
compound or to the most active fraction or extracts have been discovered. Finally, high
priority should be given towards the studies on the mechanisms of action, interactions
with antibiotics, other medicinal plants or compounds and the pharmacokinetic profile of
the extracts..
2.5.2 Wound Healing Bioassay The injury of the body which is typically involves in breaking of the membrane and
damages the dermis of the skin is called a wound. It is of two types; open and closed and are
based on the object that caused the wound. Open types included Incisions or incised
wounds, Lacerations, Abrasions, Puncture wounds, Penetration wounds and Gunshot
wounds while closed types includes Contusions, Hematomas and Crushing injuries
(Barbara et al., 1999).
A series of actions take place when the body is subject to any wound which is
collectively known as the wound healing process. Proper healing of wounds is essential
for the restoration of disrupted anatomical continuity and disturbed functional status of
the skin (Kuman, 2007). Wound healing is a natural process of regeneration of dermal
and epidermal tissues. Restoration of wound is categorized in four or more stages
19
including the inflammatory, proliferative, and remodeling phases (Glynn, 1981; Clark,
1996; Martin, 1996). In the inflammatory phase clotting takes place to obtain the
homeostasis or stop the blood loss. Different types of factors are released to attract those
cells that help in the process of phagocytosis of debris, bacteria, and damaged tissue and
other factor also released that initiate the proliferative phase of wound healing. After
inflammatory phase, Clotting cascade occurs. When tissue is firstly wounded, blood
comes in contact with collagen and triggering blood platelets to begin the secretion of
inflammatory factors (Buffoni et al., 1993). Platelets also express certain glycoprotein on
the surface of cell membranes that allow them to interconnect with one another and
aggregates. In the process of blood clotting Fibrin and fibronectin cross-link together and
form a plug that traps proteins and particles and prevents further blood loss (Kunicki,
1989). This fibrin-fibronectin plug is also the main structural support for the wound until
collagen is deposited. About two or three days after the wound occurs, fibroblasts begin
to enter the wound site, marking the onset of the proliferative phase even before the
inflammatory phase has ended. As in the other phases of wound healing, steps in the
proliferative phase do not occur in a series but rather partially overlap in time. When the
levels of collagen production and degradation equalize, the maturation phase of tissue
repair is said to have begun (Lawrence, 1998). The maturation phase can last for a year or
longer, depending on the size of the wound and whether it was initially closed or left
open. During Maturation, type III collagen, which is prevalent during proliferation, is
gradually degraded and the stronger type I collagen is laid down in its place Originally
disorganized collagen fibers are rearranged, cross-linked, and aligned along tension lines.
With the passage of time, the tensile strength of the wound increases, to 50% that of
normal tissue in three months after injury and ultimately becoming as much as 80% as
20
strong as normal tissue if the person is medically normal. Since activity at the wound site
is reduced, the scar loses its erythematous appearance.
Treatment of wound includes the administration of drugs either by locally or
systemically, to achieve the wound repair (Savanth and Shah, 1998). The current agents
used include desloughing agents (chemical debridement, for example, hydrogen peroxide,
eusol and collagenase ointment) (Savanth and Mehta, 1996), antiseptics and antibiotics
(Chulani, 1996), wound healing promoters (e.g. honey, Tretinoin, comfrey, aloe vera
extract, benzoyl peroxide, dexpanthenol, chamomilia extract, clostebol acetate,
tetrachlordecaxide solution and the experimental cytokines (Raina, 2008). Pants or plant
derived chemical entities need to be identified and formulated for treatment and
management of wounds. A number of medicinal plants especially those mentioned in
conventional systems of medicine have been well explored and their usefulness has now
been well established. Various herbal products have been used in management and to
cure wounds over the years.
Herbal drugs used in wound healing process include Alae vera for healing minor
burns (Schmidth and Greenspoon, 1991), Azardica indica used for skin diseases, anti
inflammatory, anti bacterial and antifungal (Chopra et al, 1986), Lantana camara antimalarial,
anti-inflammatory and wound healing (Kurian, 1995), Tridax procumbens used as
antagonized anti-epithelization and tensile strength depressing effect of dexamethasone
without affecting its anti-contraction and anti-granulation action. (Diwan et al, 1983),
Abrus precatorius L used in cuts and wounds (Bhatt et al., 2002; Katewa et al., 2004),
Bergenia ciliate (Haw.) Sternb used in wounds treatment (Punjani, 2002; Sindhi et al.,
2003), Calycopteris floribunda Lam. Used in cuts and wounds (Bhandary and
Chandrasekhar, 2002; Kshirsagar et al., 2003), Melastoma malabathricum L. used in cuts
and wounds (Begum and Nath, 2000; Bharadwaj and Gakhar, 2005) and Pergularia
21
daemia (Forsk) Chiov for wounds and leprotic wounds (Ramadas et al., 2000; Kshirsagar
et al., 2003). The compounds that’s are involved in wound healing process includes
Linarin(acacetin-7-O-rutinoside), Luteolin, 6-hydroxyluteolin, Curcumin, Echinacoside,
Verbascoside, Madecassic acid, Asiatic acid, Asuaticoside, Caffeic acid and Ferulic a c id
(Habbu et al., 2007).
Traditional wound healing remedies research falls into many categories including
herbal, use of animal/ insect products as wound healing agents and the use of organisms
to effect wound healing. Injuries due to working in the fields, leg ulcers resulting from
treated wounds, burns from cooking and sleeping near fires and increasing injuries
resulting from traffic accidents has led to receive attention towards herbal research used
traditionally. There are many models in which medicinal plants have been reported for
the acitivity and the extract showed activity. These all plants which have been reported
pharmacologically are being used traditionally as well. Research has been focused on
Acalypha indica (Reddy et al., 2002), Aegle marmelos (Jaswanth et al., 2001), Allmanda
cathartica (Nayat et al., 2006), Anogeissus latifolia (Govindarajan et al., 2004,
Bryophyllum pinnatum (Khan et al., 2004), Butea monosperma (Sumitra et al., 2005),
Cyperus rotundus (Puratchikody et al., 2006), Flaveria trinerva (Umadevi et al., 2006),
Laura nobilis (Nayak et al., 2006), Leucas hirta (Manjunatha et al., 2006), Plagiochasma
appendiculatum (Singh et al., 2006), Lavandula x allardii honey (Lusbey et al., 2006),
Vitex trifolia L. and Vitex altissima L (Manjunatha et al., 2007) and Lantana camara L.
(Nayak et al., 2008). .
2.5.3 Anti diabetic Bioassay
Diabetes mellitus is one of the world’s major multifactorial diseases. It
currently affects an estimated 143 million people worldwide and the number is increasing
22
day by day. The estimated direct and indirect costs of diabetes exceed US 132 billion
dollars annually. Diabetes mellitus is a chronic condition characterized by major
derangements in glucose metabolism and abnormalities in fat and protein metabolism.
There are several forms of diabetes but spontaneous diabetes is the major form in the
west, whereas malnutrition related diabetes is a major form in Africa and Asia.
Spontaneous diabetes is classified in into type I and type II diabetes. Type I diabetes, also
called as insulin dependent diabetes is inherited and usually occurs early in life. In this
disease very little insulin or none at all, so glucose accumulates in the blood serum unless
insulin is supplied. Type II diabetes mellitus is usually called as adult onset diabetes.
Treatment of diabetes, especially Type II, is complicated due to the inherent
patho- physiological factors related to this disease and elevated post prandial
hyperglycemia is one of the risk factors (Horowitz, 2002). Plant based medicinal plants
has been known since ancient times and several medicinal plants and their products have
been used to control diabetes in the traditional medicinal systems of many cultures
worldwide. Plants have been the primary source of drugs and many of the currently
available drugs have been directly or indirectly derived from plants.
About 800 plant species have been reported to have antidiabetic activity. A wide
range of plant –derived principles belonging to compound, mainly alkaloids, glycosides,
galactomannan gum, polysaccharides, hypoglycans, peptidoglycans, guanidine, steroids,
glycopeptides and terpenoids have shown bioactivity against hyperglycemia (Mentreddy,
2007). Several plant species have been used for prevention of diabetes by the Native
Americans, Chinese, South Americans and Asian.
Among 45 medicinal plants and their products that have been mentioned in the
Asian Traditional System of medicine called Ayurveda, the plants that have shown
23
antidiabetic activity are Allium cepa (Lata et al., 1991), Allium sativum (Lata et al.,
1991), Aloe vera (Rajendran et al., 2007), Cajanus cajan (Jaiswal et al., 2008), Coccinia
indica (Khan et al., 1980), Caesalpinia bonducella (Chakrabarti et al., 2003) , Eugenia
jambolana (Ravi et al., 2005),, Ficus bengalenesis (Shukla, 1994), Gymnema sylvestre
(Shanmugasundaram, 1990), Momoridica charantia (Hu et al., 2006), Murraya koenigii
(Yadav et al., 2002), Ocimum sanctum and Pterocarpus marsupium (Halim et al., 2006)
are considered the most effective against diabetes and have been most extensively in
relation to diabetes and its complications. Active natural principles and crude extracts of
these plants have been used during the study.
Aloe barbadensis has been used for centuries as an oral treatment for type 2
diabetes and hyperlipidaemia. Similarly flavonoids, gallic acid, ellagic acid and tannins
of Eugenia jambolana has been most extensively researched for its hypoglycemic and
antihyperglycaemic properties than many other plants species with known antidiabetic
properties. The vicine, polypeptides and charantin of seeds of Gymnema sylvestre
suppresses an individual’s ability to taste anything sweet that’s why it is used to reduce
blood glucose level in diabetic patients. Momordica charantia is frequently used as an
antidiabetic and Antihyperglycemic agent because its fruit has been used to shown to
enhance the uptake of glucose by cells, to promote insulin release and to potentiate the
effect of insulin (Mentreddy, 2007).
It is concluded that the majority of plants with blood glucose lowering activity
contain polysaccharides because this chemical class lowers blood glucose level by
impeding glucose absorption from the gastrointestinal tract and thus reducing
postprandial hyperglycemia. There are some measures which are proposed as means of
increasing the probability of successful isolation of novel antidiabetic compounds and
these are identifying the molecular mechanism of action, performing acute, sub acute and
24
chronic toxicological testing, monitoring general body parameters, focusing on the
antidiabetic approach and developing in to new trends.
