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CHAPTER 2 LITERATURE REVIEW

Ph.D. Thesis (2015), Integral University 0

CHAPTER 2

LITERATURE REVIEW

2.1 Bauhinia racemosa Lam

2.1.1 Background

Bauhinia racemosa Lam, belonging to the family Caesalpiniaceae is a small

deciduous tree used in the indigenous system of medicine [1,2]. Its synonym

is Bauhinia vahlii Wight & Arn [3]. It is named as Mountain ebony in

English, Kachnal/Kanchanara/Sonpatta in Hindi, Gul-e-anehnal in Urdu and

Sona/Sonpatta or Apta in Marathi [4]. It is of religious significance. It is being

considered to be as valuable as gold for its medicinal values. Therefore, there

is a ritual of exchanging Sonpatta (leaf of gold) leaves during Dussehra in

India. It teaches to enjoy the joy of giving [5,6]. It is having antifilarial

activity, abortifacient, anti-anxiety, anthelmintic, antimicrobial,

antihistaminic, anti-inflammatory, analgesic, and antipyretic effects,

antimalarial, anti-oxidant, anticarcinogenic, antitumor, anti-ulcerogenic,

hepatoprotective, and various other pharmacological activities. It is

traditionally used in for the treatment of various ailments like diarrhoea,

dysentery, headache, jaundice, etc.

Flowers are white and appear in axillary or terminal racemes.

Flowering takes place in March to June. Fruit is a pod, oblong, compressed,

often twisted and dark green. Fruiting takes place through the season. Bark is

rough and black. Leaves are orbicular, bifoliate and alternate distichous with

entire margins. Their apices are mucronate and bases are chordate (Figure

2.1).

Bauhinia (Caesalpiniaceae) is mostly present in lowland and drier

forest types of northwestern South America, extending to Brazil and

Argentina, recorded in cerrado [7]. B. racemosa is very common in foothills

upto 1000 m in India and Srilanka. It is inhabited in semi-arid region of


Ph.D. Thesis (2015), Integral University Page 11

Rajasthan in India along with various fodder trees like Prosopis cineraria,

Acacia nilotica, Albezia lebbek, Ailanthes excelsa, Azardirachta indica [8]. It

is a tropical dry thorn found in transects in the Nilgiri landscape of Western

Ghats in India [9,10].

Mean wood specific gravity (WSG, g cm-3

), height range (m) and mean

basal area (BA, m2 ha

-1), carbon density (CD, kg-C ha

-1), carbon accumulation

rate per unit basal area (CAb, kg-C m-2

yr-1

) and carbon accumulation rate per

unit ground area (CAa, kg-C ha-1

yr-1

) of the tree B. racemosa on Hathinala

site of tropical dry forest were found to be 0.57, 3.4-4.5, 0.08, 430, 523, 41.8

respectively [11]. Density, basal area (m2 ha

-1) and importance value index

(IVI) of woody species B. racemosa in the valley of the Slopka forest were

10±10, 0.06±0.06 and 1.9 respectively. It’s IVI on different aspects of hill

slopes (valley, east-facing slope and south-facing slope) in the undisturbed

Slopka forest (the average of 30 quadrats of 100 m2) were 1.96, 2.99 and 3.18

respectively. Density, basal area (cm2 100 m

-2) and IVI in relation to human

disturbance in the Sariska Tiger Project (based on 130 quadrats of 100 m2

each in each of the study site Slopka forest-undisturbed, Kalighati forest-

protected and Bharthari forest-partially disturbed) were 0.30, 18.96, 1.63;

0.13, 8.06, 2.92 and 0.07, 1.32, 1.23 respectivrly [12].

It is found in highly disturbed stand of dry deciduous forest of Western

Ghats, India too along with Albizia amara and Pleiospemium alatum [13]. It

is one of the many new seedlings emerging indicating better regeneration

potential in the low lantana cover site of Vindhyan tropical dry deciduous

forest of India [14]. Its seeds, in Vindhyan hill tract in the Sonebhadra district

of Uttar Pradesh, India have the weight of 0.124 g/seed and are relatively

shade-intolerant [15]. It is propagated easily from seed and can grow in poor

and even very harsh climatic conditions [5].

