Chapter 3 Introduction to Plants and Review of Literature 3....

Chapter 3 Introduction to Plants and Review of Literature

KLE University’s College of Pharmacy, Belgaum-590010. 9

3. INTRODUCTION TO PLANTS AND REVIEW OF LITERATURE

3.1 Introduction to Hemidesmus indicus R. Br.

Family: Asclepidaceae

Fig. 3.1: Photograph of Hemidesmus indicus R. Br.

[Inset showing pieces of dried roots]

Taxonomic Classification:14-16

Kingdom : Plantae

Phylum / Division : Magnoliophyta

Subphylum / Subdivision : Magnoliophytina

Class : Magnoliopsida

Subclass : Magnolidae

Order : Gentianales

Suborder : Gentianineae

Family : Periplocaceae

Subfamily : Asclepiadoideae

Genus : Hemidesmus

Species : Indicus

Synonym : Periploca indica

Distribution:16

H. indicus is a climbing twiner plant, found throughout India, more commonly in

Bengal, Bombay presidency and extending to Travancore and Ceylon.



Vernacular names of Plant:14-17

English : Indian Sarsaparilla

Hindi : Magrabu

Marathi : Lahankavali

Kannada : Namada-beru

Sanskrit : Anantmul

Malayalam : Nannari

Tamil : Arakkam

Morphologial Characteristics:14,16,17

Leaves Leaves are variable, from elliptic-oblong to linear lanceolate, 5 to 10

cm long, apiculate. Narrow leaves are acute and broad ones are often

obtuse at the apex, dark green and with reticulate veins.

Stem It has numerous slender stems, which are terete, glabrous or pubescent

and thickened at the nodes.

Flowers Flowers are crowded in subsessile cymes in the opposite axils.

Seeds Seeds are black, 6 to 8 cm long, ovate-oblong, flattened with silvery

white, 2.5 cm long coma.

Roots Roots are cylindrical in shape, irregularly bent, curved or slightly

twisted.

Chemical constituents:14-20

Roots of H. indicus are reported to contain chemical constituents like - an essential

oil containing 80% of 2-hydroxy 4-methoxy benzaldehyde, a ketone, fatty acids, saponin,

tannins, resinal fractions, resin acids, sterols, -sitosterol, stigmasterol and sarsapic acid.

Hemidesmin 1, hemidesmin 2, alpha-amyrin, beta-amyrin, lupeol and 2-hydroxy-

4-methoxy benzoic acid have been isolated and identified from roots of H. indicus.

Therapeutic uses:14-20

Root is sweet, cooling and demulcent. It is used as tonic, diuretic and aphrodisiac.

Whole root and root-bark are useful in syphilis, leucoderma, hemicrania, rheumatism and

in diseases of liver and kidney. There is plethora of evidence that many Indian tribes use

roots of H. indicus as a traditional medicine for a wide range of ailments including

nutritional disorders, skin diseases, gravel and other urinary problems. Powdered root



mixed with cow’s milk is given with much benefit in cases of scanty and highly coloured

urine and in those of gravel and strangury.

Review of literature (till date):

A thorough literature survey on H. indicus was carried out from chemical

abstracts, biological abstracts, textbooks, national and international journals, herbal

databases from Internet and other published research materials. Till date, following

phytochemical and pharmacological studies have been carried out on H. indicus.

Fig. 3.2: Phytoconstituents isolated from dried twigs of H. indicus [Desinine (1), its

deacetylated product (2), Desinine monoglycoside (3) and hydrolytic products of

desinine including acetyl genin (4), deacetyl genin (5), sugar moiety identified as D-

Oleandrose (6) and its lactone (7) and D-Oleandronic acid phenylhydrazide (8)]



Oberai K et al. (1985) isolated a new pregnane ester diglycoside, Desinine

(C37H58O12, m.p. 115-118 degrees) from the dried twigs of H. indicus. Its structure was

identified as drevogenin B-3-O-beta-D-oleandropyranosyl-(1-4)-beta-D-

oleandropyranoside21

.

Rao RVK et al. (1989) carried out a comparative study of constituents of

Sarsaparillas (H. indicus and Decalepis hemiltonii) from Indian market and reported that

there were clear differences in their chemical and anatomical characters. D. hemiltonii

contains beta-amyrin, 2-hydroxy, 4-methoxy benzaldehyde and ferulic acid, whereas,

ferulic acid is absent in H. indicus; but, it contains flavonoids22

.

Prakash K et al. (1991) isolated two new pregnane glycosides (indicin and

heminine) from the dried stems of H. indicus and elucidated their structures as calogenin-

3-O-beta-D-digitoxopyranoside and calogenin-3-O-beta-D-boivinopyranoside,

respectively23

.

Mandal S et al. (1991) isolated and identified a new coumarino-lignoid,

Hemidesminine from H. indicus24

.

Das P et al. (1992) isolated and identified two new coumarino-lignoids

hemidesmin-1 and hemidesmin-2 from the roots of H. indicus25

.

Desh D et al. (1992) isolated and elucidated a new pregnane glycoside, hemidesine

from chloroform and chloroform-alcohol 4:1 extracts of H. indicus. The structure of

hemidesine, a diglycoside (C36H58O11, m.p. 148 degree C) was confirmed as (20-O-acetyl-

calogenin-3-O-beta-D-oleandropyranosyl(1 to 4)-O-beta-D-digitoxopyronoside26

.

Desh D et al. (1992) isolated and elucidated a novel pregnane triglycoside namely

emidine, (C39H64O12, m.p. 192-196 degree C) from chloroform and chloroform-alcohol 4:1

extracts of H. indicus. The structure of emidine was confirmed as (20-O-acetyl-calogenin-

3-O-beta-D-digitoxopyronosyl(1 to 4)-O-beta-D-digitoxopyronoside27

.

Banerji A (1992) reported the studies on several Indian medicinal plants belonging

to the genera Piper, Ferula, Dioscorea and Hemidesmus for the isolation and

characterization of their constituents. A number of new amides, lignans,

coumarinolignoids, phenanthrene derivatives and sesquiterpenoid coumarin have been

isolated and identified28

.

Chandra R et al. (1994) isolated two novel pregnane glycosides, hemidescine and

emidine, from dried stem of H. indicus and elucidated their structures as, 20-O-acetyl

calogenin 3-O-beta-D-digitoxopyranosyl(1 to 4)-O-beta-D-oleandropyranoside and



calogenin 3-O-beta-D-digitoxopyranosyl(1 to 4)-O-beta-D-digitoxopyranosyl(1 to 4)-O-

beta-D-digitoxopyranoside, respectively29

.

Mandal S et al. (1995) described the detailed investigation in the isolation of three

new and novel coumarinolignoids viz., hemidesminin, hemidesmin-1 and hemidesmin-2.

The basic structure of these coumarinolignoids consists of a coumarin moiety which is

linked with a phenyl propanoid unit through a 1,4-dioxan bridge30

.

Desh D et al. (1995) isolated a novel pregnane oligoglycoside (Indicusin) from

Chloroform-Ethanol (3:2) fraction of H. indicus and elucidated it’s structure as 11alpha,

12beta-dO-acetyl-orgogenin-3-O-beta-D-cymaropyranosyl(1 to 4)-O-beta-D-

cymaroyranosyl(1 to 4)-O-beta-D-cymaropyranoside31

.

Desh D et al. (1997) isolated and identified structures of three new pregnane

oligoglycosides, medidesmine, hemisine and desmisine from H. indicus. The plants

belonging to Asclepiadaceae family are reported to be rich in pregnane and cardiac

glycosides32

.

Sigler P et al. (2000) isolated two novel pregnane glycosides viz. denicunine and

heminine, from dried stem of H. indicus. Chemical transformations and spectroscopic

evidences revealed their structures as calogenin 3-O-3-O-methyl-beta-D-fucopyranosyl-(1-

4)-O-beta-D-oleandropyranoside and calogenin 3-O-beta-D-cymaropyranosyl-(1-4)-O-

beta-D-digitoxopyranoside, respectively33

.

Fig. 3.3: Phytoconstituents isolated from dried stem of H. indicus [Denicunine (1),

calogenin (2) and its acetylated derivative (3), Heminine (4) and its acetylated

derivative (5)]

Roy SK et al. (2002) carried out phytochemical studies on roots of H. indicus and

isolated a acyclic triterpenic acid, acyclic diterpenic ester and monocyclic sesterpene ester

and elucidated their structures based on spectral and chemical data34

.



