2.0 LITERATURE REVIEW Literature Review
Literature search was carried out for types of DNA markers and their appHcations in herbal drug technology. Detailed description for RAPD-PCR and SCAR markers has been provided. Further, monograph of P. emblica and brief description for 6 Phyllanthus
species under study has been given.
2.1 DNA Markers^'
DNA markers are based on genetic polymorphism, which can be defined as the
simultaneous occurrence of a trait in the same population of two or more discontinuous
variants or genotypes. Although DNA sequencing is a straightforward approach for
identifying variations at a locus, it is expensive and laborious. A wide variety of
techniques have, therefore, been developed for visualizing DNA sequence polymorphism.
The techniques produce bar-code-like DNA fragment patterns generated by multilocus
probes after electrophoretic separation of genomic DNA fragments. The emerging
patterns make an unique feature of the analyzed individual and are currently considered
to be the ultimate tool for biological individualization. This technique so called DNA
fingerprinting has found its utility in characterization of genetic variability, genome
fingerprinting, genome mapping, gene localization, analysis of genome evolution,
population genetics, taxonomy, plant breeding, and diagnostics.
15 Literature Review
2.1.1 Properties Desirable for Ideal DNA Mariners .52
• Highly polymorphic nature
• Co-dominant inheritance (determination of homozygous and heterozygous states
of diploid organisms)
• Selective neutral behavior (the DNA sequences of any organism are neutral to
environmental conditions or management practices)
• Easy access (availability)
• Easy and fast assay
• High reproducibility
• Easy exchange of data between laboratories.
2.1.2 Types of DNA Markers used in Plant Genome Analysis .33
Various types of DNA based molecular markers are utilized to evaluate DNA polymorphism and are generally classified as hybridization-based markers and
Polymerase Chain Reaction (PCR)-based markers and sequencing based markers.
a. Hybridization-based Markers
Hybridization-based markers include Restriction Fragment Length Polymorphism
(RFLP) and Variable Number Tandem Repeats (VNTR) loci where probes such as random genomic clones, cDNA clones, probes for microsatellite and minisatellite sequences are hybridized to filters containing DNA which has been digested with restriction enzymes.
16 Literature Review b. PCR-based Markers
These are fingerprinting techniques that use the polymerase chain reaction (PCR) to specifically amplify a multipHcity of target sites in one or more nucleic acid molecules.
PCR-based markers can be random or specific depending upon the type of primer used, the stringency of the PCR conditions and the method of fragment separation and detection. Random PCR markers include Random Amplified Polymorphic DNA
(RAPD), Arbitrarily Primed PCR (AP-PCR) and DNA Amplification Fingerprinting
(DAF). Specific PCR-based markers include Inter Simple Sequence Repeats (ISSRs),
which is a PCR based marker for genome analysis where a terminally anchored primer
specific to a particular SSR is used to amplify the DNA between two opposed SSRs of
the same type. A recent approach known as Amplified Fragment Length Polymorphism
(AFLP) is a technique that is based on the detection of genomic restriction fragments by
PCR amplification and can be used for DNAs of any origin or complexity.
c. Sequencing -based Markers
DNA sequencing is a definitive means for identifying TCM. Further, variation due to
transversion, insertion or deletion can be assessed directly and information on a defined
locus can be obtained.
2.2 Applications of Molecular Markers in Herbal Drug Technology:
DNA based molecular markers have proved their utility in the fields like taxonomy,
physiology, embryology, genetics etc. As the science of plant genetics progressed
researchers have tried to explore these molecular marker techniques for their applications
17 Literature Review
in commercially important plants such as food crops, horticultural plants etc and recently
in pharmacognostic characterization of herbal medicine.
2.2.1 Genetic Variation / Genotyping
It has been well documented that geographical conditions affect the active constituents of
the medicinal plant and hence their activity profiles . Many researchers have studied
geographical variation at the genetic level. Estimates of genetic diversity are also
important in designing crop improvement programs, for management of germplasm and
evolving conservation strategies. RAPD based molecular markers have been found to be
useful in differentiating different accessions of Taxus wallichiana^^, neem^^, Juniperus
communis L. , Codonopsis pilosula , Allium schoenoprasum L. , Andrographis paniculata Scutellaria baicalensis Morinda officinalis Tinospora cordifolia (Willd.)