CHAPTER 2

Ph.D. Thesis (2015), Integral University

Figure 2.1: (A) Whole tree of

flower of Bauhinia racemosa.

length.

2.1.2 Traditional Uses

Bauhinia racemosa is one of the plants commonly used by Ayurvedic doctors

for the prevention and treatment of cancer [4]. Traditionally, mixture of its

bark and Bridelia retusa

area of southern Rajasthan and act as co

and the locales of the Shiwalik Himalaya of Uttarakhand utilize it in the form

of different products as ethnomedicine, fodder, food, fibre etc. It is

commercially exploited by drug dealers and it come in threatened cate

Therefore, there is an urgent need of its conservation for sustainable

development [18]. This is one of the plants eaten by the Rhesus monkey

(Macaca mulatta) and Hanuman langur (

Pradesh [19].

Its stem bark is kept i

two people and air is blown in patient’s ear, after which a glass of water is

given to drink to cure scorpion bite [20]. Pounded bark powder of Kachnal

2 LITERATURE REVIEW

Thesis (2015), Integral University

(A) Whole tree of Bauhinia racemosa. (B) Twig with white

racemosa. (C) and (D) Twigs with leaves broader than

Traditional Uses

is one of the plants commonly used by Ayurvedic doctors


Bridelia retusa is given orally to women to develop sterility in tribal

area of southern Rajasthan and act as contraceptive [16,17]. The folk people



commercially exploited by drug dealers and it come in threatened cate



) and Hanuman langur (Presbytis entellus) in Himachal

Its stem bark is kept in house as snake repellent. Leaves are chewed by



LITERATURE REVIEW

Page 12

(B) Twig with white

(C) and (D) Twigs with leaves broader than

is one of the plants commonly used by Ayurvedic doctors


is given orally to women to develop sterility in tribal

ntraceptive [16,17]. The folk people



commercially exploited by drug dealers and it come in threatened categories.



) in Himachal

n house as snake repellent. Leaves are chewed by





Tree, in 10 g quantity, or decoction with water in 20 ml quantity, is used

twice per day for chronic dysentery and diarrhoea in the tribals of Sonaghati

of Sonbhadra district, Uttar Pradesh, India [21,22]. It is used in headache also

[22].

Whole plant juices either wild or cultivated, administered with

vegetable oil orally is used in leucorrhoea by 5% people of Sariska region of

Rajasthan [23]. For the treatment of jaundice, water extracts of its bark, leaves

and roots are taken two times daily after meal for 2-4 weeks in the region of

Jalgaon, Dhuleand, Nandurbar districts of Maharashtra and also the stem bark

of Woodfordia fructicosa (L.) Kurz ground with its bark and Oroxylum

indicum are taken as poultice administered in two spoonfuls banana fruit

twice a day for 5-7 days in Adilabad district of Andhra Pradesh [24].

Leaves of B. racemosa, Aloe vera and P. murex crushed together and mixed

with water given to animals three times a day can relief food poisoning in

cattle [25].

2.1.3 Phytochemistry

Phytochemical screening of the plant leaves reveals the presence of

carbohydrates [26-29], alkaloids [26-29], steroids [26,28,29], glycosides

[28,30], tannins [26-30], saponins [27,28,30], phenolic compounds

[27,28,30], flavonoids [26,27,28,30,31], protein [27,30], oil and fats [27,28].

Bioassay guided fractionation of ethanolic extract of the leaves led to the

isolation of galactolipid and catechin class of the compounds (A-G) (Figure

2.2) from the most active n-butanol fraction [31]. The screening of the

heartwood reveals the presence of polycyclic phenolics, racemosol and

pacharin [31-33]. The screening of the stem bark reveals the presence of

triterpenoids and sterols [31]. Phytochemical screening of the seed reveals the

presence of palmitic and stearic acids [34]. It also contains phenolics,

flavonoids, saponins, glycosides, tannins and proteins [30].