Sen T et al. (2003) isolated and elucidated the structure of new pentacyclic

triterpene ester, alpha-Amyrin-3-acetate from roots of H. indicus R. Br35

.

Gupta MM et al. (1992) isolated and characterized a new terpene lactone, 3-keto-

lup-12-ene-21 to 28-olide from the hexane soluble fraction of ethanol extract of stem of H.

indicus. Further, lupanone, delta12-dehydrolupanyl-3beta-acetate, delta12-dehydrolupeol

acetate, hexadecanoic acid, 4-hydroxy-3-methoxybenzaldehyde and 3-hydroxy-4-

methoxybenzaldehyde were also isolated for the first time from this plant36

.

Fig. 3.4: 2-hydroxy-4-methoxybenzaldehyde isolated from H. indicus roots

Nagarajan S et al. (2001) reported that volatile oil constituents from H. indicus

roots were obtained by steam distillation (yield, 0.25%) as 2-hydroxy-4-

methoxybenzaldehyde (91%) and (-)-ledol (4.5%), isolable in pure form as major

constituents. About 40 minor constituents from the residual oil were identified including

nerolidol (1.2%), borneol (0.3%), linalyl acetate (0.2%), dihydrocarvyl acetate (0.1%),

salicylaldehyde (0.1%), isocaryophyllene (0.1%) and traces of alpha-terpinyl acetate and

1,8-cineol37

.

Gomes A (2004) isolated and identified two pure compounds (2-OH-4-MeO-

benzoic acid) from roots of H. indicus and one compound (beta-sitosterol) from root

extract of Pluchea indica and studied these compounds for their snake venom

neutralization properties (lethal, haemorrhage, oedema, PLA2, cardiotoxic and neurotoxic).

These compounds potentiated the action of the polyvalent snake venom antiserum in

experimental animals and showed antioxidant property through lipid peroxidation and

SOD activity38

.

Banerji A et al. (1992) studied extensively Indian Sarsaparilla, (H. indicus) for its

chemical constituents and pharmacological properties as blood purifier and anti-rheumatic

agent39

.

Alam MI et al. (1996) evaluated methanol extracts of roots of H. indicus and

Pluchea indica for neutralization of snake venom (Viper russellii) activity. Maximum

neutralization was achieved by H. indicus root extract40

.



Hiremath SP et al. (1997) evaluated in vitro antimicrobial activity of different

extracts of Striga sulphurea (Scrophulariaceae) and H. indicus (Asclepiadaceae) against

Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Aspergillus niger.

The chloroform and ethanol (95%) extracts of H. indicus showed antifungal activity

against A. niger41

.

Pandey KK et al. (2001) conducted clinical trials of “RENALKA” syrup

[containing extracts of Tribulus terrestris, Crataeva magna, H. indicus, Cyperus rotundus,

Vetiveria zizanoides, Asparagus racemosus and Elletaria cardamomum and Trikatu] for

effectiveness in curing and relieving the symptoms associated with UTI. The drug was

found to be safe and effective against E. coli, B. proteus, Klebsiealla and Pseudomonas42

.

Austin A et al. (2003) studied antimicrobial activity of hot and cold aqueous and

acetone, chloroform and methanol extracts of flowering and vegetative period samples of

Hemidesmus indicus var. indicus R. Br. against human isolates of Helicobacter pylori.

Extracts from samples collected during flowering period were better than that of

vegetative43

.

Das S et al. (2003) studied effect of methanolic extract of H. indicus roots against

Salmonella typhimurium, Escherichia coli and Salmonella flexneri, in vitro and in

experimentally induced diarrhea in albino rats, in vivo. It showed significant

antienterobacterial and antidiarrhoeal effects44

.

Prabhakaran M et al. (2000) evaluated the protective effect of ethanolic extract of

H. indicus roots (100 mg/kg, orally, for 15 days) in rats against rifampicin and isoniazid-

induced hepatotoxicity. The significant activity has been attributed to a free radical

scavenging activity of the coumarino 73 lignoids present in the extract45

.

Ravishankara MN et al. (2002) evaluated antioxidant activity of methanolic extract

of H. indicus root bark in several in vitro and ex vivo models (like radical scavenging

activity by DPPH reduction, superoxide radical scavenging activity in riboflavin/light/NBT

system, nitric oxide radical scavenging activity in sodium nitroprusside/greiss reagent

system and inhibition of lipid peroxidation induced by iron-ADP-ascorbate in liver

homogenate and phenyl hydrazine induced haemolysis in erythrocyte membrane

stabilization study)46

.

Anoop A et al. (2003) evaluated the antiulcerogenic property of aqueous ethanolic

extracts of the roots of H. indicus var. indicus in animal models like, modified pyloric

ligated (Shay) rat model and aspirin induced ulcerogenesis in pylorus ligated rat model.

Evaluation of parameters like, gastric volume, ulcer score, pH, free and total acidity,



sodium and potassium ion output and biochemical estimations like total proteins, total

hexoses, hexosamines, facose, sialic acid and pepsin were carried out47

.

Austin A et al. (2003) evaluated the antiulcerogenic property of 50% ethanolic

extracts of the roots of H. indicus var. pubescens R. Br. (Perioplocaceae) in animal models

as a muco-protective agent48

.

Austin A et al. (2002) conducted toxicity studies of crude aqueous, ethanolic

extracts (1000 to 4000 mg/kg dose level) of the roots of H. indicus var. pubescens by oral

and intraperitoneal administration. It was found to possess nonspecific changes in liver49

.

Evans DA et al. (2004) studied the antidiarrhoeal property of aqueous extract (5 or

10 mg/sac) of roots of H. indicus. The effect of aqueous extract was not affected by heat at

100 degree fro 30 min. The study indicates that H. indicus root powder or its aqueous

extract can be incorporated in oral rehydrating salt solution for increasing its anti-

diarrhoeal efficacy50

.

Verma PR et al. (2005) studied the ethanolic extract of roots of H. indicus for its

antinociceptive effect in mice. Oral administration of H. indicus root extracts exhibited a

dose dependent antinociceptive activity in all models and it blocked both neurogenic and

inflammatory pain51

.

Shetty TK et al. (2005) studied the radioprotective effect of H. indicus (Anantmul)

root extract on lipid peroxidation in rat liver microsomes and plasmid DNA. Anantmul

root extract was found to protect microsomal membranes as evident from reduced lipid

peroxidation and it could also protect DNA from radiation induced strand breaks52

.

Lakshman K et al. (2005) studied the comparative anti-inflammatory activity of

four species of Sariva [Decalepis hemiltonii (Asclepiadaceae), Cryptolepis buchnanii

(Asclepiadaceae), Ichnocarpus frutescens (Apocyanaceae) and H. indicus

(Asclepiadaceae) in carrageenan-induced rat paw oedema. The ethanolic extracts of roots

of various species of sariva exhibited significant anti-inflammatory activity at a dose of

350 mg/kg p.o. as compared to control group53

.

Sowmia C et al. (2007) reported that administration of H. indicus root (40 mg/g

body wt./day) for four weeks significantly decreased the serum cholesterol, triglyceride,

free fatty acids and phospholipid showing antihyperlipidemic activity. It also showed

significant hypoglycemic effect of in alloxan induced diabetic rats54

.

Nambier K et al. (1996) reported a pharmacognostic studies on H. indicus

including its macro and microscopic characters which will help in identification of the



correct raw drug from various adulterants like Ichnocarpus frutescence, Cryptolepis

buchnanii and Decalepis hemiltonii55

.

Kotnis M et al. (2003) reported the pharmacognostic characteristics of H. indicus

Linn. var. pubescens. Also, the data from efficacy study suggest that the drug holds

promising future for the treatment in kidney disorders56

.



3.2 Introduction to Vitis vinifera Linn.

Family: Vitaceae

Fig. 3.5: Photograph of Vitis vinifera L. [Inset showing dried raisins]

Taxonomic Classification:14-16

Kingdom : Plantae

Phylum / Division : Magnoliophyta

Subphylum / Subdivision : Magnoliophytina

Class : Magnoliopsida

Subclass : Magnolidae

Order : Vitales

Suborder : Vitineae

Family : Vitaceae

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

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

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



Subfamily : Vitoideae

Genus : Vitis

Species : vinifera

Synonym : -

Vernacular names of Plant:14-16

English : Common Grape vine, Grapes

Hindi : Angur

Marathi : Draksha

Kannada : Drakshi

Sanskrit : Amrutphala

Malayalam : Gostani

Tamil : Kodimundi-rigai

Distribution:16

Vitis vinifera L. (Grapes) are largely cultivated in North-Western India, in Punjab,

Kashmir, Baluchistan and Afghanistan.