Miers''^ collected fi"om different geographical regions. Similarly, different accessions of
Cannabis sativa^^ have been discriminated using ISSR markers and those of Arabidopsis
thaliana L. Heynh.^'* have been differentiated using Cleaved Amplified Polymorphic
Sequence (CAPS) and ISSR markers. Inter and intra species variation has also been
studied using DNA based molecular markers. Interspecies variation has been studied
using RFLP and RAPD in different genus such as Glycerrhiza^^, Echinacea^^, Curcuma
^^ and Arabidopsis^^. RAPD and RFLP have also been applied for characterization of
Epimedium ^^ species at the genetic level. Members of three different species of
Scutellaria^'^, Chinese medicinal plants and three subspecies of Melissa officinalis have
been discriminated using RAPD. Varietal characterization of Kenaf {Hibiscus cannabinus
L.) ^^has been done with the help of agronomical and RAPD data. Varietal identification
18 Literature Review and genetic purity test in Pepper and Capsicum annuum were carried out using RAPD markers . RPLP technique was used for interspecific genetic variation within the genus
Capsicum and also for DNA fingerprinting of pepper cultivars^"*. RAPD has served as a tool for the detection of variability in Jojoba {Simmondsia chinensis L. Schneider)^^ Vitis
vinifera L. and tea {Camellia sinesisf. Attempt has been made to understand population structure of Podophyllum peltatum to establish commercial level propagation of useful secondary metabolites using molecular markers^^. Also, high genetic diversity has been shown in Podophyllum hexandrum species fi'om Himachal Pradesh, India^'^.
Genetic variation and relationships among and within Withania species^" Chinese
O 1
Carthamus tinctorius L. and genetic relationships among Papaya and its wild relatives
(Caricaceae) have been revealed using AFLP markers. Genetic variation within
Brassica campestris cultivars has been studied using AFLP and RAPD markers^''.
Phylogenetic relationship has been studied among citrus and its relatives using SSR
markers . RAPD has been used to construct genetic linkage maps of Eucalyptus grandis
and Eucalyptus urophylla . An attempt has been made to develop a physical AFLP map
of the complex Arabidopsis genome by combining gel-based AFLP analysis with in silico
restriction fi-agment analysis using the published genome sequence^^.
2.2.2 Authentication of Medicinal Plants
DNA based techniques have widely been used for authentication of plant species of
medicinal importance. This is especially useful in case of those that are frequently
substituted or adulterated with other species or varieties that are morphologically and/or
phytochemically indistinguishable.
19 Literature Review
Dried fruit samples of Lycium barbarum were differentiated from its related species using
RAPD markers . RAPD technique has also been used for determining the components of a Chinese herbal prescription, yu-ping-feng san. In this study presence of three herbs
{Astragalus membanaceus (Fisch.) Bge., Ledebouriella seseloides Wolff, and
Atractylodes macrocephala Koidz) in the formulation have been detected using a single
RAPD primer^^. Gradient PCR technique was recently used identification of Panax species in multi component herbal formulation^^.
Three RAPD primers have been identified that could successfully discriminate between three species oi Atractylodes, from Chinese formulation purchased from local markets'*^.
In another study, three random primers were used to reveal the genetic variability of
Astragalus medicine materials sold in Taiwan market. SSCP analysis was also conducted on PCR products from the ITS-1 region of ribosomal DNA in order to differentiate the two Astragalus species^°. DAF has been used to identify the Chinese traditional
Medicine, Magnoliae officinalis, its counterfeits and substitutes^'. ISSR have been employed to authenticate eight populations of Dendrobium officinale ^^. A RAPD primer that is selective for an elite strain Aizu K-111 of Panax ginseng including its cultured tissues has been identified^^. RAPD and PCR-RFLP analysis have been used for authentication of Panax ginseng among ginseng populations'*''. Some researchers have used a new approach called Direct Amplification of Length Polymorphism (DALP) for authentication of Panax ginseng and Panax quinquefolius . Recently, RFLP markers
20 Literature Review have been developed for authentication of Plectranthus species, an herbal medicine popular in Brazil for its analgesic and dyspeptic activities'^.