De-O-methylracemosol (Figure 2.3) was obtained from the column

chromatographic separation of a benzene extract of the roots, recrystallized

from benzene as brown crystals [35]. Racemosol and de-O-methylracemosol

were synthesized by Sae-Lao et al (2006) [36]. The key steps involved were

the lateral lithiation reaction of O-methyl tolulate and the pyran formation via

a tandem demethylation-cyclization reaction. The structure of pacharin,

previously isolated from the heartwood of B. racemosa Lamk has been

established as 1,7-dihydroxy-3-methoxy-2-methyl-dibenzo(2,3-6,7)oxepin by

a study of its chemical and spectroscopic properties, including X-ray analysis

[33,37]. Resveratrol (trans-3,5,4’-trihydroxystilbene), a phytoalexin is

obtained from several plants and also from B. racemosa [38].

Crude fibre, fat and carbohydrate contents were found to be 1.91%,

15.53% and 36.12% respectively in B. racemosa seeds. Its saponification and

acid values were found to be 343.72. 5.64 [39]. Crude proteins, crude lipids,

ash and nitrogen free extractives constitute 19.84%, 9.52%, 3.31%, and

60.65%, respectively in Ayyanarkoil Forest germplasm of the tribal pulse, B.

racemosa Lamk; whereas, in Mundanthurai Wildlife Sanctuary germplasm

they constitute 19.31%, 8.94%, 3.81% and 61.30% respectively. The caloric

values were found to be 407.64 KCal (Ayyanarkoil Forest germplasm) and

402.90 KCal (Mundanthurai Wildlife Sanctuary germplasm). Essential amino

acids like isoleucine, tyrosine, phenylalanine and lysine were found to be high

in the seed proteins of both the germplasms. The fatty acids, palmitic, oleic

and linoleic acids were found to be relatively higher in the seed lipids of both

the germplasms. Both the germplasms seemed to be a rich source of calcium,

potassium, magnesium, zinc, manganese and iron. Antinutritional substances

like total free phenols, tannins, L-DOPA and phytohaemagglutinating activity

were also investigated [40].



Dahikar et al. (2011) showed that petroleum ether extract of B.

racemosa (PEEBR) had only alkaloids and steroids occurring in high

concentration [26]. The chloroform extract (CEBR) and ethyl acetate extract

(EAEBR) of B. racemosa had similar photochemical constituents, alkaloids

and steroids in higher concentration, carbohydrates, flavonoids, glycosides

and saponins. Methanol extract of B. racemosa (MEBR) had alkaloids,

steroids, flavonoids, saponins and tannins, but no protein. Kumar et al. (2010)

found the presence of protein, oil and fats, phenolic compounds, flavonoids,

alkaloids, saponins, tannins and carbohydrates as major phytochemical groups

in crude MEBR and aqueous extract of B. racemosa (AEBR) leaf [27].

Manohar et al. (2011) found the presence of carbohydrates, glycosides,

alkaloids, phytosterols, saponins, tannins, phenolics, flavanoids, fixed oils and

fats as phytochemicals in crude MEBR and AEBR leaf [28]. Anjaneyulu et al.

(1986) reported a new tetracyclic phenol, racemosol from the hexane extract

(HEBR) of the heart-wood of B. racemosa Lamk [32]. Racemosol was

crystallized as deep red prisms from chloroform. It is soluble in alkali giving

yellow colour which reverts to the red colour on acidification. Racemosol

diacetate and racemosol dimethylether was synthesized by him. Prakash and

Khosa (1976) reported octacosane, β-amyrin and β-sitosterol in PEEBR stem

bark from column chromatographic resolution over Brockmann alumina [41].

Elution of the column with petroleum ether (60°-80°) furnished octacosane,

with benzene β-amyrin and with benzene:chloroform (3:1) β-sitosterol.



Figure 2.2: Isolated phytoconstituents (A-H) from the leaves of Bauhinia

racemosa [Galactolipids: (2S)-1,2-di-O-linolenoyl-3-O-α-

galactopyranosyl(1→6)-O-β-galactopyranosyl glycerol (A), (2S)-1-O-



linolenoyl-2-O-palmitoyl-3-O-α-galactopyranosyl(1→6)-O-β-

galactopyranosyl glycerol (B), (2S)-1-O-oleoyl-2-O-palmitoyl-3-O-α-

galactopyranosyl(1→6)-O-β-galactopyranosyl glycerol (C); Catechins: (−)-

epiafzelechin (D), (−)-epicatechin (F), (−)-catechin (G), protocatechuic acid

(E); and Long chain fatty acid alcohol: octacosanol (H)].