Morphologial Characteristics:14, 16, 20

Leaves Leaves of are 7.5 to 15 cm long, orbicular-cordate, 3 or 5 lobed,

margins irregularly and coarsely toothed, and glabrous.

Flowers Flowers are green with 5 petals cohering at the apex.

Fruits Berries are variable in different sizes; bluish black or greenish

yellow (fresh), yellowish-brown (raisins) in colour.

Seeds Seeds are pear shaped with a discoidal tubercle on the back.


Fresh fruits contain grape-sugar (glucose), gum, tannin, tartaric, citric, racemic and

malic acids, chlorides of potassium and sodium, sulphate of potash, tartarate of lime,

magnesia, alum, iron, some albumin, ozotized matters and acid tartarate of potassium.

Raisins contain calcium, magnesium, potassium, phosphorous and iron in an

assimilable form; besides gum and sugar. Seeds contain a dense fixed oil or fat and tannic

acid (5%). Skins contain tannin.




The ripe fruits are demulcent, laxative, stomachic, diuretic and cooling and used

in cases of dysuria. Leaves are used as diuretic and ashes of stem are useful for pain in

joints and stones in bladder.

Fermented juice of grapes, with the flowers of Woodfordia floribunda and sugar

popularly known as “Drakshasava”, taken in doses of ½ to 2 tolas twice a day after food is

very useful as stimulant, tonic and diuretic.

Grapes are useful in certain cases of bilious dyspepsia, haemorrhage, dysuria,

ardour urinae and strangury.


A thorough literature survey on V. vinifera has been carried out from chemical


database from internet and other published research materials. Till date, following

phytochemical and pharmacological studies have been carried out on V. vinifera.

Diaz-Lanza AM et al. (1989) isolated and identified hyperin, isoquercitrin and

quercetin-3-O-beta-D-glucuronic acid from the leaves of three cultivars of V. vinifera var.

tinctoria57

.

Ohnishi M et al. (1990) analyzed total lipids from five varieties of grape seeds for

their chemical compositions. The major molecular species of triglycerol (TG) were shown

to be trilinolein (40%), oleoyldilinolein (21%) and palmitoyldilinolein (18%)58

.

Teissedre PL et al. (1996) extracted, isolated and purified catechin oligomers and

procyanidin dimmers (B2, B3, B4, B6, B8) and trimers (C1, C2) from grapes (V. vinifera)

seeds and evaluated these compounds for their inhibition of LDL oxidation along with

other monomeric wine phenolics. The procyanidin dimmers B2 and B8 and trimer C1 and

monomers catechin, epicatechin and myrecetin showed the highest antioxidant activity59

.

Ito J et al. (1997) isolated and identified structures of six lupane-type triterpenes

as, lupenone, lupeol, 3beta-hydroxy-30-norlupan-20-one, betulin, 3beta,28-dihydroxy-30-

norlupan-20-one and 3beta,29-dihydroxy-30-norlupan-20-one60

.

J. Fausto Rivero-Cruz et al. (1997) isolated eight known compounds, oleanolic

acid (1), oleanolic aldehyde (2), linoleic acid (3), linolenic acid (4), betulin (5), betulinic

acid (6), 5-(hydroxymethyl)-2-furfural (7), and b-sitosterol were isolated from an

hexanesoluble partition of a methanol extract of Thompson seedless raisins (Vitis vinifera)



and evaluated for antimicrobial activity. From an EtOAc soluble partition rutin (8) and b-

sitosterol glycoside were isolated. In an attempt to increase the resultant antimicrobial

activity of oleanolic acid (1), a series of acylation and etherification reactions were

performed on oleanolic acid to obtain derivatives 1a–1f. All the compounds isolated and

the derivatives 1a–1f were evaluated for their antimicrobial activity against two oral

pathogens, Streptococcus mutans and Porphyromonas gingivalis associated with caries and

periodontal disease, respectively. Compounds 1, 2, 7 and 1f inhibited the growth of the test

bacteria with concentrations ranging from 3.9 to 500 mg/mL. Derivative 1f showed greatly

enhanced antimicrobial activity when compared with oleanolic acid (1).61

Fig. 3.6: Structures of oleanolic acid and its derivatives isolated from Vitis vinifera

Korhammer S et al. (1995) isolated and identified the structure of a new tetramer

of resveratrol (3,5,4’-trihydroxystilbene), r-2-viniferin from roots of species and hybrids of

the genus Vitis62

.

Ourtoule JC et al. (1996) isolated a biologically active stilbenoidic polyphenol (E-

viniferin) and a new resveratrol tetramer showing a symmetrical bicycle {6.6.0}

tetradecane framework, from the stalks of V. vinifera63

.

Teguo PW et al. (1996) isolated and identified structures of stilbene glycosides,

(E)-piceatannol(3,5,3’,4’-tetrahydroxystilbene)3-O-beta-glucoside and (Z)-resveratrol

(3,5,4’-trihydroxystilbene) 3-O-beta-glucoside accumulated in suspension cultures of V.

vinifera grown in an inductive polyphenol synthesis medium64

.



Foo LY et al. (1998) isolated two novel biphenyl linked biflavonoids from

Chardonny grape pomace. Their structures were established as epicatechin {6’ to 8}-

epicatechin and epicatechin {6’ to 8}-catechin65

.

Patil SG et al. (1998) studied eleven Vitis species and their cultivars for their

phytochemical constituents and revealed the presence of polyphenols, catechol tannins,

lignins, tannins, flavonoids, raphides and absence of cyanogenic and syringin glycosides,

saponins, mucilaginous and aucubin like substances. Presence of leuco-anthocyanins and

raphides has taxonomic significance in separation and systematic relationships among Vitis

species and their cultivars66

.

Fig. 3.7: Phytoconstituents isolated from dried stembark of Vitis vinifera

Teguo PW et al. (1998) isolated novel stilbene glucosides, viz. (E)- and (Z)-

resveratroloside and (Z)-astringin, from suspension cultures of V. vinifera. The free radical

scavenging activity was evaluated using 1,1-diphenyl-2-picryl-hydrazyl and antioxidant

effects were assessed by their capacity to prevent copper ions induced lipid peroxidation in

human low density lipoprotein67

.



Berthier L et al. (1999) isolated and identified a lectin fraction from grape juice by

affinity chromatography on a column of p-aminopyranosyl beta-D-glucose derivatized

agarose. Isolectins seemed to be glycoproteins since they were bound on a concanavalin A-

Sepharose column68

.

Gabetta B et al. (2000) reported detection of monomeric flavan-3-ols and dimeric

proanthocyanidins. The analysis revealed presence of approx. 15% of (+)-catechin and (+)-

epicatechin, 80% of (-)-epicatechin 3-O-gallate, dimmers, trimers, tetramers and their

gallates and 5% of pentamers, hexamers, heptamers and their gallates69

.

Souquet JM et al. (2000) reported polyphenolic compounds from grape stem

(including tannins, phenolic acids, flavonols, flavononols like astilbin).Tannins consisted

of (-)-epicatechin units alongwith smaller amounts of (+)-catechin, (-)-epicatechin gallate

and (-)-epigallocatechin. Phenolic compounds consisted of quercetin-3-glucuronide,

catechin, caffeoyltartaric acid and dihydroquercetin-3-rhamnoside (astilbin)70

.

Baltenweck-Guyot R et al. (2000) isolated and characterized eight glycosides and

a phenylpropanoid glycerol from V. vinifera cv. gewurztraminer wine. Cis-1-(5-Ethenyl-5-

methyltetrahydrofuran-2-yl)-1-methylethyl-O-beta-D-apiofuranosyl-(1 to 6)-beta-D-

glucopyranoside, (E)-3,6,9-trihydroxymegastigm-7-ene-9-O-beta-D-glucopyranoside, 2-

phenylethyl-O-beta-D- apiofuranosyl-(1 to 6)-O-beta-D-glucopyranoside and 2-{4-(3-

hydroxypropyl)-2-methoxyphenoxy}propane-1,3-diol were reported for the first time as

wine components71

.

Skouroumounis GK et al. (2000) isolated Alangioside J and a new norisoprenoid

beta-D-glucopyranoside by serial separations using different chromatographic techniques.

The later compound was found to be C-9 conjugate of the same (3S, 5R, 6S, 9R)-

megastigmane-3,9-diol as the C-3 conjugated former one72

.