2.2.3 Detection of Adulteration/Substitution
RAPD technique was adopted to identify eight types of dried Coptis rhizomes and one type of Picrorrhiza rhizome, a substitute for the former in the Chinese herbal market^^.
Panax ginseng is often substituted by P. quinquefolius (American ginseng). SCAR, AP-
PCR, RAPD and RFLP markers have been successfully applied for differentiation of these plants and to detect substitution by other closely related species'^' '^' ^'.
Characterization of Echinacea species and detection of possible adulterations has been
done by using RAPD technique'*^ DNA fingerprinting and polymorphism in the Chinese drug "Ku-Di-Dan" (Herba elephantopi) and its substitutes were studied using AP-PCR
and RAPD. The results were used for authentication of "Ku-Di-Dan" and its
substitutes"^*^. DNA fingerprinting of Taraxacum mongolicum (Herba taraxaci) and its
adulterants of six species of Compositeae was demonstrated using AP-PCR and
RAPD'°'. Bulb o{Fritillaria cirrhosa, an official drug of Chinese Pharmacopoeia (1995)
is commonly used as an antitussive and expectorant. It has often been adulterated with
similar bulbs of other related species. Specific DNA based primers have been designed
for authentication of F. cirrhosa at the genomic level'°^. Molecular marker that is
specific to medicinal Rhubarb based on chloroplast tmL/tmF sequence that is absent in
its adulterants has been identified'"^. DNA sequence of 5S-rRNA spacer domains was
used for genetic distinction of radix adenophorae, a traditional Chinese medicine fi-om its
adulterants' .
21 Literature Review
DNA sequence analysis of rDNA internal transcribed spacer (ITS) and PCR-RFLP were explored for their applications in differentiating four medicinal Codonopsis species from their related adulterants, Campanumoea javania and Platycodon grandiflorus. The technique allowed effective and reliable differentiation of Codonopsis from the adulterants^^.
2.2.4 Marker Assisted Selection of Desirable Chemotypes
Along with authentication of species identity, prediction of the concentration of active phytochemicals may be required for quality control in the use of plant materials for pharmaceutical purposes. Identification of DNA markers that can correlate DNA fingerprinting data with quantity of selected phytochemical markers associated with that particular plant would have extensive applications in Quality Control of raw materials.
AFLP analysis has been found to be useful in predicting phytochemical markers in cultivated Echinacea purpurea^^^ germplasm and some related wild species. RAPD fingerprint has been developed to support the chemotypic differences in oil quality of three different genotypes of Pelargonium graveolens^^^ and flavonoid composition of
Aconitum '°^ species. DNA profiling has been used to detect the phylogenetic relationship
1 OR among Acorus calamus chemotypes, differing in their essential oil composition .
Artemisia annua, source of antimalarial compound artemisinin shows variation in artemisinin content all over India. These chemotype variants of Artemisia annua L. have been characterized using RAPD markers. This study also revealed existence of very high levels of genetic variation in the Indian population despite geographical isolation and
22 Literature Review opens out a possibility of further genetic improvement for superior artemisinin content.
Attempt has also been made to study variation in essential oil components and interspecific variations using RAPD technique'°^. Morphological, chemical and genetic differences in twelve basil (Ocimum gratissimum L.) accessions were studied to determine whether volatile oil and flavonoids can be used as taxonomical markers and to examine the relation between RAPDs to these chemical markers''°.
2.2.5 Medicinal Plant Breeding
ISSR-PCR has been found to be a very efficient and reliable technique for the identification of zygotic plantlets in citrus interploid crosses'''. Molecular markers have been used as a tool to verify sexual and apomictic offspring of intraspecific crosses in
Hypericum perforatum, a well known antihelminthic and diuretic"^. An attempt has been made towards marker-assisted selection of fertile clones of garlic with the help of RAPD markers' . RAPD markers have been successively used for selection of micropropogated plants oi Piper longum for conservation .