Figure 2.3: Other isolated phytoconstituents from Bauhinia racemosa

[pacharin (A), racemosol (B), de-O-methylracemosol (C), resveratrol (D),

octacosane (E), β-amyrin (F), β-sitosterol (G)].

2.1.4 Pharmacology

Bauhinia racemosa is having antifilarial activity [31], abortifacient [42], anti-

anxiety [43], anthelmintic [44], antimicrobial [26,27,28,29,45,46],

antihistaminic [47], anti-inflammatory [48], analgesic [48-50], and antipyretic

effects [48,51], antimalarial [52], anti-oxidant [46,53,54,55], anticarcinogenic

[56], antitumor [54], anti-ulcerogenic [57,58], hepatoprotective [55,59], and

various other pharmacological activities [60].



It does not induce any toxic effects [61]. Its seed mucilage is used as a

binder for pharmaceutical dosage forms. It was found to be useful for the

preparation of uncoated tablet dosage form in 8% w/w binder concentration

[62].

2.1.4.1 Anti-anxiety activity

The extract MEBR, administered orally (po) in two different doses of 150

mg/kg and 300 mg/kg body weight (b wt) in adult male Swiss albino mice,

was able to increase the time spent and the number of arm entries in the open

arms of the elevated plus-maze. It also increased the time spent by mice in the

illuminated side of the light-dark test, showed significant increase in nose

poking and decrease locomotion in hole board test, as well as caused

significant reduction in freezing (immobility with rigid body posture) time in

comparison with control animals. These effects were comparable to that of the

diazepam (2.0 mg/kg b wt, po) indicating that MEBR is an effective

anxiolytic agent [43].

2.1.4.2 Antifilarial activity

The n-butanol fraction of ethanolic extract of B. racemosa (EEBR) showed

promising adulticidal (IC50 5.46 mg/mL) and microfilaricidal (IC50 4.89

mg/ml) activities with Minimum Inhibitory Concentration (MIC) of 15.6

mg/ml. Among the active galactolipids, 1 emerged as the lead molecule which

was active on both forms of lymphatic filarial parasite, Brugia malayi. It was

found to be better than the standard drug ivermectin and diethylcarbamazine

(DEC) in terms of dose and efficacy [31].

2.1.4.3 Anthelmintic activity

AEBR, EEBR and PEEBR whole plant of B. racemosa have anthelmintic

activity. The crude EEBR significantly demonstrated paralysis and also

caused death of earthworm (Pheretima posthuma) in dose dependent manner



(50, 75 and 100 mg/ml) in in vitro study, while AEBR and PEEBR showed

weak anthelmintic effect compared to albendazole [44].

2.1.4.4 Antihistaminic activity

EEBR leaves inhibited clonidine-induced catalepsy in male Swiss albino mice

at a dose of 50 mg/kg b wt, admistered intraperitoneally (ip). But, there was

no effect on haloperidol-induced catalepsy suggesting that the inhibition is

through an antihistaminic action and that there is no role of dopamine. It has

role in the treatment of asthma [47].

2.1.4.5 Anti-inflammatory, analgesic and antipyretic effects

MEBR possesses potent anti-inflammatory, analgesic and antipyretic

activities. In acute phase of inflammation, a maximum inhibition of 44.9,

43.2, 44.8 and 45.9% (p<0.001) was noted at the dose of 200 mg/kg b wt of

Wistar albino rats after 3 h of treatment with MEBR in carrageenan, dextran,

histamine and serotonin-induced paw oedema, respectively. Administration of

MEBR (200 mg/kg b wt) and indomethacin (10 mg/kg b wt) significantly

(p<0.05) decreased the formation of granuloma tissue induced by cotton pellet

method at a rate of 50.4 and 56.2%, respectively. The extract also inhibited

peritoneal leukocyte migration in mice. The MEBR also produced significant

(p<0.01) analgesic activity in mice in the models, acetic acid-induced

writhing and hotplate tests. Further, the MEBR potentiated the morphine- and

aspirin-induced analgesic action. Treatment with MEBR showed a significant

(p<0.01) dose-dependent reduction in yeast-induced hyperpyrexia in rats [48].