Decendit A et al. (2002) reported that suspension cultures of V. vinifera were

found to produce catechins and stilbenes when grown in a medium inducing polyphenol

synthesis. (-)-epicatechin-3-O-gallate, dimeric procyanidin B-2 3’-O-gallate and two

resveratrol diglucosides were isolated together with a new cis-resveratrol-3,4’-O-beta-

diglucoside by spectroscopic methods73

.

de Pinho PG et al. (2001) identified beta-carotene and six xanthophylls (lutein,

neoxanthin, violaxanthin, cryptoxanthin, echinenone) and semiquantitatively or

quantitatively determined in musts and port wines. Some experiments were performed to

follow carotenoid content from grapes to wines74

.



Pirniyazov A et al. (2003) studied the chemical composition of polyphenols from

grape (V. vinifera) seeds. They were found to contain four catechins and two

proanthocyanidins75

.

Cao X et al. (2003) reported the Supercritical fluid extraction of grape seed oil and

subsequent separation of free fatty acids by high speed counter current chromatography

coupled with evaporative light scattering detection. The separation of 1.0 g of oil can yield

about 430 mg pure linoleic acid at 99% purity76

.

Mendes-Pinto MM et al. (2004) reported seven known carotenoids (neochrome,

neoxanthin, violaxanthin, flavoxanthin, zeaxanthin, lutein and beta-carotene), one more

type of neochrome and two geometrical isomers of lutein and beta-carotene in grapes from

three cultivars by analyzing using normal and reversed phase HPLC-DAD77

.

Han Y (2003) examined the effect of grape seed extract against Candia albicans

under in vivo and in vitro conditions. The results indicated that grape seed extract has

prophylactic effect but not therapeutic effect against disseminated candidiasis78

.

Abulrob AN et al. (2004) isolated and characterized grapefruit oil components (4-

{(E)-5-(3,3-dimethyl-2-oxiranyl)-3-methyl-2-pentenyl}oxy}-7H-furo{3,2-g}chromen-7-

one (2) and studied the antimicrobial activity against Methicillin-resistant and Methicillin

Susceptible Staphylococcus aureus (MRSA and MSSA) strains. The results suggested that

grapefruit oil enhances susceptibility of test MRSA strains to antimicrobial agents

(Ethidium bromide and Norfloxacin) to which these microorganisms are normally

resistant79

.

Fig. 3.8: Resveratrol diglucoside isolated from Vitis vinifera cell culture [Arrows

indicate HMBC correlations]

Larronde A et al. (2005) isolated and reported three new monomeric stilbenoid

glucosides, (Z)- and (E)-resveratrol 3,5-O-beta-diglucosides and (Z)-resveratrol 3,5,4’-O-



beta triglucoside from an extract of V. vinifera cell suspension cultures (cv. Cabernet

Sauvignon) together with known (E) and (Z)-piceids and (E) and (Z)-resveratrol 3,4’-O-

beta diglucosides which have already been identified in a Gamay cell culture extracts80

.

Kallithraka S et al. (2007) reported the differentiation of young red wines based on

chemometrics of minor polyphenolic constituents. The polyphenols analyzed belonged to

two categories: benzoic acid derivatives (including gallic acid, protocatechuic acid, vanillic

acid and syringic acid) and stilbenes (including astringin, piceid and resveratrol) [all trans

isomers]81

.

Castillo Munoz A et al. (2007) reported the flavonol profiles of V. vinifera red

grapes and their single cultivar wines. In addition to the main flavonols like 3-glucosides

and 3-glucuronides of myricetin and quercetin, 3-glucosides of kaempferol and

isorhamnetin found in 7 widespread cultivars of red grapes, the methoxylated trisubstituted

flavonols (laricitrin and syringetin) were predominantly found as 3-glucosides, alongwith

minority flavonols like, 3-galactosides of kaempferol and laricitrin, 3-glucuronide of

kaempferol and 3-(6”-acetyl glucosides of quercetin and syringetin82

.

Girre L et al. (1991) studied the in vitro antiherpetic activity of leaves of V.

vinifera in various galenical forms, against HSV-1. The differences of activity between the

extracts of same galenical form and between the three galenical forms studied showed the

importance of the aqueous extraction and of a low temperature treatment83

.

Tripathi D (2001) evaluated different extracts of V. vinifera roots for antifungal

activity against some human pathogenic fungi like Chrysasporium tropicum and

Aspergillus niger. Chloroform and acetone extracts exhibited better zone of inhibition84

.

Han Y (2007) reported a synergic effect of grape seed extract (V. vinifera)

combined with Amphotericin B against Candida albicans. The results indicated that the

combination therapy can reduce more than 75 percent of Amphotericin B dose, implying

that the grape seed extract has a synergistic effect with Amphotericin B85

.

Therese Meunier M et al. (1987) evaluated the procyanidolic oligomers from V.

vinifera and Cupressus sempervirens and the monomers, (+)-catechin and (-)-epicatechin,

for their effects on angiotensin I converting enzyme (ACE) activity. Compared to

monomers, the oligomers showed more activity86

.

Toukairin T et al. (1991) isolated new 5’-nucleotidase inhibitors (designated as

NPF-88BU-IA, NPF-88BU-IB, NPF-88BU-IIA and NPF-88BU-IIB, respectively) from

the seeds and skin of wine grape “Koshu” (V. vinifera). These polyphenolic substances



strongly inhibited 5’-nucleotidase activities of snake venom and rat liver membrane and

showed significant therapeutic activity against Ehrlich ascites carcinoma. They also

showed inhibitory effects on the growth of Streptococcus mutans MT148(c), a primary

cariogenic bacterium87

.

Liviero L et al. (1994) assessed procyanidins obtained from V. vinifera seeds for in

vitro antimutagenic effect. This effect could be due, at least in part, to the antioxidant

properties of procyanidins88

.

Meyer AS et al. (1997) reported that the inhibition of human low density

lipoprotein (LDL) oxidation by fresh grapes ranging from 22% to 60% at 10 microM gallic

acid equivalents (GAE) of total phenols and from 62% to 91% at 20 microM GAE89

.

Tebib K et al. (1997) reported that compared with a normal vitamin E diet aortic,

cardiac, hepatic, intestinal, muscular and renal catalase, glutathione peroxidase and

superoxide dismutase activities were significantly lower in rats receiving the deficient

vitamin E diet. Polymeric tannins (71mg/kg), but not monomeric tannins, were able to

restore all these enzymatic activities90

.

Bagchi D et al. (1998) assessed the hydrogen peroxide-induced oxidative damage

in macrophages J774A.1 and neuroactive adrenal pheochromocytoma PC-12 cells, and

concentration dependent ability of grape seed proanthocyanidin extract (GSPE) to protect

these cells. GSPE exhibited significantly protection in these models91

.

Fontecave M et al. (1998) reported that resveratrol found in grapes has shown to

be a remarkable inhibitor of ribonuclease reductase and DNA synthesis in mammalian

cells. It might have further applications as an antiproliferative or a cancer chemopreventive

agent in humans92

.

Fremont L (2000) reported that resveratrol (3,4’,5’-trihydroxystilbene), has been

shown to modulate the metabolism of lipids and to inhibit the oxidation of low density

lipoproteins and the aggregation of platelets. Moreover, it may provide cardiovascular

protection and possesses anti-inflammatory and anticancer properties93

.

Carini M et al. (2001) reported the inhibitory properties of procyanidins from V.

vinifera seeds on the respiratory burst and on the release of granule components in

activated human neutrophils. Procyanidins strongly inhibit superoxide generation through

a direct scavenging of superoxide and prevent the release from calcium ionophore

activated neutrophils of beta-glucuronidase, myeloperoxidase and elastase94

.



Sugisawa A et al. (2004) studied effects of grape seed extracts (Vitis sp.) on

chromosomal damage. The results indicated that grape seed extract is not genotoxic, but

rather has an antigenotoxic effect against H2O2 via direct scavenging action of H2O295

.

Etheridge AS et al. (2007) studied the effect of extracts and individual

constituents of goldenseal, Ginko biloba and its hydrolysate, grape seed (V. vinifera), milk

thistle (Silybum marianum) and ginseng (Panax ginseng) on the activities of cytochrome

P450 enzymes and P-glycoprotein. The data suggested that the clearance of a variety of

drugs may be diminished by concomitant use of these herbs via inhibition of P450

enzymes, but less so by P-glycoprotein mediated effects96

.

Khan MA et al. (2006) studied Punica granatum (Punicaceae) and V. vinifera

(Vitaceae) for their haematinic activities in rats as mentioned in Unani classical literature97

.