2.2.6 Applications in Foods and Nutraceuticals
DNA based molecular markers have been used extensively for a wide range of
applications in food crops and horticultural plants"^. These applications include study of genetic variation, cultivar identification, genotyping, cross breeding studies, identification of disease resistant genes, identification of quantitative trait loci, diversity analysis of
exotic germplasms, sex identification of dioecious plants, phylogenetic analysis, etc.
23 Literature Review
Recently, the application of DNA based molecular markers is being explored in the field ofnutraceuticals.
According to new European Council (EC) legislation'^^, the labeling of food or food ingredients produced from, or containing licensed Genetically Modified Organisms
(GMOs) must indicate the inclusion of these ingredients where they are present at or above a level of 1%. In compliance with the labeling regulation for GM foods, several countries in Europe such as Germany and Switzerland have extensively developed PCR methods for both identification and quantification purposes.
In response to reports of unlicensed GM ingredients in foods on the international market, the Food Safety Authority of Ireland (FSAI) has completed a survey to determine the levels of GM maize ingredients in tortilla chips and taco shells on sale in Ireland using the PCR technique"^. Where sufficient GM DNA was present in the sample, quanfitative
analysis was undertaken using Real-Time PCR.
Primers specific for inserted genes in the Roundup Ready^"^ soybean have been found to be suitable for detection and discrimination of GM soybean from non-GM products . In
another study. Roundup Ready Soybeans, Bt 176 maize and Cecropin D capsicum have
been successfully discriminated from non-GM products using primers specific for
inserted genes and crop endogenous genes"^.
24 Literature Review
2.3 RAPD-PCR'^°
In 1991 Williams et al developed a new PCR-based genetic assay namely random amplified polymorphic DNA (RAPD). This method is also called as AP-PCR (Arbitrarily
Primed Polymerase Chain reaction). This procedure detects nucleotide sequence polymorphisms in DNA by using a single primer of arbitrary nucleotide sequence. In this reaction, a single species of primer anneals to the genomic DNA at two different sites on complementary strands of DNA template. Each product is derived from a region of the genome that contains two short segments in inverted orientation, on opposite strands that are complementary to the primer and sufficiently close together for the amplification to work. If these priming sites are within an amplifiable range of each other, a discrete DNA product is formed through thermocyclic amplification. The amplification products are separated on agarose gels in the presence of ethidium bromide and visualized under ultraviolet light. On an average, each primer directs amplification of several discrete loci in the genome, making the assay usefiil for efficient screening of nucleotide sequence polymorphism between individuals including plants, microbes, animals and humans''^
The application of RAPDs in quality control and standardization of medicinal plants has
largely been explored due to its several advantages. The technique is simple to perform, cost effecfive and only a small amount of DNA is required for amplification. In addition, it reveals high degree of polymorphism and does not require prior DNA sequence
information. However, due to the stochastic nature of DNA amplification with random
sequence primers, it is important to optimize and maintain consistent reaction conditions
for reproducible DNA amplification. Further, problems such as poor reproducibility,
faint or fiazzy products, and difficulty in scoring bands, which lead to inappropriate
25 Literature Review inferences limits its applications and demands development of more specific and reproducible markers such as SCAR.
2.4 SCAR
Michelmore et al}^^ and Martin et al}^^ introduced the SCAR (Sequence Characterized
Amplified Regions) for amplification of specific band. In this technique the RAPD bands are purified, cloned and sequenced (Fig.2). Based on the sequence of the amplicon specific primers that could amplify the desired DNA region are designed. Generally longer primers of length 22-24 nucleotide bases are designed for specificity. These are similar to STS markers in construction and application. The presence or absence of the band indicates variation in sequence. These are better reproducible than RAPDs. SCARs are usually dominant markers, however, some of them can be converted into co dominant markers by digesting them with tetra cutting restriction enzymes and polymorphism can be deduced by either denaturing gel electrophoresis or SSCP. Compared to arbitrary primers, SCARs exhibit several advantages in mapping studies (codominant SCARs are
informative for genetic mapping than dominant RAPDs), map-based cloning as they can be used to screen pooled genomic libraries by PCR, physical mapping, locus specificity,
etc. SCARs also allow comparative mapping or homology studies among related species,
thus making it an extremely adaptable concept in the near future.