Chapter 3
MATERIALS AND METHODS
3.1 COLLECTION AND PREPARATION OF SAMPLES
Root samples of Berberis lyceum Royal were collected from different locations of
hilly areas of Kotli sattian, Rawalpindi about 65 kilometer from Islamabad, Abbotabad,
Mansehra (North West Frontier Provience) and Bagh (Kashmir), Pakistan. Information
regarding weight, date, and locations of sampling were recorded for immediate reference.
Plant was identified by expert taxonomist as specimen was deposited in University
herbarium. Samples were washed, dried and ground to powder form and stored for
further analysis.
3.2 BIOCHEMICAL ANALYSIS OF BERBERIS LYCEUM ROYAL
3.2.1 Wet and Dry Weight Analysis
This procedure is intended to determine the amount of total solids remaining after
45oC and 105oC as described by Hames et al. (2005). For the 45oC, sample(s) were dried
in a conventional oven at 45± 3oC after placing in a dried container for 24 to 48 hours.
The weight of the sample was noted before (Wi) and after (Wf) the drying. By applying
the following equation different masse was calculated:
Where:
25
% T45 = percent total solids of a sample oven dried at 45ºC,
Wt = tare weight of freeze-drier container,
Wi = initial weight of container and sample
Wf = final weight of container and sample.
For 105oC, thoroughly mixed sample (2 gram), was placed in a oven at 105 ±3 oC for four
hours in a dried aluminum weighing dish. The samples were cooled at room temperature
in a desiccator. Weight, percentage of total solid, percentage of moisture and relative
percent difference (RPD) between two samples were calculated by the following
equations.
% Total Solids = (Weight dry pan plus dry sample – Weight dry pan) × 100
Weight sample as received
% Moisture = 100 – (Weight dry pan plus dry sample – Weight dry pan × 100
Weight sample as received
3.2.2 Carbohydrate Analysis
For analysis of Carbohydrate strong acid hydrolysis method was used. The dried
sample was treated with 1.5 ml of 72% H2SO4 in four different pyrex tubes, placed in a
water bath with a temperature of 30oC. After 1 h samples were diluted with 42 ml Milli-Q
water for the first two tubes and 43 ml with other tubes. One mL spiked solution (33 gL-1
glucose and 30 gL-1 xylose) was further added to first two tubes. The samples were then
autoclaved for 1 h at 121oC. After cooling samples were taken and analyzed by HPLC
26
(Agilent Technologies, 1200 system) equipped with an Aminex HPX-87H organic acid
analysis column (Bio-Rad) at 60◦C. The eluent was 4 mM H2SO4 at flow rate of 0.6
ml/min with detection on a Refractive Index Detector. Prior to HPLC analysis, 1 ml
samples were acidified with 10 µl of 20% H2SO4 and centrifuged at 14 000 rev./min for
10 min, followed by filtration through 0.45 µm membrane filter.
3.2.3 Protein Analysis
Protein content (nitrogen × 6.25) was determined by micro-Kjeldahl nitrogen
analysis by the AOAC methods, 1990 (979.09 and 920.87).
3.2.4 Lipid Analysis
The oil contents were analyzed by AOAC method, 920.85 (AOAC, 1990) with
Soxhlet apparatus. In the Soxhlet extraction procedure, 5 g of the crushed roots (80
mash) was packed in a thimble and the oils were extracted with diethyl ether for 6 hrs.
3.2.4.1. Analysis of fatty acids
The lipid extracted from the B. lyceum root samples were mixed with boron
trifluoride (BF3)-methanol reagent (20%) and fatty acids were converted into the methyl
ester derivatives (Morrison and Smith, 1964). The methyl esters of the fatty acids were
dissolved in CHCl3 and analyzed by GC and GC-MS.
3.2.4.2. Gas chromatography (GC) conditions
GC analysis was performed on an Agilent 6890N Network GC system, under the
following conditions: column, HP Innowax Capillary; 60.0 m x 0.25 mm x 0.25 µm;
oven temperature programme, the column held initially at 60ºC for 3 min after injection,
then increased to 185ºC with 10ºC/min heating ramp for 1 min and increased to 200ºC
with 5ºC/min heating ramp for 10 min. Then the final temperature was increased to
220ºC with 5ºC/min heating ramp for 20 min; injector temperature, 250°C; detector (FID)
27
temperature, 275°C; carrier gas, He; inlet pressure, 40.65 psi; linear gas velocity, 39
cm/s; column flow rate , 2.7 ml/min; split ratio, 40:1; injected volume, 1 µL.
3.2.4.3. Gas chromatography-mass spectrometry (GC-MS) conditions
GC-MS analysis was performed on an Agilent 6890N Network GC system
combined with Agilent 5973 Network Mass Selective Detector. MS conditions were
regulated as follows; ionization energy: 70 eV, ion source temperature: 280°C; interface
temperature: 250°C; mass range: 35-450 atomic mass units. Identification of the
components was assigned by comparison of their retention times and mass spectra with
corresponding data from reference compounds and by comparison of their mass spectra
with Wiley and Nist libraries.
3.3 ESSENTIAL METAL ION ANALYSIS
A mineral ions study was carried out by wet digestion method using HNO3 –
HCLO4 according to soil and plant analysis laboratory manual (Ryan et al., 2001). For
wet digestion, 0.5 gram powdered plant material was taken in 100 ml pyrex digestion
tube and digested with 10 ml 2:1 nitric-perchloric acid mixture and allowed to stand over
night. After preliminary digestion tubes were placed in a cold block digester, heated at
150°C for 1 hour. After one hour temperature was increased up to 235oC until the white
fumes appeared in the tubes, indicating complete digestion. Samples were then brought at
room temperature and allowed to cool. After vapors condensation, digested residues were
dissolved in deionized water and final volume made up to 100 ml. Sodium and
Potassium were analyzed by Flame photometer (FP) (Jenway PFP-7) while the remaining
elements (Magnesium, Calcium, Zinc, Copper, Iron and Manganese) were analyzed by
Atomic Absorption Spectrophotometer (AAS) (GBC 932 plus, Australia). All analysis
was conducted in triplicate.
28
3.4 QUANTIFICATION OF ALKALOIDS FROM ROOT OF BERBERIS LYCEUM ROYAL
Extraction of alkaloid(s) was done by n-hexane, petroleum ether, ether,
chloroform, ethanol and methanol. Extraction was initially performed on shaking
apparatus with a 10ml:1g solvent to dry weight ratio. This procedure was repeated three
times and all extracts were combined, filtered and extracts were concentrated in rotary
evaporator at 60oC.
3.4.1 Isolation of Alkaloid(s) by Column Chromatography
Column Chromatography (CC) using silica gel was done to determine the
quantity of possible alkaloid(s) present in root samples of Berberis lyceum Royal. After
solvent – solvent fractionation, the fractions were dried to determine the mass extract in
each solvent. Initially hexane fraction was chosen for CC due to its relatively low
complexity. Then chloroform fraction was also applied.
3.4.2 TLC Analysis of Alkaloid
In order to select the best mobile phase for eluting the hexane fraction, 5 µl of a
100 mg/ml solution was spotted on TLC and was run with combination of different
solvents. In this way the solvent system that was exhibited the most favorable separation
of compounds was chosen. Further analysis was done by high performance liquid
chromatography (HPLC).
3.4.3 HPLC analysis of alkaloids
The quantification of berberine and palmatine from the root extract of Berberis
lyceum was carried out by using HPLC (Gilson, Anachem, Luton, UK) attached to a
UVD340 S DAD (diode array detector), and Chromeleon vs 6.10 software (Dionex,
Macclesfield, UK). The analytical C18 column was eluted with methanol: phosphoric
acid (0.1%) 70:30; gradient from 5:95 to 100 over 20 min, flow rate of 1.5 mL/min. UV
29
traces were measured at 225nm. Analysis were conducted triplicate and berberine and
palmatine two major alkaloids with high quantity of berberine was found.
3.4.3. NMR analysis of alkaloids
3.4.3.1 Preparation of Samples.
Total 15 mg of powdered root samples of Berberis lyceum were exactly weighed
into a NMR tube (0.3 mm i.d.) and added 0.5 mL of MeOH-d4 (contained 85.2 µg
anthracene). The sample was sonicated at room temperature for 25 min and used for 1H
NMR measurement.
3.4.3.2 NMR spectra analysis
1H NMR spectra were recorded in methanol-d4 (99.9 %) using a Varian UNITY
plus 400 MHz spectrometer. For each sample, 100 scans were recorded i.e
0.187 Hz/point; spectra width, 14400 Hz; pulse width, 4.0 µs; relaxation delay, 2 s. For
quantitative analysis, peak area was used and the start and end points of the integration of
each peak was selected manually.
3.4.3.3 Recovery
The recovery samples were also to check the efficacy of the results. Pure
berberine and palmatine were spiked into 15 mg of powered root extract of Berberis
lyceum. A blank recovery sample was prepared and analyzed for the comparison. Limit
of detection (LOD) was evaluated at a signal-to-noise ratio of 3. Limit of quantization
(LOQ) was evaluated at a signal-to-noise ratio of 6. The identity of alkaloids were
further confirmed by comparing with data from reference compounds and the Wiley and
Nist libraries.
30
3.5 BIOASSAYS
3.5.1 Antimicrobial Studies
3.5.1.1 Preparation of sample
Root sample of Berberis lyceum were subjected to shadow drying followed by
oven drying at 80oC for overnight and then converted into powdered form. Total 100 g of
samples was added 300 ml of methanol, ethanol and water separately and extracted in
soxhlet apparatus for 4 hours at temperature less than the boiling point of solvent. The
extract was further concentrated by rotary evaporator and residue was stored for further
process. Whereas in case of aqueous media same amount of sample was dissolved in
water and boiled, filtered and saved for further process.
3.5.1.2 Microorganism
A total 18 microbial culture belonging to 35 bacterial species and 4 fungi and
yeast were used in this study. The identified microorganisms were obtained from
Pakistan Institute of Medical Sciences (PMIS), Microbiology laboratory Quaid-i-Azam
University, Islamabad and National Institute of Health, Islamabad.