Panico AM et al. (2006) studied in vitro effects of lyophilized extract of wine

obtained from Jacquez grapes (V. aestivalis-cinerea x V. vinifera, Vitaceae). Data showed

significantly greater protective effect of extract in cartilage, alteration than that elicited by

Indomethacin (a standard NSAID drug)98

.

Bilgrami KS et al. (1993) assessed cortisone, mercurious corrosives (a

homeopathic drug) and aqueous extract of V. vinifera for controlling nephrotoxicosis

caused by citrinin in mice for 20 weeks. The results showed significant positive effect and

upto 41% recovery was achieved99

.

Shastry CS et al. (2002) studied the diuretic activity of aqueous and alcoholic

extracts of V. vinifera leaves in rats. Both extracts showed increase in urine volume, cation

(Na+ and K

+) and anion (Cl

-) excretion

100.

Waterhouse AL (1995) reported that wine phenolics are beneficial nutrients that

can reduce congestive heart disease mortality. Nutritional and pharmacological

significance of resveratrol and flavonoids (epicatechin, malvidin-3-glucoside, quercetin

and procyanidin B1), absorption of these phenolics in human body and dietary resources of

these compounds have been discussed101

.

Facino RM et al. (1996) studied effects of highly purified, high molecular weight

fraction of oligomeric procyanidines isolated from V. vinifera seeds on myocardial

reperfusion injury after 40 mins of low flow (1ml/min) ischemia102

.

Rao Padma GM et al. (1999) evaluated the hepatoprotective activity of grape seed

oil in paracetamol-induced rats for 3 days. GSO exhibited significant hepatoprotective

activity which may be attributed to very high levels of vitamin E in it103

.



Orhan DD et al. (2007) reported the hepatoprotective effect of ethanolic extract

and its four fractions (CHCl3, EtOAc, n-BuOH and Water fraction) of V. vinifera leaves

using CCl4 induced acute hepatotoxicity in rats. The n-BuOH fraction in 83 mg/kg dose

possessed remarkable antioxidant and hepatoprotective activities104

.

Mallikarjuna Rao C et al. (1998) assessed effects of grape seed oil (GSO) on

collagenation, wound contraction and epithelization phases of wound healing in male

Wistar rats. GSO significantly promoted the collagenation phase by healing despite

causing significant reduction in wound granulation and collagen content. In excision

wound it promoted wound contraction105

.

Folts JD (1998) evaluated the platelet inhibitory effects of red and white wine and

purple grape juice in 10 healthy human subjects. The antiplatelet / antioxidant

polyphenolic compounds in red wine or purple grape juice were found to reduce the rate of

progression of the atherosclerotic process and reduce the incidence of acute occlusive

platelet mediated coronary thrombosis106

.

Jang M et al. (1998) examined the chemopreventive potential of resveratrol on

cyclooxygenase (COX) metabolites monitored by HPLC analysis. Resveratrol was found

to inhibit generation of arachidonic acid metabolites catalyzed by both COX-1 and COX-2.

In addition, resveratrol significantly inhibited malignant transformation induced by

chemical carcinogens in the mouse C3H10T1/2 cell culture system107

.

Saito M et al. (1998) evaluated antiulcer activities of grape seed (V. vinifera)

extracts (GSE-I and GSE-II) and proanthocyanidins using rats. GSE-I (with low flavonol

content), GSE-II (with high flavonol content) and proanthocyanidins at a dose of 200

mg/kg strongly inhibited stomach mucosa injury induced by 60% ethanol containing 150

mM Hydrochloride. Oligomers longer than tetramers showed a strong protective effect

against gastric mucosal damage108

.

Castillo J et al. (2000) evaluated the antioxidant activity of grape seed (V. vinifera)

extract and other reference compounds by measuring their ability to scavenge the ABTS+

radical cation (TEAC). Most effective compounds were in order: Grape seed extract >

rutin > (+)-catechin > diosmin ≥ ascorbic acid. The radioprotective effects of grape seed

extracts and other reference compounds were determined by using the micronucleus test

for anticlastogenic activity. Most effective compounds were in order: Grape seed extract >

rutin > DMSO > ascorbic acid > 6-n-propyl-2-thiouraci-6e (PTU) > diosmin109

.

Basley JP et al. (2000) evaluated the estrogenic/antiestrogenic effects on human

breast cancer cell lines and scavenging properties of (E)- and (Z)-resveratrol (a natural



compound found in grapes and wine) based on structural similarity to the synthetic

estrogen diethylstilbesterol. Both isomers exhibited cytotoxic activity at higher

concentrations and they were found to be as free radical scavengers or pro-oxidant

compounds110

.

Spagna G et al. (2004) studied the in vitro antioxidant activity and in vivo

photoprotective effect of extract obtained from Jacquez grapes (V. aestivalis-cinerea, V.

vinifera) grapes. The significant level of proanthocyanins together with low levels of

anthocyanins and hydroxyl cinnamic acids contributed for strong in vitro antioxidant / free

radical scavenging effects and in vivo protection against UVB light-induced skin

erythema111

.

Yilmaz Y et al. (2004) studied the total antioxidant capacity of major monomeric

flavonols and phenolic acids in grape seed and skins. Peroxyl radical scavenging activities

of phenolics present in grape seeds and skins in increasing order were resveratrol >

catechin > epicatechin > gallocatechin > gallic acid > ellagic acid, indicating that dimeric,

trimeric, oligomeric or polymeric procyanidins account for most of the superior

antioxidant capacity of grape seeds112

.

Orhan N et al. (2006) studied the acute and subacute hypoglycaemic and

antihyperglycaemic effect of aqueous extract of leaves V. vinifera L. (250, 500 mg/kg) and

its different fractions. The results showed that Ethyl acetate fraction (25 mg/kg) of

aqueous extract was rich in polyphenolics and possessed a significant antihyperglycaemic

and antioxidant activity equipotent with reference hypoglycaemic drug (Tolbutamide) in

diabetic rats113

.

Sondhi SM et al. (1995) carried out determination of mineral elements in 32

medicinal plants (including V. vinifera). Concentrations of Na, K, Cu, Ni, Mn, Co, Zn, Cd,

Fe, Pb, Mg, Cr, Ca, Hg, Al, Ag, Sr, Mo and V were determined using flame photometer

and atomic absorption spectrometer and inductively coupled plasma respectively114

.

Aagte VV et al. (2003) reported the study result for micronutrients and antioxidant

potentials of grapes available in India for their nutritional values. The level of

micronutrients from 100 g of grapes will be equal to 11 % RDA for vitamin C, 10 % RDA

for riboflavin, 6 % RDA for thiamine and 3 to 4 % for Mn and Se. Grapes however, seem

to be poor sources for beta-carotene, iron and zinc115

.

Larrauri JA et al. (1997) studied the effect of drying temperature (60, 100 and 140

degree C) on the polyphenols’ content and antioxidant activity of red grape (V. vinifera)

pomace peels, Freeze-dried samples were used as reference. At higher temperatures (100



and 140 degree C) significant reduction in extractable polyphenols and condensed tannins

was observed along with a significant decrease in antioxidant activity116

.

Saucier C et al. (2001) reported a rapid separation method that permits separation

of grape seed proanthocyanidins (condensed tannins) according to their polymerization

degrees. This method was based on liquid/liquid extraction and relative solubility of these

compounds in different solvents (water, ethyl acetate, methanol and chloroform)117

.

Torres JL et al. (2001) reported that a new family of antioxidants has been

obtained from a residual fraction of polymeric polyphenols of grape origin. Among these

polyphenols oligomeric proanthocyanidins were particularly significant118

.

Ghosh N et al. (2003) isolated and identified anthocyanin pigments from skin of

Indian varieties of grapes. Glucosides of malvidin constituted the major pigment in all

varieties of grapes, while acyl anthocyanins alongwith glucosides of delpinidin, petunidin

and peonidin were also identified in most of them119

.

Sauro-Calixo FD et al. (1999) have patented in PCT Int. Appl. WO 99/25209/A, a

process patent, where-in naural oxidant dietary fibres in powder form from grapes is

described120

.

Murad H (2001) has patented in US patent No. 6,296,880, some pharmaceutical

compositions (containing V. vinifera constituents) useful for skin conditions such as acne

and psoriasis121

.