26 Literature Review
RAPD-SCAR
5, I I I I I I I I I I I I I I I I I I I I I 3' 5' I I I I I I I I I I I I I I 3' 3 5' 3' 5' i -3' 5' _5'
"5' 3' "3' i
B B
M 1 2
Fig 2. Schematic diagram of the RAPD-SCAR marker development.
27 Literature Review
2.5 Plants under study
2.5.1 Phyllanthus emblica Linn.
i ; • ••... - ^ --? ; • •
* i
Fig. 3 a. Twig of the P. emblica plant Fig. 3 b. Fresh fruits of P. emblica
A. Definition^"
Amla (Indian gooseberry) consists of dried fruit rinds of Phyllanthus emblica Linn (Syn-
Emblica officinalis Gartn.) Family- Euphorbiaceae
B. Selected Vernacular names^^
English- Indian gooseberry
Sanskrit- Amalaka, dhatri, adiphala
Marathi- Avala, Hindi- Amla, aonla
C. Plant description^^'*
The tree is deciduous normally reaches a height of 60 ft. Its fairly smooth bark is pale grayish brown and peels off in thin flakes. The miniature, oblong leaves 3 mm wide and
1-2 cm long distichously deposited on very slender branchlets, giving a misleading impression of finely pinnate foliage. Its flowers are monoecious having greenish yellow
28 Literature Review color. They grow in auxiliary clusters and start appearing in the beginning of spring
season.
D. Habitat'^^
It is found in both wild and cultivate state. It is common in the mixed deciduous forests of
India, 1300m on the hills.
E. Plant Part of Interest: Dried fruit rinds
F. Morphology'^^
Fresh fruit globose, depressed, shine yellowish green when ripe. Six vertical furrows are
distinct. Seeds trigonous, two each in three crustaceous cocci. The dried fruit shows a
broad, highly shriveled and wrinkled external convex surface, lateral surface transversely
wrinkled, external surface exhibit few white specks, occasionally some pieces show a
portion of stony testa.
G. Microscopic characteristics'^^
The epicarpic cells are rectangular in shape and their outer and radial walls are highly
cuticularized. In surface view the epicarpic cells appear polygonal in outline with thick
walls. Few anomocytic stomata can be located. Collateral fibrovascular bundles are
scattered through out the inner mesocarp. Pitted and helical tracheids with tapering ends
are seen. At places in the phloem, large cavities filled with crystal mass are present.
H. Cultivars (See Tablel)
29 i-o 60 S e c O in o 2 ON o •3 so o ^ ^ S <^ 't 'c3 00 ON — ON < O ^ o rn r^ ro OH (N Of c O B O in ^ o 13 c^ m m ^ 00 fN ^ '3 S "* (A R U R B o -a B in o ^ o in 3 't l-H f^ (N i «« >n ^ d 'S C3 r-~ o 00 ^ o d O f^ ^ in \D OH d '^ '^ (N 't H B o _2 O lO 1—< R so r<^ m •^ > X) OS a\(N p "(SU t-- ON rS •c3 ^ m SO 1^ ^ d d O O r R O o (U r<~i ro r<-) ^3 00 O o • »-H )-H VO ^ o R m r<-i . o IZ ^ d d U ro '*" SO OH d '^ ro (N in S T3 o O 3 I m 13 ^ r-i ;-H r- TT 'O so O p '3 ON > X) in •c3 r-- r4 . (N Z ^ d, ci O O rn -^ ro SO OH d Tf m I. Purity tests Foreign matter: Not more than 3% Total ash: Not more than 5% Acid soluble ash: Not more than 2% Alcohol soluble extractive: Not less than 40% Water-soluble extractive: Not less than 40% J. Chemistry'" Chemical constituents Major Vitamin C (L-(+) - threo-ascorbic acid, (-2%). Tannins (-5%) Viz., gallic acid, ellagic acid, phyllembUc acid and emblicol Minor Alkaloids viz., phyllantidine and phyllantine; pectin and minerals. Recently, six new phenolic constituents, L-malic acid 2-0- (1), mucic acid 2-0- (5), mucic acid 1,4-lactone 2-0- (6), 5-0- (8), 3-0- (10), and 3,5-di-O- (11) gallates, were isolated from the fioiit juice of P. emhlica together with their methyl esters . Six elagitannins, phyllanemblinins A-F are also been identified recently'^^.Other known constituents are listed in the table. 