AEBR stem bark at the dose of 200 mg/kg b wt produced significant

analgesic activity by ‘Tail Immersion Method’ in Wistar rats whereas

100mg/kg dose did not produce significant results when compared with

control receiving only normal saline (p<0.01). EEBR produced significant

results at both the doses (p<0.01) [49].



EEBR stem bark shows marked antipyretic activity in a dose dependent

manner in healthy Wistar albino rats with 50% sex ratio where the pyrexia

was induced by injecting a suspension of 15% of brewer’s yeast and 2% gum

acacia in normal saline subcutaneously below the nape of neck in the volume

of 1 ml/100 gm of animal weight. The temperature was brought back to

normal after 4 hrs of post administration of extracts. AEBR in a dose of 200

mg/kg b wt and EEBR in the doses of 100 mg/kg and 200 mg/kg b wt was

found to have significant effect and was found significant at 5% level of

significance [51].

2.1.4.6 Antimalarial activity

Racemosol and de-O-methylracemosol exhibited cytotoxicity against KB cell

line (EC50 at 15.0 µg/ml and 5.6 µg/ml, respectively) and BC cell line (EC50 at

6.1 µg/ml and 3.6 µg/ml, respectively) exhibiting moderate antimalarial

activity against parasite Plasmodium falciparum (EC50 at 0.9 µg/ml and 2

µg/ml, respectively) [52].

2.1.4.7 Antimicrobial activity

In an attempt to develop a new pharmaceutical drug from natural origin for

prevention of enteric infection, antibacterial activity of various extract of the

leaves was carried out by disc diffusion method against certain enteric

bacterial pathogens such as E. coli, S. aureus, Klebsiella pneumonia,

Enterobacter aerogenes, P. aeruginosa, Salmonella typhimurium, S. typhi,

Staphylococcus epidermidis and Proteus vulgaris and found that MEBR had

wide range of antibacterial activity against enteric bacterial pathogens than

PEEBR, where as EAEBR was of slightly higher antibacterial activity than

CEBR [26].

MEBR leaves showed significantly higher inhibitory effect compared

to AEBR on tested organisms. The MEBR showed a broad spectrum of

antimicrobial activity in agar well diffusion method as it inhibited Gram



negative bacteria (E. coli, Micrococcus luteus and P. aeruginosa), Gram

positive bacteria (Bacillus subtilis) on Mueller hinton agar and fungi

(Candida albicans and A. niger) on Sabouraud dextrose agar. Both the

extracts showed maximum relative percentage inhibition against A. niger.

MIC values determined by modified agar well diffusion method for MEBR

varied from 1.5-25 mg/ml [27].

The antimicrobial studies of AEBR and MEBR leaves were carried out

by cup-plate agar diffusion method against bacterial pathogens B. subtilies, S.

typhi, S. aureus (zone of inhibition of MEBR: 6 mm, 6 mm, 7 mm

respectively and that of AEBR: 4 mm, 4 mm, 5 mm respectively). It

concluded that MEBR was having good antimicrobial activity [28]. MEBR

bark had wide range of antimicrobial activity against enteric bacterial

pathogens such as E. coli, S. aureus, B. subtilis, P. aeruginosa and fungi A.

niger and C. albicans than EEBR, where as EEBR had slightly higher

antibacterial activity than AEBR in agar well diffusion method [29].

As compared to HEBR, the MEBR extracts of all the examined sixteen

plants of the family Caesalpiniaceae, collected around Karachi, Pakistan,

showed stronger growth inhibitions against both bacteria and fungi. HEBR

showed antibacterial activity against Corynebacterium diptheriae;

Escherichia coli; Salmonella typhi; Staphylococcus aureus; Staphylococcus

pyogenes while MEBR against Bacillus cereus; Shigella sonii; Pseudomonas

aeruginosa; S. typhi; Shigella boydii; S. aureus; S. pyogenes. HEBR also

showed antifungal activity against Trichophyton longifuses; Pseudallescheria

boydii; Aspergillus niger; Microsporum canis while MEBR against T.

longifuses; P. boydii; M. canis; Trichophyton mentagrophytes; Trichophyton

simii; Fusarium solani; Macrophomina phaseolina; Trichophyton

schoenleinii [43].