3.3 Introduction to Bombax ceiba Linn.

Family: Bombacaceae

Fig. 3.9: Photograph of Bombax ceiba L. showing flower and young fruits

Taxonomic classification:14-16

Kingdom : Plantae

Order : Malvales

Suborder : Gentianineae

Family : Bombacaceae

Subfamily : Malvaceae

Genus : Bombax

Species : ceiba

Synonyms : Bombax malabaricum D.C.

Salmalia malabarica Schott and Endl.

Distribution:16

This tropical tree is found throughout the hotter forest regions of India, common in

Bengal, Bombay presidency.



Vernacular names :14-17

English : Silk Cotton Tree

Hindi : Shimal

Marathi : Shaalmali, Saanwari

Kannada : Kempuburuga

Sanskrit : Rakta Shalmali

Malayalam : Mullilavu

Tamil : Elevam

Morphologial characteristics:14,16,17

Leaves Leaves are large with 3-7 leaflets, 7.5-18 cm long, glabrous,

reticulately veined, lanceolate and acute at the base.

Flowers Flowers are numerous, near the end of branches, appearing before

new leaves.

Calyx Calyx is thick, 3-lobed. Corolla is bright red, tomentose on outside.

Petals Petals are elliptic-oblong, recurved, with close parallel veins.

Stamens Stamens are more than 60, arranged in bundles of 9-12 each. Filament

is flattened.

Ovary Ovary is conical and glabrous.

Capsule Capsules are 10-12.5 cm long, ovoid, 5-valved, lined with white silky

hairs.

Seeds Seeds are 9 mm long, numerous, ovoid, packed in white cotton


Stem, root, flower, fruit, and leaves of B. ceiba have been reported to contain

many important phytoconstituents including alkaloids, glycosides, phytosterols, and

triterpenoids (lupeol and β-sitosterol), proteins, phenolic compounds (naphthalene

derivatives, mangiferin, shamimin, kaemferol, and quercetin) and tannins. Seeds yield

good non-drying oil. Gum called “mocharas”, contains tannin and gallic acid.


An ethnobotanical study reported the use of B. ceiba as a traditional anti-

inflammatory agent. The dried young fruits of B. ceiba are given in calculus affections

and chronic inflammation and ulceration of the bladder and kidneys including strangury



and other forms of dysuria.


A thorough literature survey on B. ceiba L. has been carried out from chemical


databases from internet and other published research materials. Till date, following

phytochemical and pharmacological studies have been carried out on B. ceiba.

Gopal and Gupta (1972) reported a study on chemical constituents of Salmalia

malabarica Schott and Endl. Flowers122

. Agarwal GD et al. (1972) isolated and

characterized a polysaccharide from the stamens of Bombax malabaricum flowers123

. In

another study, Niranjan GS et al. (1973) isolated and reported some anthocyanins from the

flowers of Bombax malabaricum124

. Rizvi and Saxena (1974) reported the presence of new

glycosides, terpenoids, colouring matters, sugars and fatty compounds from the flowers of

Salmalia malabarica125

. Dhar DN et al. (1976) studied and reported the chemical

examination of the seeds of Bombax malabaricum126

.

Sankaram AKB et al. (1981) reported that the hemigossypol-6-methyl ether, to be

present in the root bark of Bombax malabaricum, has been shown to be isohemigossypol-l-

methyl ether. Isohemigossypol-1,2-dimethyl ether, 8-formyl-7-hydroxy-5-isopropyl-2-

methoxy-3-methyl-l,4-naphthaquinone, 7-hydroxycadalene and an unidentified phenolic

compound have also been isolated. Long range couplings in the 1H NMR spectrum of

isohemigossypol-l-methyl ether have been established by decoupling experiments127

.

Faizi and Ali (1999) isolated shamimin (a new flavonol C-glycoside) as a pale

yellow powder from the ethanolic extract of fresh, undried leaves of Bombax ceiba. Its

structure has been elucidated as 2-(2,4,5-trihydroxyphenyl)-3,5,7-trihydroxy-6-C-

glucopyranosyloxy-4H-1-benzopyran-4-one through extensive spectroscopic methods (IR,

mass, 1H- and

13C-NMR), and 2D-NMR experiments. Shamimin showed antimicrobial

activity against a few bacteria and fungi128

.

Kotoky R et al. (2001) carried out phytochemical analysis for seed oils of

Garcinia xanthochymus, Phoebe attenuate, Polyalthia jenkensii, Pyrus pashia, S.

malabarica and reported that these contain 5 to 23% fatty oil content. Gas liquid

chromatography analysis of methyl esters of fatty acids indicated that oleic acid was

predominant ranging from 39 to 71%129

.

Sreeramulu K et al. (2001) isolated a new naphthoquinone together with 7-

hydroxy-cadalene and 8-formyl-7-hydroxy-5-isopropyl-2-methoxy-3-methyl-1,4-



naphthoquinone from methanolic extract of heartwood of B. malabaricum. The new

naphthoquinone was characterized as 7-hydroxy-5-isopropyl-2-methoxy-3-methyl-1,4-

naphthoquinone based on spectral and chemical studies130

.

Shahadat AA et al. (2003) isolated a xanthone, Mangiferin from n-BuOH fraction

of 70% EtOH of the dried leaves of B. malabaricum. It was found to be identical to

Shamimin, a compound for which originally a flavonol structure was proposed and the

structure of later has been revised131

.

Saleem R et al. (2003) isolated a novel constituent, shamimicin, 1"',1"""'-bis-2-

(3,4-dihydroxyphenyl)-3,4-dihydro-3,7-dihydroxy-5-O-xylopyranosyloxy-2H-1-

benzopyran along with lupeol from Bombax ceiba stem bark and it was found to possess

potent hypotensive activity. BCBMM - one of the most active hypotensive fractions has

revealed its adverse effects on heart, liver and kidneys of mice at the dose of 1000

mg/kg/d132

.

Vijaya BRM et al. (2003) reported a new sesquiterpene lactone from Bombax

malabaricum133

.

Dar A et al. (2005) isolated mangiferin, 2-beta-D-glucopyranosyl-1,3,6,7-

tetrahydroxy-9H-xanthen-9-one, directly from methanolic extracts of Bombax ceiba leaves

in substantial amounts and demonstrated strong antioxidant activity (EC(50) 5.8+/-0.96

mug/ml or 13.74 muM) using DPPH assay comparable to rutin, commonly used as

antioxidant for medical purposes. The acetyl and cinnamoyl derivatives were found to be

less active than mangiferin whereas, methyl and 3,6,7-trimethylether tetraacetate

derivatives were inactive implying that for antioxidant activity, free hydroxyl groups and

catechol moiety are essential. Moreover, mangiferin showed hepatoprotective activity

against carbon tetrachloride induced liver injury further supporting the free radical

scavenging property in the in vivo system. Additionally, plant extracts and mangiferin

failed to exhibit acute anti-inflammatory activity whereas, it displayed significant analgesic

effect in acetic acid-induced writhing and hot plate tests in mice. Using naloxone, it was

revealed that plant extracts induced analgesia was independent of opioid receptor, whereas,

mangiferin demonstrated significant interaction with it at peripheral site with a slight

contribution at the neuronal level134

.

Faizi S et al. (2006) reported that by employing concerted 1 and 2D NMR

techniques, exact NMR spectral assignments have been made of the acyl and methyl

derivatives of mangiferin isolated from the leaves of Bombax ceiba. Derivatives have been

reported in literature alongwith some new compounds. The acetates were found to be



unstable and were converted into the same penta-acetate at room temperature. Extensive

NMR studies on mangiferin and its derivatives showed that H-4 exchanges with deuterium

of the solvent molecule more easily. This exchange under acidic conditions occurred at

that position (C-4) where electrophilic substitution reactions can easily take place. This is

the first report describing the exchange of C-4 proton of mangiferin, or any other xanthone,

with deuterium of solvent molecules135

.

Fig. 3.10: Structures of Mangiferin and its derivatives isolated leaves of Bombax ceiba

Zhang X et al. (2007) reported that the phytochemical investigation of the

chemical constituents of the roots of Bombax malabaricum afforded nine cadinane

sesquiterpenoids, including five new compounds (bombamalones A-D; bombamaloside),

and four known compounds (isohemigossypol-1-methyl ester; 2-O-methylisohemigossylic

acid lactone; bombaxquinone B; and lacinilene C). The structures of five new compounds

were identified by spectroscopic methods and comparison with literature values. These

compounds were evaluated against the HGC-27 human gastrointestinal cancer cell line, but

all were inactive (IC(50) >10 microM)136

.