3.5.1.3 Antimicrobial activity
The root extracts of Berberis lyceum prepared above were again dissolved in
methanol, ethanol and water separately (30mg/ml) and sterilized by filtration though
0.45 um Millipore filters. The antimicrobial activity test was carried out by disk diffusion
(Barnabas and Nagarajan, 1988 ) by using 100 µl/ml of suspension containing 104
spore/ml of fungi spread on nutrient agar (NA), Saboured dextrose agar (SDA) and potato
dextrose agar (PDA) media respectively. The disks (6 mm) containing 10 µl of extracts
(300 µg/disk) with the concentration of 30 mg/ml were impregnated in the inoculated
31
agar. Negative control was prepared by using similar plants extracts of solvents. Whereas
Ofloxacin (10 µl/disk), Sulbactum (30 µg) Cefoperazone (75 µg), (105 µg/disk) and or
netilnicin (30µg/disk) were used as positive control to determine the sensitivity of each
strain/isolate for each microbial species tested. The inoculated plates were incubated at
37oC for 24 hours in the case of clinical bacteria strains, 48 hours for yeast and 72 hours
for fungi isolate. Antimicrobial activity was assessed by measuring inhibition zones in
reference to test organisms and each process was repeated to get accurate results
(Barnabas and Nagarajan, 1988; Davis, 1994).
3.5.1.4 Micro dilution Assays
The minimum inhibitory concentration (MIC) values were determined for
microorganism those were sensitivity to Berberis lyceum extract in disk diffusion assay.
The inocula of microorganisms were prepared from 12 hours breath cultures and
suspension was adjusted to 0.5 McFarland culture, turbidity. The concentration of 100
µg/ml of plant extract was prepared in 10 % dimethylsulfoxide (DMSO) and serial
dilution was made ranging from 10 µg/ml in 10 sterile test tubes (containing nutrient
broth), therefore on the basis of micro dilution assays activity of plant extract against
bacterial strains were determined (Clark, 1996). The plates were prepared by dissolving
95 µl of nutrient broth and 5 µl of the inoculums, 100 µl of plant extract and 100 µl from
serial dilution was taken in each plates. For negative control 195 µl of nutrient broth and
5 µl of the inoculums was used. Maxipime (Bristol-Myers Squibb) at concentration
ranging from 8.5 – 500 µg/ml was prepared in nutrient broth and selected as positive
control. The plates were covered with sterile plate sealer. The contents of each plate were
mixed on a shaker at 2500 rpm for 25 second and incubated at suitable temperature for 24
hours. Microbial growth was determined by measuring absorbance by micro titer (US) at
32
600 nm, which was further confirmed by apply 5 µl of samples from each plates on
nutrient agar media. The plant extracts in this study was tested twice for each organism.
The minimum inhibitory concentration was defined as lowest concentration of the
compound to inhibit the growth of microorganisms (Karman et al., 2003).
3.5.2 WOUND HEALING ACTIVITY OF Berberis lyceum
3.5.2.1 Preparation of Plant material
The methanol extract was prepared from 300 g of root powder added to 300 mL
of methanol and refluxed by soxhlet for about 1 hours. The extracts were filtered and
concentrated under reduced pressure. The aqueous extract was made by macerating 300 g
of root powder with 1000 ml of distilled water for three days with intermittent stirring;
this was filtered and again concentrated under reduced pressure.
3.5.2.2 Drug formulation
For topical administration a 5 % w/w gel was made in 2 % sodium alginate
solution, and for oral administration, a suspension of 30 mg/ml of the extracts in 1 % gum
tragacanth was prepared.
3.5.2.3 Experimental animals
Swiss Wistar rats of either sex (200-300g) were maintained under standard animal
house conditions, fed with commercial rat chow (Feed Mills, Islamabad) and allowed
water ad libitum. Animal study was conducted by fallowing all described rules for
experimental animals.
33
3.5.2.4 Wound healing activity Berberis lyceum
3.5.2.4.1 Excision wounds
A circular wound of about 5.0 sq mm was made on the depilated, ethanol-
sterilized dorsal thoracic region of rats under light ether anesthesia (Leite et al., 2002).
The animals were divided into 4 groups of 6. Group 1 was untreated as the control; group
2 was treated with 1% w/w nitrofurazone ointment and served as a reference standard
(positive control); group 3 was treated topically with the gel prepared from aqueous
extract of Berberis lyceum and group 4 was treated with the gel prepared from the
methanol extract. The gel was topically applied once daily until epithelialisation was
complete. The parameters studied were wound closure and epithelialisation, by tracing
the outline of each wound on graph paper on the 3rd , 6th , 9th, 15th, 12th and 18th days,
until healing was completed (Pital et al., 2001). The percentage of wound closure and the
area of epithelialisation were recorded.
3.5.2.4.2 Incision wounds
Incision wounds 6 cm long, through the full thickness of the skin on either side of
the vertebral column of rats (Para vertebral incisions), were made (Ehrlich and Hunt,
1969; Kapoor et al., 2004). The wounds were closed with interrupted sutures 1 cm apart.
The animals were divided into 4 groups of 6 animals each and the treatment of the
experimental animals was similar to that for the excision wound experiments. The
ointment gel was applied topically once daily. The sutures were removed on the 8th day
of post incision. The skin breaking strength of the wound was measured on the 10th day
after treatment with a continuous water flow technique (Nadkarni and Nadkarni, 1996).
34
3.5.2.4.3 Dead wound space
Animals were divided into 3 groups of 6 rats. Group 1, the control, received 1 ml
of vehicle (1 % gum tragacanth) per kg). Animals in groups 2 and 3 received the oral
suspensions of the aqueous and methanolic root extracts of Berberis lyceum in doses of
30 mg/kg respectively. Under light ether anesthesia, dead space wounds were created by
subcutaneous implantation of a sterilized, shallow metallic ring, 2.5 cm x 0.3 cm (known
as a cylindrical pith), on each side of the dorsal paravertebral skin surface (Putil and
Kulkarni 1984). Granulation tissue formed on the outside and inside of the pith was
excised on the 10th day of post wounding. The dry weight of the granulation tissue and
the breaking strength were measured, and the amount of hydroxyproline, which indicates
collagen turnover (Kapoor et al., 2004; Salah et al., 2004) was estimated using a
colorimeter (Nadkarni and Nadkarni, 1996). Histopathological examination was used to
assess the extent of collagen formation.
3.5.2.5 Acute toxicity and selection of dose
Acute toxicity studies were conducted for both extracts using the method of
Saipurana et al. (1995) in order to select a suitable dose for evaluation of wound healing
activity. The LD 50 of both aqueous and methanol extracts were 300 mg/kg, therefore
1/10 of LD 50 dose and 30 mg/kg was selected for testing.
3.5.3 ANTI DIABETIC ACTIVITIES OF Berberis lyceum ROOTS EXTRACTS
3.5.3.1 Preparation of plant extracts
The root samples were ground in a Waring blender and sifted through a wire
screen (mesh size, 2mm x 2mm). The roots were exhaustively extracted with ethanol
(root to solvent ratio 1:5). The extracts were filtered and concentrated on a rotary
evaporator
35
3.5.3.2 Experimental Animals
Wistar rats of either sex (200-300 g) were maintained under standard animal
house conditions, fed with commercial rat chow (Feed Mills, Islamabad) and allowed
water ad libitum. Fasted animals were deprived of food for at least 16 hours, but allowed
free access to water. The animals were carefully monitored and maintained in accordance
with ethical recommendation to experimental animals. Fasted animals received 65 mg/kg
body weight of alloxan by a single dose by intravenous injection. Only diabetic rats were
included in the experiment, their body weights and serum glucose levels were assessed
after zero to 5 days. Insulin was administrated by intra peritoneal route and all other
treatment was made orally by gavage.
3.5.3.3 Acute toxicity and selection of doses
Acute toxicity studies were conducted for both extracts in order to select a
suitable dose for evaluation of anti diabetic activity. The LD50 values of both extracts
were calculated using the method described by Litchfield and Wilcoxon (1949).
3.5.3.4 Effect of root extract on different animal models
3.5.3.4.1 Effect of the ethanol extracts of Berberis lyceum on serum glucose levels in
normal fasted rats.
Animal fasted overnight were randomly allocated into 3 groups of 6 rats (n=6);
Group 1 (vehicle) received 2 % ethanol/ H2O mixture (0.5 ml); Group 2 and 3 received
the ethanolic extract of Berberis lyceum at 50 and 100 mg/kg doses. The serum glucose
was measured at 0, 1, 3 and 5 hours of treatment.
36
3.5.3.4.2 Effect of the ethanol extracts of Berberis lyceum on serum glucose level in
alloxan- induced diabetic rats.
Diabetes was induced by a single intravenous injection of 5 mg/kg of alloxan
monohydrate (dissolved in 0.9 % NaCl) to overnight fasted rats. A serum glucose range
of 400-500 mg/dl was used for the experiment. Hyperglycemic was confirmed in animals
after 72 hours of alloxan injection. Animals were divided into 5 groups of 6 animals
(n=6).
Group 1 diabetic animals received insulin (0.5IU); Group 2 diabetic animals (vehicle)
received 2 % ethanol/ H2O (0.5ml); Group 3 and 4 diabetic animals received ethanol
extracts of 50 and 100 mg/kg ; Group 5 diabetic animals received glibenclamide (20
mg/kg). Serum glucose level was measured on 0, 1, 2 and 5 day following the treatment.
3.5.3.4.3 Effect of the ethanol extracts of Berberis lyceum on glucose tolerance test
Fasted rats were divided into 5 group of 6 animals (n=6) to each treatment ;
Group 1 rats received glucose (3g/kg) and insulin (0.5IU); Group 2 hyperglycemic rats
received glucose (3g/kg); Group 3 and 4 rats received glucose (3g/kg) and ethanol
extract (50 and 100 mg/kg) and Group 5 hyperglycemic rats received glibenclamide (20
mg/kg). Blood samples were collected at 0, 30, 60, 120 and 180 minutes, after the
glucose loading and quantity of glucose in serum was measured.
37
3.5.3.4.4 Effect of aqueous extracts of Berberis lyceum on serum glucose level (mg/dl) in alloxan diabetic rats.
Animals were divided into 5 group of 6 each (n=6). Group; 1 diabetic rats got
insulin (0.5IU); Group 2 diabetic animals (vehicle) received 0.5 ml of 2 % ethanol/ H2O
(0.5ml); Group 3 and 4 animals received 50 and 100 mg/kg of aqueous root extracts.
Whereas dose of 20 mg/kg of glibenclamide was given to animals of group 5. The level
of glucose in the experimental animals was quantified on 0,1,3 and 5 days following the
treatment.
3.5.3.4.5 Effects of aqueous extracts of Berberis lyceum on serum glucose level (mg/dl) in oral glucose tolerance test.
Fasted animals were divided into 5 groups of 6 each (n=6); Group 1 was given
insulin (0.5IU); Group 2 received a dose of glucose 3 g/kg Group 3 and 4 got aqueous
root extract of 50 and 100 mg/kg and group 5 received 20 mg/kg of glibenclamide. The
glucose level was quantified at zero, 30, 60,120 and 180 minutes, after the treatments.