The anti-oxidant activity of the MEBR bark increased in a

concentration dependent manner. About 50, 100, 250 and 500 µg/ml MEBR

inhibited the peroxidation of a linoleic acid emulsion by 62.43, 67.21, 71.04

and 76.83%, respectively. Similarly, the effect of MEBR on reducing power

increased in a concentration-dependent manner. The IC50 values were found

to be 152.29 µg/ml, 78.34 µg/ml and more than 1000 µg/ml for DPPH radical-

scavenging, nitric oxide-radical scavenging and hydroxyl radical-scavenging

assays, respectively. MEBR scavenged also the superoxide generated by the

PMS/NADH-NBT system. Total phenolic content was also determined and

64.7 µg pyrocatechol phenol equivalent was detected in MEBR (1 mg).

MEBR showed broad-spectrum antimicrobial activity determined by disc

diffusion against all tested microorganisms; four Gram positive bacteria S.

aureus, Streptococcus pneumonia, M. luteus, S. epidefrmidis, five Gram

negative bacteria P. aeruginosa, E. coli, S. typhi, Shigella dysenteriae, Vibrio

cholerae and four fungal species C. albicans, A. niger, Aspergillus flavus, and

Alternaria solani. The results obtained indicate that MEBR can be a potential

source of natural antioxidant and antimicrobial agents [46].

2.1.4.8 Anti-oxidant activity

AEBR leaves showed good anti-oxidant activity in different systems of

assays. IC50 values found were 1739, 536, 216 and 797 µg/ml for hydroxyl

radical-scavenging, superoxide radical-scavenging, DPPH radical-scavenging

and lipid peroxidation preventive activities, respectively. Total antioxidant

capacity was found to be 16.5 and 58.1 gallic acid equivalent, GAE and

amino acid equivalent, AAE (µg/mg plant material) respectively. Phenol and

flavonoid content was found to be 150 GAE and 13 Catechin Equivalent

(µg/mg plant material) respectively [53].



2.1.4.9 Antitumor and anticarcinogenic activity

Administration of MEBR bark was able to suppress neoplastic nodule

development/hepatocellular lesion formation in .-nitrosodiethylamine

induced hepatocarcinogenesis in male Wistar albino rats. The extract

treatment increased the level of antioxidants vitamin C, vitamin E, reduced

glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT), and

dramatically decreased lipid peroxidation levels indicated by low level of

malondialdehyde (MDA). MEBR also produced a protective effect by

decreasing the levels of serum enzymes (SGOT, SGPT and ALP) and

bilirubin, and increasing the protein and uric acid levels. It suggested that

MEBR exert chemopreventive effects by suppressing nodule development

and decreasing lipid peroxidation and enhancing the levels of antioxidants in

NDEA carcinogenesis by reducing the formation of free radicals [56].

The MEBR bark (at the doses of 50, 100, and 200 mg/kg b wt) showed

decrease in transplantable murine tumor volume, packed cell volume and

viable cell count, and increase in nonviable cell count and mean survival time

thereby increasing life span of Ehrlich Ascites Carcinoma (EAC) tumor

bearing mice. Hematological profile (hemoglobin content, red blood cell and

white blood cell counts) reverted to more or less normal levels in extract

treated mice. The level of lipid peroxidation was decreased and that of GSH,

SOD and CAT was increased. Thus, MEBR bark exhibits antitumor effect by

modulating lipid peroxidation and augmenting antioxidant defense system in

EAC bearing mice. Gupta M et al proposed that the additive and synergistic

antioxidant activity of phytochemicals such as flavonoids, triterpenoids,

steroids, etc, present in MEBR bark are responsible for its potent antitumor

activity [54].