31 Literature Review Table2: <[Chemica l constituents isolated from different parts of P. emblica Sr. Class of Compounds Name of compounds Occurrence No. 1. Alkaloid Phyllantine Leaves, fruit Phyllantidine Zeatin Leaves Zeatin nucleotide Fruit Zeatin riboside 2. Benzenoid Chebulic acid Leaves Chebulinic acid Chebulagic acid Gallic acid Leaves, fruits EUagic acid Amlaic acid Corilagin 3-6-di-O-galloyl-glucose Ethyl gal late 6 -glucogallin Putranjivain A Digallic acid Phyllemblic acid Emblicol Music (=galactaric) acid 3. Furanolactone Ascorbic acid Fruit, leaves 4. Diterpene Gibberellin A-1 Fruits Gibberellin A-3 Gibberellin A-4 Gibberellin A-7 Gibberellin A-9 5. Triterpene Lupeol Fruit, leaves 6. Flavonoid Leucodelphinidin Kaempherol Kaempherol-3-glucoside Quercetin Kaempherol-3-O-B-DgIucoside Quercetin-3-O-B-D-glucoside 7. Sterol 6-sitosterol Leaves 8. Carbohydrate Acidic and neutral Leaves polysaccharides 32 Literature Review K. Pharmacology a. Experimental Pharmacology i. Antioxidant Activity Extract of fresh P. emblica fruits inhibits Upid peroxidation induced by gamma radiation in rat Uver microsomes. It also inhibited radiation-induced damage to the antioxidant enzyme superoxide dismutase in rat Uver mitochondria'^°. In an another in vitro study, tv^o low-molecular weight hydrolysable tannins, emblicanin A and B, were shown to provide dose-dependant protection against rat peripheral blood erythrocyte haemolysis induced by oxygen radicals'^'. P. emblica extract containing hydrolysable tannins (embUcanin A 37%, emblicanin B 33%, punigluconin 12% and pedunculagin 14%) has demonstrated antioxidant activity in an animal model'^^. It also ameliorates hyperthyroidism and hepatic lipid peroxidation in mice'^^. Fresh fruit homogenate causes myocardial adaptation and protects against oxidative stress in ischemic-reperfrision injury in rats'^^ ii. Antimicrobial and Anti-HIV activities Ether and alcoholic fioiit extracts acidified with hydrochloric acid have antibacterial activity. They inhibits growth of M. pyogenes var. S. typhosa and S. paratyphi at a concentration of 0.21 mg /ml and that of M pyogenes var. albus; S. schottmmellari and S. dysenteriae at a concentration of 0.42mg/ml'^^' '^''. The fruit of P. emblica contains a number of compounds of which ellagitannin, putranjivain have shown potent inhibitory activity against HIV reverse transcriptase (IC50=3.9mcM) 33 Literature Review iii. Cardioprotective and antiatherogenic activity Alcoholic extract of dried fruits (Ig/kg) has shown an increase in the cardiac glycogen and a decrease in serum GOT, GPT and LDH in isoprotenol pretreated rats, suggesting a cardioprotective action. It showed a reduction in serum cholesterol levels and a significant antiatherogenic effect '^^' '^^. The lipid lowering and antiatherosclerotic effects of amla fresh juice were evaluated in cholesterol fed rabbits (rendered hyperlipidemic by atherogenic diet and cholesterol feeding). Amla fresh iuice significantly reduces serum cholesterol, Triglycerides, phospholipid and Low-density lipoprotein levels. Similarly, the tissue lipid level showed a significant reduction following amla juice administration. Aortic plaques were regressed. Amla juice treated rabbits exerted more cholesterol and phospholipids, suggesting that the mode of absorption be affected. Amla juice is an effective hypolipidemic agent and can be used as a pharmaceutical tool in hyperlipidemic subjects'^°''^'. iv. Antidiabetic activity Relieving oxidative stress and improving glucose metabolism are the probable mechanisms for antidiabetic activity of amla'"*^. Aqueous extract and tannoid principles show inhibition of aldose reductase, a drug target involved in development of secondary complications of diabetes including cataract in experimental system' ' v. Anti tumor activity Aqueous extract of P. emblica fruit was shown to protect mice against the chromosome damaging effects of the well-known carcinogen 3, 4-benzo (a) pyrene . An anti-tumor effect of a P. emblica aqueous fruit extract was demonstrated in tumor-bearing mice, 34 Literature Review resulting in a 35 percent increase in life span. The anti-tumor activity was shown to be