3.5.3.5. Determination of the serum glucose concentration
Blood samples (100 µl), from the tail vein of the anesthetized rats were collected
and centrifuged. The serum was used to determine the glycemia by the glucose oxidase
method.
3.5.3.6 Data and statistical analysis
38
Data were analyzed by one way ANOVA using the Newman–Keuls test for
multiple comparisons. A value of p<0.05 was considered statistically significant.
39
Chapter 4
RESULTS AND DISCUSSION
4.1 BIOCHEMICAL ANALYSIS OF BERBERIS LYCEUM ROYAL
4.1.1 Proximate Analysis
Proximate analysis of root samples of B. lyceum collected from different areas of
Pakistan was analyzed and results are shown in Table 4.1 which showed statistically
significant results (P≤ 0.05). A variation of 4.4 to 6.24 % was found, considering protein,
among the varieties. Comparison of means revealed that the protein contents were found
to be maximum in Bagh sample (6.24 %), where as sample from Mansehra exhibited
minimum protein contents (4.4 %). Non-significant difference in contents of samples
from Abbotabad and Mansehra sample was found. The difference in protein contents
might be due to different factors like climate and soil types, which affected the chemical
composition and nutrient value (Ebadi et al., 2005).
Fat contents showed no variation among the root samples collected from different
areas and only 0.5 percent crude fat was obtained in all samples, indicating low level of
fat in hard roots of B. lyceum Royal. Crude ash also showed less variation among Bagh,
Mansehra and Kotli-Sattian samples but root sample from Abbotabad contained high
level of crude ash. Statistical analysis showed significant difference among the crude
fiber contents of all the samples (Table 4.1). A variation of 14.96 to 16.40 % was found
and comparison of means revealed that the crude fiber contents were found to be
maximum in Abbotabad sample i.e. 16.40 %, where as Bagh sample, exhibited minimum
40
Table 4.1 Proximate analysis of different Berberis lyceum Royal samples collected from different areas of Pakistan (%)*
Sample Collection
Area Protein Crude fat Crude Fiber Crude Ash
Abbotabad 4.55±0.16ab 0.5±0a 16.40±0.1d 6.99±1.2a Bagh 6.24±0.32c 0.5±0a 14.96±0.5c 3.79±0.62bc Mansehra 4.4±0.14ab 0.5±0a 15.83±0.24b 4.06±0.27bc KotliSattian 5.82±0.31d 0.5±0a 16.10±0.19a 4.5±0.98bc * Means of three replication on dry weight basis ±Standard error Means values in the column sharing a common letter are not statistically significant according to Duncan’s Multiple Range Test (DMRT) (P≤0.01)
41
protein contents i.e. 14.96 percent. The determination of structural components will
enable us to understand the nutrient component of the material that can be used/intake in
addition with the compounds present in the samples to cure different diseases.
4.1.2 Determination of oil from root samples
Analysis of total oil contents of B. lyceum Royal collected from different areas
during study and their analysis of saturated and unsaturated fatty acids by GC-MS are
given in Table 4.1a and 4.1b respectively. The principal saturated and unsaturated fatty
acid of B. lyceum Royal oil were Palmitic (16:0), Oleic (18:1) and Linoleic (18:2) acids.
It has been noticed that octanedioic acid, azaleic acid and palmitoleic acid were absent in
all samples. Polyunsaturated fatty acids (PUFAs) were greater than monounsaturated
fatty acids (MUFAs) however, when comparing MUFAs and saturated fatty acids (SFA),
MUFAs were greater than SFA except in Kotlisattian sample where SFA was greater
than MUFAs (Table 4.1a and 4.1b).
It has also been noticed that the concentration of PUFAs especially linolenic acid,
a omega 3 fatty acid, was present in higher concentration (5.71±0.19 %) of total fatty
acid analyzed in present study. This omega 3 fatty acid cannot be synthesized by the
human and animals but mostly synthesized by the plants. High Carbon (20-22)
unsaturated fatty acids can be synthesized by linolenic acid however, these conversions
occur competitively with n−6 fatty acids which are closely related chemical analogues.
42
Time (min) Fig. 4.1 Spectra of representative GC–MS for fatty acid analysis of B. lyceum Royal oil collected from different areas of Pakistan
10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
650000
43
Both linolenic acid and linoleic acid are essential fatty acid which must be obtained from
food for energy purposes.
Study of fatty acid is the most prominent area of the research now days. It is
because of the consideration of critical balance of different fatty acids through
supplementation and their role in cure of different diseases. There is almost no system of
the body that does not require reduction of specific fatty acid substrate and coenzymes to
health maintenance and body tissue repair. Role of lipids and their metabolism is being
explored as a new means to unearth some of the complexities of disease, as lipid
imbalance and their deficiency have a close link to the endocrine, hepatic, renal,
pulmonary, gastrointestinal, musculoskeletal and cardiovascular systems (Marjorie et al.,
2006).
The physiological function ascribed to omega 3 fatty acids includes relaxation and
contraction of muscles, movement of calcium and other substances, secretion of digestive
enzymes and hormones, inhibition and promotion of clotting, cell division and growth
and also control of fertility (Innis, 1996). It has also been reported by Tamas et al. (2005)
that significantly higher plasma values of the essential fatty acids are present in diabetic
children but significantly lower values of their longer-chain metabolites. The onset of
one major disease called dyslipidemia caused due to abnormality in blood lipids are
associated with type 2 diabetes. This is because of the combination of
hypertriglycerdemia, low levels of high density lipoprotein (HDL), cholesterol and
44
abnormal low density lipoprotein (LDL). High level of LDL and low levels of HDL
cholesterol resulted in cardiovascular diseases (Hartweg et al., 2008).
Diabetes incidence was also significantly associated with the proportions of total
saturated fatty acids in plasma cholesterol ester (CE) and phospholipid (PL) (Wang et al.,
2003). They concluded that proportional saturated fatty acid composition of plasma is
positively associated with the development of diabetes. It was also reported that the
intake of Omega-3 fatty-acid reduces the risk of Islet autoimmunity in children which are
at high risk for diabetes (O'Riordan, 2007). Trans fatty acids (TFA) possibly will affect
cell membrane functions which may influence peripheral insulin sensitivity and
developmental risk of type 2 diabetes (Riserus, 2006). It is also important to recognize
whether low amounts of TFA consumed during long periods which may promote insulin
resistance and have clinically relevant effects on diabetes risk. As the results of Berberis
lyceum Royal fatty acids indicated that relative percentage of unsaturated is much higher
than that of saturated fatty acids. Total unsaturated fatty acids in different samples of B.
lyceum Royal are 86.76, 84.25, 83.32 and 62.91 from Abbotabad, Bagh, Mansehra and
Kotlisattian respectively. This much higher concentration of unsaturated fatty acid not
only involve in reducing the risk factor of onset of type 2 diabetes but also involved in
other musculoskeletal diseases like wound healing.
The role of essential fatty acids in wound healing is not yet clear, but as they are
involved in the synthesis of new cells, reduction would certainly delay wound healing.
Omega-3 fatty acids are anti-inflammatory, which helps in wound healing, but also
45
Table 4.2a Percentage* (%) of saturated fatty acids of Berberis lyceum Royal oil analyzed by GC–MS and their retention times
Sample Collection area
Octanedioic acid (17.33 min)**
Azaleic acid (18.71 min)
Palmitic acid (19.77 min)
Stearic acid (23.56 min)
Total SFA
Abbotabad - - 11.73±0.08 1.09±0.05 12.82 Bagh - - 15.36±0.01 2.66±0.26 18.02 Mansehra - - 11.83±0.95 1.45±0.14 13.28 KotliSattian - - 32.04±0.99 2.12±0.26 34.17 SFA: saturated fatty acid. *Values represent the average of three replicates ± standard deviation (SD). **Retention time in parenthesis.
Table 4.2b Percentage* (%) of unsaturated fatty acids of Berberis lyceum Royal oil analyzed by GC–MS and their retention times
Sample Collection area
Oleic acid (24.13 min)
Palmitoleic acid
(20.16 min)
Linoleic acid (25.37 min)
Linolenic acid (27.24 min)
Total MUFA
Total PUFA
Total Unsaturated
fatty acid
Abbotabad 39.67±0.61 - 45.21±0.58 1.88±0.03 39.67 47.09 86.76 Bagh 35.12±0.75 - 47.43±2.21 1.70±0.89 35.12 49.13 84.25 Mansehra 37.52±0.07 - 42.59±2.86 3.21±0.32 37.52 45.80 83.32 KotliSattian 12.01±0.54 - 45.19±5.54 5.71±0.19 12.01 50.90 62.91 MUFA: monounsaturated fatty acid. PUFA: polyunsaturated fatty acid. * Values represent the average of three replicates ± standard. **Retention time in parenthesis.
46
inhibit clotting which may be disadvantageous (Williams and Leaper 2000). It has been
reported by Hulsey et al. (1980) that essential fatty acid deficiency may cause delayed
healing. Polyunsaturated fatty acids can control prostaglandin synthesis and hence induce
wound healing ( Bowman and Rand, 1980; Gibson, 1983).
4.1.3 Metal ion analysis
The micro and macro elements of different samples of B. lyceum Royal were
analyzed by AAS and FP and results are shown in Figure 4.2 to 4.5. Data was subjected
to statistical test CRD two factors ANOVA which shows significant results. The results
showed that the higher mineral ion contents under investigation were found in Mansehra
sample i.e. 599.12 μg /g, whereas Abbotabad had the lowest content, 242.63 μg/g. The
total mineral ion contents was in the sequence of Mansehra> Kotlisattian> Bagh>
Abbotabad. Calcium (Ca2+) was the highest, ranging from 456 to 187.33 μg/g and copper
(Cu2+) was the lowest, ranging from 0.37 to 0.013 μg/g.