Lin CC et al. (1992) evaluated the anti-inflammatory and liver protective effect of

bark, xylem of stem and root of Bombax malabarica DC. and Ceiba pentandra GAERTN.

with carrageenan-induced paw edema and CCl4-induced hepatotoxicity in rats,

respectively. The statistical analysis shows that all of the treatment used exhibited

significant anti-inflammatory activity against carrageenan-induced edema. Furthermore,

the administration of root and xylem of stem of B. malabarica showed the activity even



better than indomethacin group did. However, only three used parts of B. malabarica

significantly alleviated liver injury induced by CCl4. Meanwhile, the histological changes

in rat hepatic tissues such as fatty change, ballooning degeneration, cell necrosis,

lymphocytes and Kupffer cells were also observed137

.

Sikawar RLS (1994) reviewed and reported around 35 plant species under 33

genera and 27 families, which have been used by tribal and non tribal rural folks for

treatment of various kinds of ailments for their domestic animals. B. ceiba has been

reported to be used in bone dislocation138

.

Fig. 3.11: Phytoconstituents (Shamimicin and Lupeol) isolated from leaves of Bombax

ceiba

Saleem R et al. (1999) evaluated the hypotensive, hypoglycaemic and

toxicological studies on aqueous and methanolic extracts of B. ceiba leaves and one of its

fractions. Shamimin, a C-flavonol glucoside showed significant potency as a hypotensive

agent at the doses of 15 mg/kg, 3 mg/kg, 1 mg/kg and significant hypoglycaemic activity at

500 mg/kg in Sprague-Dowley rats139

.

Babu R et al. (2001) evaluated a polyherbal formulation “RV08” containing

Asparagus racemosus, Mucuna pruriens, Withania somnifera, B. malabaricum,

Sphaeranthus indicus, Butea frondosa, Cleodendrum serratum and Sida cordifolia for its

ability to potentiate both specific and nonspecific host defense responses. The results



indicated possible involvement of RV08 as a line of defense through significant

immunomodulation140

.

Bafna P et al. (2003) evaluated “MEBARID”, an ayurvedic formulation containing

extracts of Holarrhena antidycenterica, Berberis aristata, Aegle marmalos, Punica

granatum, Myristica fragrans, Salmalia malabarica for its anti-diarrhoeal, antiulcer and

anti-motility activities in animals. MEBARID was found to have significant activity in all

three models141

.

You YJ et al. (2003) reported that the methanol extract of the stem barks of

Bombax ceiba was found to exhibit a significant antiangiogenic activity on in vitro tube

formation of human umbilical venous endothelial cells (HUVEC). Bioactivity-guided

fractionation and isolation carried out on this extract afforded lupeol as an active principle.

At 50 and 30 microg/mL lupeol showed a marked inhibitory activity on HUVEC tube

formation while it did not affect the growth of tumor cell lines such as SK-MEL-2, A549,

and B16-F10 melanoma142

.

Nam NH et al. (2003) reported that 7 of 58 plant materials from Vietnamese

medicinal plants showed strong to moderate inhibitory activity on the tube-like formation

induced by human umbilical venous endothelial cells in the in vitro angiogenesis assay.

These plant materials include the herb of Ephedra sinica, leaves and stem of Ceiba

pentandra, seed of Coix lachryma-jobi, rhizome of Drynaria fortunei, fruits and stem of

Illicium verum and stem of Bombax ceiba. Of these, the methanol extracts of the herb of

Ephedra sinica and stem of Ceiba pentandra exhibited the strongest activities with

inhibition percentages of 89.3% and 87.5% at 30 and 100 microgram/mL, respectively143

.

Ravichandran G et al. (2004) studied the efficacy and safety of “Acne-N-Pimple

cream” in the management of Acne vulgaris in 26 patients. Significant reduction in number

of blackheads and whiteheads, inflamed pustules and overall inflammation, significant

improvement in healing without scar formation and enhanced exfoliation were observed.

These effects might be due to the antioxidant, anti-inflammatory, antiandrogenic and

antimicrobial properties of the ingredients like powders of Lens culinaris and alum with

extracts of Aloe barbadensis, Vitex negundo and Salmalia malabarica144

.

Rani and Khullar (2004) screened some plants of importance in the Ayurvedic

system of traditional medicine used in India to treat enteric diseases. Fifty four plant

extracts (methanol and aqueous) were assayed for their activity against multi-drug resistant

Salmonella typhi. Strong antibacterial activity was shown by the methanol extracts of

Aegle marmelos, Salmalia malabarica, Punica granatum, Myristica fragrans, Holarrhena



antidysenterica, Terminalia arjuna and Triphal (mixture of Emblica officinalis, Terminalia

chebula and Terminalia belerica). Moderate antimicrobial activity was shown by

Picorhiza kurroa, Acacia catechu, Acacia nilotica, Cichorium intybus, Embelia ribes,

Solanum nigrum, Carum copticum, Apium graveolens, Ocimum sanctum, Peucedanum

graveolens and Butea monosperma145

.

Wang and Huang (2005) reported that 95% ethanolic extracts of Paederia

scandens (Lour.) Merr., Plumbago zeylanica L., Anisomeles indica (L.) O. Kuntze, Alpinia

speciosa (J. C. Wendl.) K. Schum. and Bombax malabaricum DC. were examined and

screened for anti-Helicobacter pylori activity. All extracts demonstrated strong anti-

Helicobacter pylori activities. The minimum inhibitory concentration values of the anti-

Helicobacter pylori activity given by the five ethanol herb extracts ranged from 0.64 to

10.24 mg ml(-1). Twenty-six herbs, including Artemisia argvi Levl. et Vant, Phyla

nodiflora (Linn.) Greene and others showed moderate anti-Helicobacter pylori activity.

The additional 19 herbs, including Areca catechu Linn., Euphorbia hirta Linn. and

Gnaphalium adnatum Wall. ex DC., possessed lower anti-Helicobacter pylori effects146

.

Review of database search on patents at various sites like www.uspto.com,

www.espacenet.com was conducted. However, no patent database was found for B. ceiba.



3.4 Summary and justification for proposed research protocol

The literature survey revealed that H. indicus contains various phytoconstituents

like coumarinolignoids, pregnane glycosides, acyclic triterpenic acid, acyclic diterpenic

ester, monocyclic sesterpene ester, terpene lactones, hexadecanoic acid, 4-hydroxy-3-

methoxy benzaldehyde, 2-hydroxy-4-methoxy benzaldehyde, 2-hydroxy-4-methoxy

benzoic acid etc. Since, most of these compounds are usually present in glycosidic form

within the plant; these have been isolated using single or binary mixtures of various highly

polar to moderately polar solvents like purified water, alcohol, methanol, chloroform etc.

Using steam distillation method, various volatile oil constituents from H. indicus roots

have been isolated. 2-hydroxy-4-methoxy benzaldehyde (91%) and (-)-ledol (4.5%) were

found to be chief constituents in volatile oil alongwith other minor constituents in traces.

Roots of H. indicus have been evaluated for different pharmacological activities.

The toxicity study on crude aqueous and alcoholic extracts has been carried out in dose

range of 1000 – 4000 mg/kg body weight. LD50 cut-off dose was found to be 4000 mg/kg

body weight by oral route for both extracts.

Antioxidant (in vitro), hepatoprotective, antiulcer, hypoglycaemic and

antihyperlipidemic activities of roots of H. indicus have been proved scientifically. These

activities suggest that roots of H. indicus may prove efficacious against oxidative cellular

damage in nephrolithiasis. Urinary tract infection (UTI) is observed frequently in many

cases of lithiasis; wherein, Klebsiealla, Proteus and Pseudomonas species have been found

to contribute for deposition of staghorn calculi. However, antienterobacterial,

antidiarrhoeal, antifungal and antimicrobial activities of roots of H. indicus justify the

inclusion of roots of H. indicus in a multidrug formulation “RENALKA” against UTI. In

addition, antinociceptive, anti-inflammatory and antirheumatic activities of roots of H.

indicus support for its usefulness in relieving the associated pain in lithiatic patients.

In another plant, (fruits of V. vinifera) the literature survey reveals that flavonoids,

tannins and phenolic compounds constitute principle secondary metabolites. Xanthophylls

like beta-carotene, violaxanthin, neoxanthin, zeaxanthin and lutein have been isolated from

grapes and wines. Various proanthocyanidins have been identified from extracts of fruits

of V. vinifera as monomer, dimmer, trimer, tetramer, pentamer, hexamer, heptamer forms

of their gallates. Benzoic acid derivatives and stilbenes constitute the polyphenolic

constituents of fruits of V. vinifera. These polar phytochemical substances have been found

to contribute for their strong to moderate antioxidant properties depending upon their



structural complexity. Oligomeric compounds have been found to be superior to

monomeric compounds in scavenging free radicals.