meditated primarily through enhanced natural killer cell activity and antibody-dependent cellular cytotoxicity Another study showed an aqueous extract of P. emblica to significantly reduce induced solid tumors in mice in a manner suggesting interaction with cell cycle regulation"*^. Extracts of P. emblica fruit inhibited the proliferation of four human tumor cell lines in vitro^'^^. Pyrogallol was identified as an active component of the extracts. An aqueous extract of the dried fruits of P. emblica protected mice against the effects of nickel chloride'''^. Nickel is a major environmental pollutant with carcinogenic potential. Fruit extract displays anti-proliferative activity on breast cancer cell lines. It also induces an increase of ER alpha mRNA in MCF7 cells''*^. The fruits have considerable anticancer against chromium (IV) induced oxidative injury in murine macrophage'^ . vi. Immunomodulation 25mg/kg IP of water decoction shows immunomodulatory properties evaluated in adjuvant induced arthritic (AIA) rat model"*^. Also, ethanolic extract shows significant antioxidant and immunomodulatory properties in-vitro'^'" '^^. Researchers have also shown that it is a potent immune-stimulator, through its activation of natural killer (NK) cell activity'"*^. 35 Literature Review vii. Nervous system Phyllembin, isolated from the ethanolic extract of the fiaiit pulp has been found to potentiate the action of adrenaline in vitro and in vivo. It showed a mild depressant action on Central Nervous System and also had a spasmolytic activity. The drug also revealed mild stimulant action on isolated frog heart, short and insignificant rise in cat's blood pressure, contraction of the nictitating membrane, the reduction of outflow of the perfusate in the hind limb of the rat and ear of rabbit, mild cerebral depressant action and anti-spasmodic activity. Of the indirect actions, potentiation of the action of adrenaline on the blood pressure of cat, isolated frog heart, and nictitating membrane of cat and the prolongation of the hypnosis were observed'^^ Further studies on the action of phyllemblin revealed that the drug antagonized the spasmogenic effect of acetylcholine, bradykinin and serotonin on the guinea pig ileum. It also antagonized serotonin and acetylcholine-induced contractions of oestrogenised rat uterus. It increased the amplitude of cardiac contraction and heart rate transiently. An increase in coronary flow was followed by persistent decrease. On perfused rat hind limb and rabbit ear preparation, phyllemblin in small doses, increased the amount of perfusate whereas in larger doses it decreased the flow significantly. A triphasic response that is initial transient rise, followed by a transient fall and then sustained rise in blood pressure was seen in anaesthetized albino rats. The sustained rise was blocked by phentolamine (Img/kg.). The drug produced 80 percent protection against leptazol seizures in mice. It protected effectively against tremors and tonic convulsions induced by nicotine. It also antagonized tremorine-induced tremors and other cholinergic symptoms'^'*. 36 Literature Review viii. Other activities antitussive activity'^^ anti-peptic ulcer'^%odifying the acute cytotoxicity of cadmium'" and arsenic'^^ Anti-inflammatory activities'^^. The methanoHc root extracts was explored for antisnake venom activity'^'^. b. Clinical Pharmacology The effect on total serum cholesterol and its lipoprotein fractions of supplementation of the diet with amla was studied in normal and hypercholesterolaemic men aged 35-55 years. Both normal and hypercholesterolaemic subjects showed a decrease in cholesterol levels . During a clinical trial, an Ayurvedic preparation (amalaki rasayana (chiefly amla fruits) on 39 cases of duodenal ulcer and 21 cases of non-ulcer dyspepsia was found to be very effective in 72 per cent patients with no recurrence of signs and symptoms for a period of 6 months to three years of follow up observations'^^. L. General Uses Spasmlytic, anti-inflammatory, hypolipidaemic, antiulcer. M. Toxicity Phyllemblin was well tolerated by mice in doses upto 100 mg/kg when given intraperitoneally in mice and up to 500 mg/kg when given orally. At doses above 500 mg/kg animals appeared drowsy and dull soon after the injection and were active again after 1-3 hours. 