Elemental studies of medicinal plants are now becoming the focused area of
research as these elements are contained in enzymes which activate them. In this way
they are influencing the biochemical processes in the cells (Narendhirakannan et al.,
2005). Among the micronutrients, Mg2+ plays an important role in cellular physiology as
its intake bestows protection against the frequency of metabolic syndrome, diabetes,
hypertension, and cardiovascular disease. It ameliorates serum lipid profiles, insulin
resistance, and lowers inflammation, oxidative stress, endothelial dysfunction, and
47
(A)
(B)
Figure 4.2: Macro element analysis of B. lyceum Royal from different areas of Pakistan. (A): Magnesium and (B): Calcium
48
(A)
(B)
Figure 4.3: Macro element analysis of B. lyceum Royal from different areas of Pakistan. (A): Sodium and (B): Pottasium
49
platelet aggregability (Spiegel, 2007; Bo and Pisu, 2008). Comparison of means revealed
that Mg2+ was found to be maximum in Abbotabad sample, 47.61 μg/g and lowest in
Bagh sample, 23.68 μg/g. The Mg2+ content was in the sequence of
Abbotabad>Mansehra>Kotlisattian>Bagh. According to DMR test (p>0.01), Mansehra
and Kotlisattian was statistically non significant but showed significant difference with
Abbotabad and Bagh which were also significant with each other (Figure 4.2).
Calcium (Ca2+) was the highest macro element observed in B. lyceum samples
under investigated (Figure 4.2). It is regulated by the parathyroid glands. The most
frequent health problem linked calcium deficiency is osteoporosis which is caused by
calcium deficiency and ultimately leading to loss of bone mass (Gennari, 2001).
Comparison of means revealed Ca2+ that was found to be maximum in Manshera sample,
456 μg/g whereas lowest in Abbotabad sample, 187.33. Sodium (Na+) concentration in
body and its balance relates to genes, sex hormones, and body fluid (Burnier, 2007). The
concentration of Na+ was less variable among all the samples. Potassium (K+) is an
essential dietary mineral and electrolyte (Peterson, 1997). Presence of K+ is required by
limited number of enzymes for their activity. The activation of sodium and potassium-
ATPase requires the presence of sodium and potassium. Pyruvate kinase, an important
enzyme in carbohydrate metabolism also required the presence of potassium for its
activity (Sheng et al., 2000). The concentration of K+ is variable among all root samples
collected from different areas of Pakistan. Mansehra sample contain higher concentration
50
(A)
(B)
Figure 4.4: Micro element analysis of B.s lyceum Royal from different areas of Pakistan. (A): Manganese and (B): Iron
51
of K+, 96.18 μg/g whereas Kotlisattian, Bagh and Abbotabad samples contain less
amount (Figure 4.3).
Macro elements such as Manganese (Mn2+), Iron (Fe2+), Copper (Cu2+) and Zinc
(Zn2+) are as essential nutrients for the maintaince of health. Manganese is an essential
nutrient and its deficiency can lead to skin problems, slowed blood clotting, lowered
cholesterol levels, changes in hair color, and other alterations in metabolism. Iron is a co-
factor in collagen synthesis, and deficiency in iron delays wound healing. Copper works
as a bactericidal and fungicidal in a very specific and simple way and act as a reservoir of
disease control. Cu2+ denatures cellular proteins and deactivates enzyme systems in fungi
and algae. Zn2+ is required for protein synthesis and is also a co-factor in enzymatic
reactions has an inhibitory effect on bacterial growth and is also involved in the immune
response (Gray and Cooper 2001). Complexes of zinc and insulin in varing ratios are
stored in pancreatic β cells and released into the blood circulation. Abnormalities in Zn2+
metabolism may have role in the pathogenesis of diabetes and its complication
(Narendhirakannan et al., 2005). The data of micro elements showed that Fe2+ was the
highest, ranging from 5.321 to 1.063 μg/g and Cu2+ was the lowest ranging from 0.37 to
0.013 μg/g. Mansehra sample contain highest concentration of Mn2+, Fe2+, and Zn2+
contents. It was also observed that these samples are lower or deficient in copper
concentration. It may be because copper is tightly bound in organic matter and may be
deficient in highly organic soil. It is not readily lost from the soil but may often be
unavailable.
52
(A)
(B)
Figure 4.5: Micro element analysis of B. lyceum Royal from different areas of Pakistan. (A): Copper and (B): Zinc
53
Over the past few decades epidemiological studies have documented the
importance of macro and micro elements in human health and disease. In this reference,
pharmaceutical companies have been marketing supplements containing combinations of
different element contents. Many studies have been reported on the elemental analysis of
Berberis spp. by Shah et al. (2003); Hill, 2003; Mohammad et al. (2005) and Parasad,
2008. These studies not only give the information regarding the bioactive ingredient of
the Berberis spp. but also give the valuable information about their elemental
constituents. This will help to isolate the bioactive compounds and making new
formulation which also contains beneficial elements used to cure different diseases.
4.2 EXTRACTION AND QUANTIFICATION OF ALKALOIDS
Berberine is an isoquinoline alkaloid with a bright yellow color that is easily seen
in most of the herbal materials that contain any significant amount of this compound
including the roots of B. lyceum. It is antimicrobial agent against a wide variety of
microorganisms including Gram-positive and Gram-negative bacteria, fungi, protozoa,
trypanosomes and plasmodia. The results obtained from the HPLC and 1H NMR studies
of berberine and palmatine are presented in Tables 4.3 and Figures 4.6 to 4.9. All the
proton and carbon signals could be detected in 1H and 13C -NMR spectra as shown in
Table 4.3.
The spectra were recorded in deuterochloroform with all shifts (δ; ppm ) referred
to the internal standard anthracene). The analysis of the NMR spectra of berberine and
Palmatine revealed that the proton H-13 resonating as a singlet (H-13 of 1: δ 8.72; H-13
54
of 2: δ 8.81) could be used for quantification. Data of berberine and palmatine recorded
in deuterochloroform can be compared with results reported by other authors including
Blask et al. (1988), Merek et al. ( 2003), Jansen et al. ( 1989) and Guinaudeau et al.
(1996). However, they have analyzed some protoberberine alkaloids not from B. lyceum
but from some other plant species.
Berberine-containing plants are used medicinally in many traditional medical
systems, including Ayurvedic herbal and Chinese herbal medicine. Berberine, produced
by a number of important medicinal plants, such as Berberis vulgaris (barberry), Berberis
aristata (tree turmeric), Berberis aquifolium (Oregon grape) or Tinospora cordifolia, has
been shown to exert potent anti-inflammatory and antitumor properties in in vitro as well
as in vivo systems. Berberine has long been used as a dye; it is currently known as
"natural yellow 18," being one of about 35 yellow dyes from natural sources. The
Berberine alkaloid appears in the roots, rhizomes and bark and major compounds of
Coptis chinensis and Phellodendron amurense is Berberine. Berberine has been
suggested to boost the immune system, as an anti-oxidant, exhibits antimalarial,
antisecretory, and anti-inflammatory as well as anticancer activities with relatively low
cytotoxicity. Berberine has also been shown to significantly decrease cholesterol levels in
mice. Berberine was isolated and used as an herbal drug in China 50 years ago (the drug
forms are usually the hydrochloride or sulfate; the chloride, as used in the dye, may have
the strongest antiseptic action). Berberine hydrochloride or sulfate is considered most
effective as an herbal treatment and appears to be most effective as an antiseptic in
chloride form. Berberine chloride from plant roots and stems also is the primary
ingredient in natural product. It has since become an
55
Figure 4.6 Peaks of HPLC (1) berberine (2) palmatine from root samples of B. lyceum
56
(1)
(2)
Figure 4.7 1H NMR spectroscopic analysis of berberine (1) and palmatine (2)
.
57
O
ON +
O CH3
O CH3
Ha
Ha
HbHb
He
H
H
H
H
H
Hc
Hc
Hd
Protons just denoted as H are labelled as Ar-H in the NMR assignment
Figure 4.8 Structure of berberine
H3CO
H3CON+
OCH3
OCH3
Ha
Ha
HbHb
He
H
Hg
Hf
H
H
Hd
Protons just denoted as H are labelled as Ar-H in the NMR assignment
Hd
Figure 4.9 Structure of palmatine
58
Table 4.3 1H NMR chemical shifts (δ; in ppm) of berberine and palmatine (Solvent
CDCl3)
Atom
H-1
H-4
H-5
H-6
H-8
H-9
H-10
H-11
H-12
H-13
2-OR
3-OR
9-R/10-R
10-R/11-R
13-OME
Berberine
7.74
7.06
3.21
4.93
9.92
-
-
8.21
8.02
8.72
6.16
-
4.12
4.03
-
Palmatine
7.73
7.12
3.24
4.92
9.93
-
8.10
8.13
-
8.81
3.91
3.86
4.14
4.01
-
59
ingredient in several Western herbal products, particularly for treatment of intestinal
infections.
A suitable internal standard should preferably be a stable compound with a signal
in a non-crowded region of the 1H NMR spectrum. Therefore for this purpose,
anthracene was utilized, and it was observed that integration of the peaks of both
alkaloids were proportional to the amount of the compound present in the root samples
Berberis lyceum (Karagian et al., 2003; Marek et al., 2002; Martin and Crouch, 1994).
The 1H NMR method used in this study was found to be simple, rapid and specific for the
analysis of protoberberine alkaloids. No reference compound was used, apart from the
internal standard, and an overall profile of the preparation was obtained directly. Using
this method the content of protoberberine alkaloids can be determined in Berberis lyceum
and other plant extracts in a shorter time than conventional method of HPLC.
The mechanism of action of the quaternary structure of berberine is attributed to
its ability to intercalate with DNA. The intercalation in combination with the inhibition of
protein biosynthesis (the major mode of action of Berberine) could be responsible for the
observed cytotoxic effect, because both targets are central to all living cells. Laboratory
studies suggested that berberine may have at least two functions in relation to reducing
blood sugar: inhibiting absorption of sugars from the intestine and enhancing production
of insulin. It was reported recently that berberine lowers cholesterol through a mechanism
different than that of the statin drugs, suggesting potential use both as an alternative to the
statins and as a complementary therapy that might be used with statins in an attempt to
gain better control over cholesterol. The apparent mechanism is increasing the production
60
of a receptor protein in the liver that binds the LDL-cholesterol, preparing it for
elimination.
4.3 BIOASSAYS
4.3.1 Antimicrobial Bioassay
Aqueous, methanolic and ethanolic root extracts of Berberis lyceum were
tested against different microorganisms. The results pertaining antimicrobial activity of
root extracts are summarized in the Tables 4.4 to 4.9. Methanol, ethanolic and aqueous
root extracts of B. lyceum were applied to control the growth of different microorganisms
and it was found that methanol and ethanol extracts have provided better results as
compared to aqueous, however, methanol root extracts have provided comparatively
better results. Therefore methanol is considered as useful solvents for assessment of
antimicrobial activities (Ahmad et al., 1998).