Some moderately polar to non-polar phytoconstituents have been found in V.

vinifera. Linoleic acid has been identified as major constituent in grape seed oil. Also,

lupane type triterpenoids (lupenone, lupeol, betulin etc.) have been identified in V. vinifera.

However, the relative proportion of highly polar and moderately polar constituents to that

of non-polar constituents seems to be more.

Extracts of fruits of V. vinifera have been exhaustively studied for the antioxidant

properties in various in vivo and in vitro models. Antifungal, antiulcer, hepatoprotective,

low density lipoprotein (LDL) inhibitory, hypoglycaemic and antihyperglycaemic

activities of fruits of V. vinifera have been scientifically proved. In addition to these, the

protective effect of V. vinifera in citrinin induced nephrotoxicosis indicates promising

effects in lithiasis.

Leaves of V. vinifera have shown to exhibit significant diuretic activity.

Procyanidolic oligomers from V. vinifera have shown effects on angiotensin-I converting

enzymes (ACE), suggesting their probable mode of action in diuresis. Thus, we presumed

that the polar to moderately polar phytoconstituents from roots of H. indicus and fruits of

V. vinifera may have a probable role in associated pharmacological potentials of the

individual plant.

From the literature reviewed, it is evident that B. ceiba contains various

phytoconstituents like sesquiterpene lactone, naphthoquinones, terpenoids, glycosides and

flavonoids. Mangiferin (2-beta-D-glucopyranosyl-1,3,6,7-tetrahydroxy-9H-xanthen-9-

one), Shamimin (2-(2,4,5-trihydroxy phenyl)-3,5,7-trihydroxy-6-C-glucopyranosyloxy-

4H-1-benzopyran-4-one), shamimicin (1"',1"""'-bis-2-(3,4-dihydroxyphenyl)-3,4-dihydro-

3,7-dihydroxy-5-O-xylopyranosyloxy-2H-1-benzopyran), lupeol have been from aerial

parts like leaves, stem bark and heartwood. While from roots, isohemigossypol-1,2-

dimethyl ether, 8-formyl-7-hydroxy-5-isopropyl-2-methoxy-3-methyl-l,4-naphthaquinone,

7-hydroxycadalene have been isolated. Since, most of these compounds are usually present

in glycosidic form within the plant; these have been isolated using single or binary

mixtures of various highly polar to moderately polar solvents like purified water, alcohol,

methanol, chloroform etc.

Many of these isolated compounds (shamimin, shamimicin) have been found to

possess significant antimicrobial properties against various bacteria and fungi. Anti-



helicobacter and anti-acne activity have also been scientifically proved. These activities

suggest that B. ceiba may prove efficacious against oxidative cellular damage in

nephrolithiasis. Urinary tract infection (UTI) is observed frequently in many cases of

lithiasis; wherein, Klebsiealla, Proteus and Pseudomonas species have been found to

contribute for deposition of staghorn calculi. B. ceiba, alone and in combination with other

plants, has been evaluated in various models for antiulcer, anti-diarrhoeal and anti-motility

activity. In addition, anti-inflammatory and hepatoprotective activities of B. ceiba may

support for its usefulness in relieving the associated pain in lithiatic patients.

Also, the toxicological profile suggests the dose dependent potency of a

hypotensive compound, shamimin at 15 mg/kg, 3mg/kg and 1 mg/kg; while, at larger dose

(500 mg/kg), it has been found to be useful as a hypoglycaemic compound.

Thus, the overall review of literature till date signifies that the crude extract of B.

ceiba with polar to moderately polar solvent may yield some important phytoconstituents

possessing pharmacological significance.

The literature survey on screening for pharmacological activities (like diuretic and

antiurolithiatic activity) reveal different methodologies opted by different researchers in

this regard. Most commonly, the diuretic activity is being studied by using model of

Lipschitz WL et al. (1943) with or without any modifications. Different reports on use of

standard diuretic drugs suggest the incorporation of Urea (100-1000 mg/kg, p.o.),

Frusemide (4-25 mg/kg, p.o.), Hydrochlorothiazide (10-25 mg/kg, p.o.) and

Spironolactone (10-50 mg/kg, p.o.) as reference diuretic drugs. The ratio of urinary output

exhibited by test extract treated rats to the urinary output exhibited by Urea treated rats

gives “Lipschitz-value”; which signifies the potency of test extract to induce diuresis in

normal rats. Also, the comparative assessment of test extract and standard diuretic treated

rats for parameters like urinary excretion of Na+, K

+ and Cl

- illustrate different constants

(like the saluretic activity, natriuretic activity, potassium-sparing effect and carbonic

anhydrase inhibition effect); which are characteristic of their diuretic potentials. These

constants are helpful in elucidating the probable mode of action of the test extract with

specific reference to the reference standard drug.

In assessment of antiurolithiatic activity, the serum levels of urea nitrogen,

creatinine and uric acid and the histopathological study of kidney elucidate the

comparative renal damage in control and test animals. Type of lithiasis and its associated

etiology can be determined by estimating various biochemical parameters like urinary



excretion of calcium, phosphate and oxalate and their accumulation in kidney (which can

be determined by evaluating the kidney homogenate). The curative and prophylactic study

of plant extracts elucidate the mechanism of action and its probable role in the inhibition of

stone formation.

Citrates, magnesium, allopurinol, thiazide and dietary modification are commonly

suggested for the treatment of various combined metabolic disturbances like

hypocitraturia, hypomagnesuria, hyperuricosuria, low or high urinary pH and

hyperoxaluria. The existing marketed polyherbal formulations, like Cystone, Calcuri and

Chandraprabha Bati are also the remedy of choice and have been widely used clinically to

dissolve urinary calculi in the kidney and urinary bladder. In this regard, till date, many

plants have been screened in search of potentially active antiurolithiatic principles and

many more are yet to be explored.

Thus, with reference to the overall literature available till date and in accordance

with the proposed research protocol (as approved by KLE University, Belgaum), diuretic

and antiurolithiatic activities were planned and conducted in two phases. In the first phase,

the diuretic activity was conducted. A dose-response study was carried out using two doses

for each extract (viz., 1/10th

and 1/5th

of LD50-cut-off dose). Based on the results of diuretic

activity, in the second phase, the antiurolithiatic activity was conducted using only the

higher dose of each individual extract of all three plants.

From the available literature, it is evident that the bioactive extract showing

diuretic potentials may yield some important phytoconstituents; that may also be effective

as antiurolithiatic agents. The results of other co-researchers ascribe the free radical

scavenging properties to polyphenolic constituents. Since, plants selected in the present

study have been reported to contain abundant of polyphenolic constituents, it can be

inferred that these polyphenolic compounds may have an unambiguous role to prevent the

nephritic tissue damage. The qualitative and quantitative determination of total

polyphenolic contents by various methods like Prussian-Blue and/or Folin-Ciocalteu

method, would ascertain the quantum of bioactive polyphenolic constituents in these

plants.

Traditionally, chromatographic techniques such as high performance liquid

chromatography (HPLC) and gas chromatography (GC) in conjunction with other

analytical techniques have been used to determine the purity and strength of a specific lot

of a compound (for the purpose of qualifying the lot to use as a reference standard).



Alternatively, an absolute method such as differential scanning calorimetry (DSC) can be

used for verifying the assigned purity of the reference standard.

Also, the use of hyphenated techniques has advanced the research methodologies

to a great extent. GC-MS have replaced the conventional GC in determination of the

volatile compounds. The advanced library search (like NIST) is proving as a helpful tool in

mass interpretation and structure elucidation. Other advances include use of HPTLC

replacing TLC for separation and isolation of individual phytoconstituent(s) of interest.

Also, the HPLC profiling studies have advanced with use of photodiode array (PDA)

detectors for determination of relative purity of isolated entity. LC-MS/MS and LC-NMR

are recently available sophisticated analytical techniques for more precise research in

phytochemistry.

The overall literature promotes the use of advanced phytochemical tools in

separation, isolation and characterization of potent molecule(s) from bioactive

extracts/fractions of plants under investigation. It was presumed that the pharmacological

activity exhibited by bioactive extracts may be solely due to the major phytoconstituent

present in it. Accordingly, the scheme of the research work was designed and carried out in

the present study.

Chapter 3 Introduction to Plants and Review of Literature 3....

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