37 Literature Review N. Commercial reports Retail market price- dried fruit powder-Rs.300/- per kg. Fresh Fruits -Rs. 60/- per kg. 2.5.2 Phyllanthus distichus Linn.'^^ Synonyms: Phyllanthus acidus L, Cicca disticha L. (Euphorbiaceae) Common name: Star gooseberry, Tahitian gooseberry, Malay gooseberry. The plant bears pale-yellow fruit, 3/4 - 1" thick and is 6-8 ridged. It is sour and astringent in taste and used as liver tonic; useful in thirst, biliousness, vomiting and constipation. Further, it has been found useful in treatment of cystic fibrosis. 2.5.3 Phyllanthus reticulatus Poir.'" Family: Euphorbiaceae. Fruit is a berry, globose to oblate, ca. 4 - 6 mm across, black with dark purplish at maturity, 4- to 12-celled. It is astringent and useful in inflammation and also used as blood purifier. The botanical also has diuretic activity. It is used in treatment of diarrhea in infants. The leaves and roots are used as medicine for the fractures and traumatic injury. I f\'\ 2.5.4 Phyllanthus urinaria Linn. Synonym:P. leprocarpus Family: Euphorbiaceae The plant is bitter, tonic, astringent and febrifuge. It is much useful as a diuretic in dropscial affections. It is also useful in gonorrhea and other genito-urinary conditions. The roots are usefril in insomnia. It is prescribed in dysentery and cystitis. The Fruits are 38 Literature Review acrid, cooling, bitter useful in thirst, bronchitis, leprosy, anemia, asthma and hiccough, geraniin and 1,3,4,6-tetra-O-galloyl-beta-d-glucose isolated from P. urinaria have shown significant antiviral activity against herpes simplex virus type 1 and type 2 infection. 2.5.5 Phyllanthus simplex Retz.'^^ Family: Euphorbiaceae The fruits and leaves of this plant have been mainly used for treatment of gonorrhea along with other constituents like cumin seeds. It is also useful in cure itching disorders. The chemical constituents namely brevifolin and 8, 9-single-epoxy brevifolin have significant hepatoprotective activity. The roots are useful in mammary abscess. 2.5.6 Phyllanthus niruri Linn. Family: Euphorbiaceae Phyllanthus niruri has a long history in traditional medicine systems worldwide. The whole plant and its aerial parts are used for many remedies, mostly biliary and urinary. Some examples are kidney and gallbladder stones, hepatitis, colds, flu, tuberculosis, and other viral infections. It has also been proven effective in liver diseases like jaundice and Hver cancer. It is sometimes used for bacterial infections such as cystitis, prostatitis, venereal diseases, and urinary tract infections. It can also assist in reducing anemia symptoms, diabetes and hypertension. It is well known for its diuretic, analgesic, stomachic, antispasmodic, febrifugal, and cell protective properties. It also helps decrease the amount of hepatitis B virus found in the blood stream. 39 Literature Review 2.5.7 Phyllanthus indofischeri Bennet.'^'* Family: Euphorbiaceae The plant specifically grows in scrub forest and stunted low altitude dry deciduous forest. It is endemic to Deccan Plateau and certain drier forest pockets of southern India. Fruits of the plant are of marble green color. Diameter ranges between 2.5 and 4 cm. Groove markings along the septa are well developed. They are often used as substitute for P. emblica. Ih\ wa^^o 40 n I 00 1^ r- r-- m -^ X) ^ -1 d '3 S (N o OH 00 ^ (N r^i in ao (U S • I-H o ^3 in o ^ in in D. "O < (^ -^ Xl C U '^ ^ ^ •n so ON ^ d d O rS -^ '^ SO ^ s d in (^ (N in