Bacterial resistant against antibiotic has been seen as a main barrier to successful
treatment. Multidrug resistant strains are now common due to the abuse of antibiotics in
daily life. The antimicrobial activity of extracts was assessed and quantified by presence
or absence of inhibition zone, zone diameter and MIC values against multidrug resistant
strains. The antimicrobial activity of methanol, ethanol and aqueous extracts were
effective against both gram negative and positive bacterial strains. The highest inhibitory
zone was observed against Bacillus cereus (28±1.5, methanolic) followed by B.
licheniformis (26±0, aqueous), P. syringae (24±2.5, aqueous), B. cereus (24±1.2,
ethanolic) and E. coli (24±0.7, methanolic). Ethanolic and aqueous extracts, in general,
provided slightly lower results but better results as compared to standard antibiotics used
61
Table 4.4 Antimicrobial activity of methanol and Ethanol root extracts of Berberis lyceum (100
µg/disk tested against bacterial strains by using disk diffusion method. Zone of inhibition in diameter (mm) around test disk*
Microorganisms (Bacteria) Methanol extracts Ethanol extracts Standard Bacillus cereus 28±1.5 24±1.2 Bacillus magaterium 20±1.4 18±1.2 Bacillus pumilus 23±0.5 - Bacillus substilis 20±1.2 21±0.5 Escherichia coli 24±0.7 22±0.3 Klebsiella pneumonia - 14±0.5 Micrococcus lutus - 14±0.8 Plesiomonas shegelloides - 20±0.5 Pseudomonas putida 16±0.5 12±0.3 Pseudononas syringae 21±0.8 20±0.6 Staphylococcus aureus 14±0.7 - Staphylococcus epidermis 22±0.4 - Streptococcus pneumonia 14±0.5 12±0.4 Streptococcus pyrogenes - * Standard antibiotic used: SCF: Sulbactum and cefoperazona, OFX: Ofloxan, NET: Netilmicin (Positive control), NC: Negative control (Methanol and Ethanol), NT: Not tested
62
in this experiments (Table 4.4 and 4.5). MICs were calculated for the test bacteria only
that had antimicrobial activity. The MIC provides a quantitative measurement of the
lowest concentration of antimicrobial agent that inhibits the growth of a bacterium. The
MIC values (Table 4.8 and 4.9) showed that methanol, ethanol and aqueous extract has
given good results against all the microorganisms tested including multidrug resistant E
.coli. E. coli, a gram negative bacterium, commonly found in lower digestive system
track can be varying widely in antibiotic resistant. Many studies have attempted to
evaluate the distribution of the resistance genes for antimicrobial agents in E. coli
populations (Bass et al., 1999; Bischoff et al., 2002; Bischoff et al., 2005). The rate of
adaptive mutations in E. coli is 10-5 per genome per generation (Perfeito et al., 2007).
Antibiotic resistant strains of E. coli may penetrate their genes responsible for antibiotic
resistant to other bacterial species (Salyers et al., 2004).
According to the antimicrobial Surveillance Program (1998) the four
microorganisms were isolated from lower respiratory track of the patients suffering from
pneumonia like Pseudomonas aeruginosa (26.8%), Staphylococcus aureus (24%),
Klebsiella pneumonia (12.1%) and Acinetobacter spp. (10.5%) (Martini, 2001). These all
microbial species are life threatening for the human beings. P. aeruginosa belongs to
gamma proteobacteria class and mostly Pseudomonas spp. are naturally resistant to
penicillin. Candida is naturally occurring yeast in human body generally found in the
mouth, throat, intestines and genitourinary tract. Most common symptom of C. albican is
vaginitis. It is a white or yellow discharge from the vagina and of the vulva (external
genital area) which causes burning and itching. The Berberis extract showed promising
63
Table 4.5 Antimicrobial activity of aqueous extracts of Berberis lyceum (100 µg/disk tested
against bacterial strains by using disk diffusion method. Zone of inhibition in diameter (mm) around test disk.*
Microorganisms(Bacteria) Berberis lyceum
extracts Negative control (NC)
Standard
Bacillus amyloliquefaciens 12±0.1 - Bacillus cereus 20±1.3 - Bacillus licheniformis 26±0 - Bacillus magaterium 24±0.3 - Bacillus pumilus 20±0.8 - Bacillus substilis 22±2.5 - Burkholdoria gladioli 16±0.4 - Eschericha coli 14±0.8 - Klebsiella pneumonia 17±1.0 - Micrococcus lutus - - Proteus vulgaris - - Plesiomonas shegelloides - - Pseudomonas putida 14±1.0 - Pseudononas syringae 24±2.5 - Staphylococcus aureus 22±0.4 - Staphylococcus epidermis 24±1.7 - Streptococcus pneumonia 12±0.2 - Streptococcus pyrogenes 26±3.2 - * Standard antibiotic used: SCF: Sulbactum and cefoperazona, OFX: Ofloxan, NET: Netilmicin (Positive control), NC: Negative control (Methanol and Ethanol), NT: Not tested
64
results against this devastating microorganism. The MIC values with both methanolic and
aqueous against C. albican are 40.5 and 35.5 μg/ml respectively.
A number of antimicrobial studies have been reported for Berberis spp. Singh et
al. (2007) reported that B. lycium, B. aristata and B. asiatica root extract showed
significant antifungal activity against Aspergillus terreus and A. flavus. B. aristata root
and B. lycium (stem) extracts gave very low MIC values with the concentration of
0.31 µg/ml as compared to other tested species. The crude methanolic extract and
alkaloidal fraction of B. aetnensis was active against Candida species (Lauk, 2007).
Berberis heterophylla leaves, stems and root aqueous extracts showed antimicrobial
activity against gram positive and negative bacteria and fungi (Freile et al., 2003).
Berberine ( alkaloid) an active ingredient of Berberis spp. has been widely used to treat
gasteroenteritis and diarrhea patients (Lin et al., 2005). Berberine exerts an anti secretory
action on epithelial cells, probably by blocking K+ channels (Taylor et al., 1999). As
some antibiotics have shown adverse effects on the host including hypersensitivity,
immune suppression and allergic reactions (Bisset, 1994). Given the alarming incidence
of antibiotics resistance in bacteria, there is a strong need for new and effective
therapeutic agents (Antimicrobial drugs) for the treatment of infectious diseases
(Almagboul et al., 1988). The development of antibiotics resistance is multifactorial as
bacteria have the genetic ability to transmit and acquire resistance to drugs (Cohen,
1992). To over this problem of antibiotics resistance medicinal plants have been
extensively studied as an alternative medicine (Ali, 2001).
65
Table 4.6 Antimicrobial activity of methanol and Ethanol extracts of Berberis lyceum (100
µg/disk tested against yeast and fungi isolates by using disk diffusion method. Zone of inhibition in diameter (mm) around test disk.
Microorganisms Methanol
extracts Ethanol Extract
Negative control (NC)
Standard
Yeast- Candida albicans 25±1.2 18±1.1 - Fungi- Alternaria alternate - - - Aspergillus flavus - - - Fasarium oxysporum - - - Penicillium spp - - - * Standard antibiotic used: SCF: Sulbactum and cefoperazona, OFX: Ofloxan, NET: Netilmicin (Positive control), NC: Negative control (Methanol), NT: Not tested Table 4.7 Antimicrobial activity of aqueous extracts of Berberis lyceum (100 µg/disk tested
against yeast and fungi isolates by using disk diffusion method. Zone of inhibition in diameter (mm) around test disk.
Microorganisms Berberis lyceum
aqueous extract Negative control (NC)
Standard
Yeast Candida albicans 10±0.5 - Fungi Alternaria alternate - Aspergillus flavus - - Fasarium oxysporum - - Penicillium spp - - Total isolates - - * Standard antibiotic used: SCF: Sulbactum and cefoperazona, OFX: Ofloxan, NET: Netilmicin (Positive control), NC: Negative control (Methanol), NT: Not tested
66
Table 4.8 The MIC (µg/ml) values of Berberis lyceum (methanol and Ethanol) tested
against microorganisms in micro dilution assays. Microorganisms(Bacteria) Methanol extract Ethanol Extract Bacillus amyloliquefaciens 200 200 Bacillus cereus 200 100 Bacillus licheniformis 100 100 Bacillus magaterium 100 100 Bacillus pumilus 200 - Bacillus substilis 200 100 Eschericha coli 200 100 Pseudomonas putida 200 200 Pseudononas syringae 100 100 Candida albicans 40.5 *Standard: Maxipime Table 4.9 The MIC (µg/ml) values of Berberis lyceum (aqueous) tested against
microorganisms in micro dilution assays. Microorganisms(Bacteria) Berberis lyceum extracts Bacillus amyloliquefaciens 200 Bacillus cereus 200 Bacillus licheniformis 100 Bacillus magaterium 100 Bacillus pumilus 200 Bacillus substilis 200 Eschericha coli 200 Pseudomonas putida 200 Pseudononas syringae 100 Candida albicans 35.5 *Standard: Maxipime
67
The results obtained during this study revealed that root of Berberis lyceum
contained some active phytochemicals those have ability to control the growth of some
microorganism (Barnabas and Nagarajan, 1988; Brantner et al., 1994). The
microorganism those was effected by plants extract could have some difference in their
cell walls or inheritance antimicrobial resistance genes as plasmids can easily be
transferred among bacterial strains. Therefore on the basis of results obtained in present
study the root extracts of this plant can be helpful for development of new and useful
drugs for different infections of human and animals.
4.3.2 Wound Healing Activity
For the assessment of wound healing activity of root extracts of B. lyceum,
extracts were prepared in methanol and aqueous media. Significant promotion of wound-
healing activity was observed with both aqueous and methanol root extracts when applied
for three wound repair models. In the excision wound repair model (Table 4.10), the
animals treated with the methanolic extract showed faster epithelialisation (1.7 ± 0.2 mm
2) than those treated with the aqueous root extract (1.9 ± 0.6 mm2). The positive control
(1% w/w nitrofurazone gel) produced an epithelialisation area of 1.5 ± 0.2 mm2, which
was slightly lower than root extracts, which shows that root extract has provided better
results.
In the incision wound repair model, the animals treated with both the methanolic
and aqueous root extracts showed an increase in breaking strength (389.76 ± 3.30 g),
(349.74 ± 3.57 g) respectively, when compared to the control (233.43 ± 2.59 g). The
68
Table 4.10 Effect of topical application of the aqueous and methanol extracts of Berberis lyceum on epithelialisation (mm2) in the excision wound repair model
Post excision day: wound size in mm2
Group/day 0 3 6 9 12 15 18 Epithelialisation area (mm2) at 18 days
Control
51.3 ±0.5 48.2 ±1.5
40.2± 1.2 35.5±0.5 27.5 ±0.7 19.2 ±0.5 8.7 ±0.4 2.3 ±0.2
Standard drug
50.2 ±1.5 40.6 ±6.0
32.5± 1.3 25.3 ±0.5 13.6 ±0.5 9.6 ±0.5 0* 1.5 ±0.2
Aqueous Extract
49.5 ±2.1 46.4 ±1.4
34.1 ± 0.5 27.3 ±0.6 14.9 ±0.5 6.7 ± 0.2 7.8 ±0.5 1.9 ± 0.6
Methanol Extract
48.7 ±7.5 44.5± 1.2
33.3 ± 0.6 26.9 ±0.8 14.3 ±0.4 1.9 ± 4 0* 1.7±0.2
Probability P
<0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
Values indicate as mean SEM, n=6 animals in each group. All results all given as percentage of wound contraction p≤ 0.001 as compared to control * = Highly significant
.
Table 4.11 Effect of the aqueous and methanol extracts of Berberis lyceum on wound breaking strength in the incision model, and granulation in the dead space model Group Weight of granulation
tissue (mg) Breaking strength (g)
Hydroxyproline (mg/100g)
Control (Vehicle) 86.9 ± 0.6 233.4 ± 2.6 1403.74 ± 1.0 Aqueous extract 148.4 ± 0.6 349.1 ± 3.6 1983.4 ±0.8 Methanol extract 186.5 ±0.5 389.7 ± 3.3 2256.0 ± 0.6 Probability P <0.001 <0.001 <0.001 Values are expressed as mean, SEM, n=6 in each group, p≤ 0.001 compared to control
69
mean breaking strength of the animals treated with the positive control was also
significant (Tables 4.10 and 4.11). In the dead space wound model, histological studies of
the granulation tissue of the control group of animals showed more aggregation of
macrophages with few collagen fibres than the treated groups. In the case of the group
treated with the aqueous root extract, moderate collagen deposition, macrophages and
fibroblasts were noticed, whereas the group treated with the methanolic extract treated
evidenced significant increase in collagen deposition showing lesser macrophages and
fibroblasts. Compared to the control group, the methanolic extract treated animals
showed a more significant increase in dry weight of granulation tissue (186.49 ± 0.52
mg/kg) compared to the aqueous extract treated group (Table 4.11).
Estimation of hydroxyproline in the granulation tissue revealed that the animal
groups treated with the methanolic extract had the highest content (2256.00 ± 0.6
mg/100g), followed by the aqueous extract treated group (1983.37 ± 0.8 mg/100g). The
control group showed a significantly lower hydroxyproline content (1403.68 ± 1.0
mg/100g). Wounding healing is a fundamental physiological response to tissue injury that
results in the restoration of integrity by the synthesis of a connective tissue matrix.
Collagen is the major protein of the extracellular matrix and ultimately contributes to
wound strength. The increase in the hydroxyproline content of the granulation tissue, the
increase in tensile strength together with the enhancement of collagen maturation shown
by increased cross linking, and the increase in dry granulation weight, shows that the
extracts of Berberis lyceum are promoting wound healing. This experiment also proved
the ethnobotanical informations collected for use of B. lyceum against wound.
70
It has previously been observed that plant constituents such as catechins and gallic
acids etc. can significantly improve the quality of wound healing, accelerate the healing
process and reduce scar formation, and could potentially be new therapeutic agents to
treat wounds (Kapoor et al. 2004). The root of Berberis lyceum contains flavonoids,
alkaloids including berberine, tannins, saponins and triterpenoids (Gulfraz et al. 2004;
Yesilada and Kupeli , 2002). Triterpenoids and saponins are thought to promote the
wound healing process due to their antioxidant and antimicrobial activities (Mahara and
Sushma 2003, Gulcin et al.2004). Their astringent and antimicrobial properties also
contribute to wound contraction and increase the rate of epithelialisation. The methanol
extract was found to have better wound healing potency than the aqueous extract, and
further studies are in progress to isolate the active compounds responsible for healing
process.
4.3.3 Anti Diabetic Studies
Root extracts of B. lyceum are being used against high sugar level in rural people
from north east of Pakistan. Therefore on basis of ethnobotanical studies this antidiabetic
study on animal model was conducted. In order to select LD 50, the acute toxicity studies
was conducted which indicated that the dose selected, 50 mg/kg for both berberine and
Berberis root extracts, was non-toxic throughout the duration of the study.
4.3.3.1 Effects of berberine and Berberis lyceum root extract on glucose tolerance, and glucose levels in normal and diabetic animals
Pure berberine produced a significant reduction in blood glucose level in all
models tested. In the glucose tolerance test (Table 4.12), a significant reduction in blood
71
Table 4.12 Blood glucose concentration on the oral glucose tolerance test after treatment with extract of B. lyceum or berberine in glucose loaded rats
Concentration (mg/dL) Group Treatment
0 min 30 min 60 min 120 min 240 min
1 Control (vehicle) 91.5±6.5 104±2.5 98.2±8.5 95.3±4.5 93.2±4.3
2 Berberis extract (50 mg/kg)
85.2±2.3 93.5±5.5 84.2±3.5* 82.5±6.5* 81.2±2.5*
3 Berberine (50mg/kg) 82.4±5.2 92.4±6.5* 80.5±5.2* 78.4±3.8* 77.3±3.6*
4 Glibenclamide (15mg/kg) 80.7±4.3 71.8±5.2 72.4±2.5 74.3±4.5 70.4±3.5
Values are expressed as means ± SE. *Significant (p <0.05) values Vs control, n = 6
Table 4.13 Blood glucose concentration after treatment with extract of B. lyceum or berberine in normal rats
Concentration (mg/dL) Group Treatment
Day 0 Day 1 Day 3 Day 5 Day 7
1 Control (vehicle) 81.5±5.5 84±2.6 82.1±8.5 75.3±6.5 68.2±4.5
2 Berberis extract (50 mg/kg)
75.2±2.4 72.4±5.7 70.3±3.6* 69.5±6.4* 64.2±2.4*
3 Berberine (50mg/kg) 76.4±6.2 71.4±6.2 68.5±5.6* 61.4±3.5* 58.2±3.7*
4 Glibenclamide (15mg/kg) 74.7±4.5 69.3±5.4 67.5±2.1 62.3±4.2 60.4±3.2*
Values are expressed as means ± SE. *Significant (p <0.05) values Vs control, n = 6
72
Table 4.14 Blood glucose concentration after treatment with extract of B. lyceum or berberine in alloxan-induced diabetic rats
Concentration (mg/dL) Group Treatment
Day 0 Day 1 Day 3 Day 5 Day 7
1 Control (vehicle) 225.4±7.8 228.4±2.8 231.2±6.4 233.5±4.6 234.2±4.5
2 Berberis extract (50 mg/kg)
238.2±2.4 173.5±5.5 164.2±3.6* 112.6±7.5* 81.2±2.4*
3 Berberine (50mg/kg) 236.2±5.8 135.4±6.7 114.5±5.2* 82.4.4±3.8* 70.3±3.5*
4 Glibenclamide (15mg/kg)
178.7±4.3 129.4±6.3 117.4±2.6* 104.2±4.3 93.4±3.5
Values are expressed as means ± SE. *Significant (p <0.05) values Vs control, n = 6
Table 4.15 Serum insulin and glycosylated haemoglobin in normal and alloxan induced diabetic rats after treatment with extract of B. lyceum or berberine Group Treatment Serum insulin
(μU/mL) Glycosylated haemoglobin (%)
1 Normal rat (vehicle) 135.4±9.5 3.8±1.2 2 Diabetic control (vehicle) 126.2±4.8 7.5±0.5 3 Berberis extract (50mg/kg) 98.2±5.4 4.5±0.4* 4 Berberine (50mg/kg) 92.3±4.5 3.5±0.8* Values are expressed as means ± SE. *Significant (p <0.05) values Vs control, n = 6
73
Table 4.16 Serum lipid profiles in normal and alloxan induced diabetic rats after treatment with extract of B. lyceum or berberine Group Treatment Triglyceride
(mg/dL) Cholesterol (mg/dL)
HDL cholesterol (mg/dL)
1 Normal rat (vehicle) 94.5±1.2 63.4±2.4 58.2±5.4
2 Diabetic control (vehicle) 192.2±2.5 153.4±5.8 32.4±3.8
3 Berberis extract (50mg/kg) 62.2±5.7* 59.4±6.5* 52.5±5.7*
4 Berberine (50mg/kg) 56.3±4.5* 48.6±6.4* 46.3±2.4*
*Significant (p<0.05) compared with control/normal animals. Values are expressed as means ± SE; n=6
Table 4.17 Body weight of alloxan induced diabetic rats after treatment with extract of B. lyceum or berberine Group Treatment Initial body weight
(g) Final body weight (g)
1 Diabetic control (vehicle) 198.5±7.5 175.2±8.4 2 Berberis extract (50mg/kg) 192.3±7.4 182.4±9.4* 3 Berberine (50mg/kg) 186.4±6.7 178.3±8.3 *Significant (p<0.05) compared with control/normal animal. Values are expressed as means±SE.
74
glucose level was observed from 30 to 240 min; the effect of berberine was comparable
to that of Berberis extract and gave lower results than the controls, but less than those of
glibenclamide (standard). In normal rats (Table 4.13) a similar phenomenon was seen
over the 7 day test period, i.e. berberine gave a slightly more potent effect than Berberis
extract, as would be expected from its lower concentration, but both were comparable to
glibenclamide. Surprisingly, in the alloxan-diabetic animals, the hypoglycaemic effects of
both berberine and the root extracts were considerably greater than those of
glibenclamide (Table 4.14).
4.3.3.2 Effects of berberine and B. lyceum root extracts on serum insulin and glycosylated Haemoglobin
Both berberine and the extract lowered serum insulin significantly (p < 0.05), as
shown in Table 4.15, and reduced glycosylated haemoglobin levels; in the case of
berberine, this was achieved at levels below those of the controls. Significant of
measuring glycosylated haemoglobin is to measures the number of glucose molecules
attached to hemoglobin, a substance in red blood cells which is very important for
smooth function of body (Ingle, 2008).
4.3.3.3 Effects of berberine and B. lyceum root extracts on lipid profiles
Non-insulin-dependent diabetes mellitus is frequently associated with premature
atherosclerosis due to the presence of unwanted triglyceride and cholesterol (Deman,
2003). Both berberine and B .lyceum root extracts prevented the increase in triglyceride,
cholesterol and HDL cholesterol blood levels observed due to induction of diabetes, and
reduced them to values below those observed in normal rats, as shown in Table 4.16.
75
4.3.3.4 Effects of berberine and B. lyceum root extracts on changes in body weight
The decrease in body weight over time, which is a consequence of diabetes (as
seen in Table 4.17, group 1) was reduced by treatment with the extract, but to a lesser
extent by berberine than by the extract. Alloxan causes diabetes by the rapid depletion of
β cells and therefore brings about a reduction in insulin release which results in
hyperglycaemia. In turn, this causes oxidative damage by the generation of reactive
oxygen species and results in the development of diabetic complications including
cardiovascular, gastrointestinal, kidney and bladder dysfunction (Scholin et al., 1999).
Protein synthesis also decreased in all tissues due to a relative insulin deficiency, and as a
result, the synthesis of haemoglobin was suppressed (Scholin et al., 1999). Increased
glycation of protein, including haemoglobin, is a consequence of diabetic complications
(Guyatt et al., 2002), and an increase in glycosylated haemoglobin has been found to be
directly proportional to fasting blood glucose levels (Lloyd and Orchard, 1999).
Therefore, it was observed that an increase in blood glucose levels in diabetic
animals was found after the induction of alloxan. This was prevented by oral
administration of berberine and Berberis extract, and a hypoglycaemic effect was seen in
normal animals and those which had been given a glucose loading. The effects were
comparable to the standard antihyperglycaemic drug glibenclamide. This effect may be
due either to the potentiation of the effect of insulin which had been secreted from the
remaining β cells, or its release from the bound form. Both Berberis root extract and
berberine-treated animals showed a significant reduction in both blood glucose levels and
glycosylated haemoglobin of diabetic animals (Table 4.15) but in the case of the treated
76
(but not fasting) animals, the reduction in blood glucose was not proportional to
glycosylated haemoglobin levels. This suggests other properties of berberine which may
be unrelated to the effects on insulin release, which is supported by the work of Zhou et
al. (2007) who suggest that activated protein kinase is involved in such metabolic effects.
Cheng et al. (2006) have reported that glucose uptake through the AMPAMPK- P38
pathway may account for some of the antihyperglycaemic effects of berberine and Lee et
al. (2006) have shown that increased GLUT4 activity is also involved however this effect
was considered in part due to stimulation of AMPK activity. Guo et al. (2003) and Pan et
al. (2003) have also found that glucose absorption can be inhibited by berberine, by
inhibition of alphaglucosidase and decreasing glucose transport through the intestinal
epithelium (Pan et al., 2003).
Ko et al. (2005) demonstrated that berberine can increase insulin sensitivity and
also increase insulin-stimulated glucose uptake, which was confirmed in present study
when insulin levels were affected by berberine (Table 4.15). The results of these various
studies indicates that more than one mechanism may be in operation for insulin
stimulation. Elevations in plasma lipid concentrations are a consequence of diabetes, with
a marked increased in serum triglyceride and cholesterol level. Serum triglyceride and
cholesterol levels decreased significantly in diabetic rats after treatment with extract of
Berberis lyceum and berberine, whereas HDL cholesterol levels were improved
compared with the control group animals (Table 4.16). The weight loss in diabetic rats
(Table 4.17) was probably associated with the lipid lowering activity of Berberis lyceum
and berberine or by its influence on various lipid regulation systems. Huang et al. (2006)
77
and Lee et al. (2006) have reported that berberine works on multiple molecular targets
and has the potential for use as a weight reducing agent, as well as for treating
hypolipidaemia and hypoglycaemia. Some of the effects of berberine, including those
related to diabetes, may be associated with its ability to scavenge free radicals (Tang et
al., 2006). Berberis extract and berberine demonstrated similar effects on all parameters
measured, and although the extract was comparable in efficacy to berberine. Furthermore
B. lyceum can not produce additional effects as compared to pure berberine by effecting
on cholesterol, total lipid as well as reducing weights. The results support the use of the
extract in traditional medicine, however important benefits that can be obtained as
compared with the pure compound is its use in a highly cost-effective means of treating
as compared to berberine and other similar drugs. Therefore, B. lyceum and similar other
medicinal plants used in rural areas of Pakistan and providing economical benefits to the
rural community as these products and lower cost easily available as well as have no or
very less side effects.
According to world health organization, medicinal plants would be the best
sources to obtain a variety of drugs. About 80% of individuals from developing countries
are using traditional medicines which has compounds derived from medicinal plants. All
these traditional medicinal systems have accumulated a great deal of knowledge on the
various medicinal plant species (Berghe and Vierinck, 1991). Therefore demand of
medicinal plants by the modern pharmaceutical industries has increased manifold because
medicinal plants now occupy a significant place in modern medicine for some important
drugs.
78
Chapter 5
SUMMARY
Medicinal plants are major source of drugs used for the treatment of various
health disorders. Berberis lyceum Royal, an indigenous plant of the Northern areas of
Pakistan study was selected to explore its medicinal value. This plant has many
therapeutic values and is being used against many diseases / infections by local
population since long. Its remedies includes swollen and sore eyes, broken bones,
wounds, gonorrhea, curative piles, unhealthy ulcers, acute conjunctive, and in chronic
ophthalmia. Biochemical, metal ion analysis, isolation and purification of alkaloids and
bioactivity of crude extract for antimicrobial, antidiabetic and wound healing have been
investigated in this study. Biochemical analysis of root samples of B. lyceum Royal from
Northern side of Pakistan showed the variation among different parameters, which
include protein contents (4.4 – 6.24 %), crude fiber (14.96 – 16.40 %) and crude ash
(3.79 – 6.99 %) on dry weight basis. No variation regarding crude fats (0.5 %) was found
in any samples under investigation. The oil contents were determined by Soxhlet method
and results revealed that the principal saturated and unsaturated fatty acid components of
B. lyceum Royal root oil were Palmitic (16:0), Oleic (18:1) and Linoleic (18:2) acids.
Palmitic acid (11.73 – 32.04 %), stearic acid (1.09 – 2.66 %), oleic acid (12.01 – 39.67
%), Linoleic acid (42.59 – 47.43 %) and linolenic acid (1.70 – 5.71) were present when
analyzed by gas chromatography-mass spectrometry. In all cases polyunsaturated fatty
acids (PUFAs) were greater than monounsaturated fatty acids (MUFAs). The micro and
macro elements of different samples were analyzed by atomic absorption spectrometry
and flame photometer. The results showed that the higher mineral ion contents under
investigation were found in Mansehra sample i.e. 599.12 µg /g, whereas Abbotabad had
79
the lowest content, 242.63 µg/g. The total mineral ion contents was in the sequence of
Mansehra> Kotlisattian> Bagh> Abbotabad. Calcium (Ca2+) was the highest, ranging
from 456 to 187.33 µg/g and copper (Cu2+) was the lowest, ranging from 0.37 to 0.013
µg/g.
Two alkaloids, berberine and palmatine were isolated and purified and proton and
carbon signals were detected in 1H and 13C -NMR spectra. The analysis of the NMR
spectra of berberine and Palmatine revealed that the proton H-13 resonating as a singlet
(H-13 of 1: δ 8.72; H-13 of 2: δ 8.81) could be used for quantification. The 1H NMR
method used in this study was found to be simple, rapid and specific for the analysis of
protoberberine alkaloids. No reference compound was used, apart from the internal
standard, and an overall profile of the preparation was obtained directly. Using this
method the content of protoberberine alkaloids can be determined in Berberis lyceum and
other plant extracts in a shorter time than conventional method of HPLC.
Bioactivity of crude extract and Berberine of B. lyceum Royle was evaluated for
antimicrobial, antidiabetic and wound healing. For antimicrobial bioassay, root extracts
of B. lyceum prepared in two different solvents, methanol and aqueous and tested against
18 bacteria, 4 fungi and yeast strains. Antimicrobial activities were assessed by using
Disc diffusion method and Micro dilution assays. It was observed that methanol and
aqueous root extracts of berberis lyceum were highly effective against different bacteria
and fungi. The methanol extracts (135-260 µg/l) have inhibited growth of
microorganisms more effectively as compared to aqueous extract (120-230 µg/l). The
results obtained in present study indicates that root of B. lyceum contained some
photochemicals having antimicrobial activity and could be useful for pharmaceutical
80
industries for development of new drugs for human and animal health. The wound
healing activities of the aqueous and methanol extracts of the root of B. lyceum were
assessed using incision, excision and dead wound space models of wound repair in rats.
After application of both extracts it was observed that the area of epithelialization
increased, followed by an increase in wound contraction, skin breaking strength, tissue
granulation, dry weight and hydroxyproline content. Histopathological studies of the
granulation tissue also indicated that there was an increase in collagen formation in those
rats treated with the methanol extract, compared with the control group animals. The
methanol extract was more effective than the aqueous extract, but both showed
significant results compared with the control.
Berberine has been shown to have hypoglycaemic activity in several in vitro and
in vivo models, although the mechanism of action is not fully known. Berberis lyceum
Royle root produces high concentrations of berberine, and in traditional medicine, the
whole extract of this plant is used widely to treat diabetes. The antidiabetic activity of the
ethanol root extract of Berberis lyceum was compared with pure berberine in normal and
alloxan-diabetic rats using similar doses of each. The purpose of the study was to
investigate the effects of berberine and a whole extract of B. lyceum on blood glucose and
other parameters associated with diabetes, to compare the effects of the crude extract with
those of pure berberine and thus validate its use as a therapeutic agent, and finally to
identify any contribution of the other components of the extract to these effects. Oral
administration of 50 mg/kg of Berberis extract and berberine to normal and experimental
diabetic rats produced a significant (p < 0.05) reduction in blood glucose levels from days
3 –7 days of treatment. Significant effects were also observed on the glucose tolerance,
81
glycosylated haemoglobin, serum lipid profiles and body weight of experimental animals.
Berberis extract and berberine demonstrated similar effects on all parameters measured,
and although the extract was comparable in efficacy to berberine, it did not produce any
effects additional to those shown by pure berberine. The results support the use of the
extract in traditional medicine, and demonstrate that apart from being a highly cost-
effective means of treating with berberine, the total extract does not appear to confer any
additional benefits or disadvantages compared with the pure compound.
82
Chapter 6
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