2.1.4.10 Anti-ulcerogenic activity

The effects of MEBR (flower buds) on the volume of gastric juice secreted,

acid output, peptic activity, mucin activity and curative ratio were recorded in

aspirin-induced gastric ulcers in Sprague-Dawley rats. It showed significant

decrease in ulcer index [57]. AEBR dried fruit powder (200 mg/kg b wt) and

EEBR (100 mg/kg and 200 mg/kg b wt) produced antiulcer activity in Wistar

albino rats when the treated rats were administered paracetamol at a dose rate

of 200 mg/kg b wt orally after one hour [58]. After 24 h, the number of ulcers,

ulcer score, percent incidence, ulcer index and healing index were recorded as

anti-ulcer parameters.

2.1.4.11 Hepatoprotective activity

The MEBR bark at the doses of 50, 100 and 200 mg/kg and silymarin at 25

mg/kg b wt produced significant (p<0.05) hepatoprotective effect by

decreasing the activity of serum enzymes (serum transaminases; SGOT,

SGPT and ALP), bilirubin and lipid peroxidation and significantly increasing

the levels of GSH, SOD, CAT and protein in a dose dependant manner in

different groups of Wistar albino rats administered with paracetamol (500

mg/kg, po once in a day for 7 days) and carbon tetrachloride (30% CCl4, 1

ml/kg b wt in liquid paraffin 3 doses ip at 72 h interval). MEBR also showed

antioxidant effects on FeCl2-ascorbate induced lipid peroxidation in rat liver

homogenate and on superoxide scavenging activity. From these results, it was

suggested that MEBR could protect the liver cells from paracetamol and

CCl4-induced liver damages perhaps, by its antioxidative effect on

hepatocytes, hence eliminating the deleterious effects of toxic metabolites

from paracetamol or CCl4 [55].

B. racemosa stem bark was found effective as hepatoprotective,

through in vivo and histopathological studies of paraffin sections of Male

Wistar albino rat’s liver and biochemical parameters. The animals treated



with EAEBR exhibited significant liver protection against the toxicant as

evident by the presence of normal hepatic cords, absence of necrosis and

lesser fatty infiltration. The hepatoprotective effect of EAEBR was

comparable to that of silymarin, a standard hepatoprotective agent [59].

2.1.4.12 Toxicological effects

MEBR bark administered ip at the doses of 100 and 200 mg/kg b wt twice a

week for thirteen weeks to Swiss albino mice did not induce any toxic effects.

Adverse effect was noted at the dose of 400 mg/kg b wt. Its administration at

the dose of 400 mg/kg b wt significantly elevated the levels of bilirubin,

serum enzymes (SGOT, SGPT and ALP) and altered the level of urea, uric

acid, creatinine, cholesterol, glucose and hematological parameters

(hemoglobin content, total count of RBC and WBC, differential count of

leucocytes such as neutrophil, lymphocyte, monocyte, hematocrit, mean cell

volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin

concentration and platelet count) [61].

2.1.5 Marketed Formulation

Masanumas2 is a tablet formulation for the second month of pregnancy. Each

coated tablet contains- Ashmantak (Bauhinia racemosa) bark extract, Black

Sesame (Sesmum indicum) seed extract, Manjishtha (Rubia cordifolia) stem

extract and Shatavari (Asparagus recemosus) root extract 60 mg each. Each

pack contains 120 tabs. Dose: 2 tablets with lunch and 2 tablets with dinner.

Indications: Habitual abortions, repeated abortions, high value pregnancies,

high risk pregnancies [63].

The extensive literature survey revealed B. racemosa to be a valuable

and important medicinal plant used for the treatment of several ailments like

headache, fever, skin and blood diseases, jaundice, chronic dysentery,

diarrhoea, and leucorrhoea, infection of malaria, boil, glandular swelling,

tumors and cancer. It is also used to cure scorpion bite, to relief food



poisoning in cattle and as contraceptive by women. Pharmacological studies

carried out on the fresh plant materials, crude extracts and isolated active

principles of B. racemosa provide a support for its numerous traditional uses.

Recent studies have been focused on evaluating the abortifacient activity,

anthelmintic, anti-anxiety, antimicrobial, antifilarial, anticarcinogenic, anti-

inflammatory, analgesic and antipyretic effects, hepatoprotective, antitumor,

anti-ulcerogenic, antihistaminic and antimalarial activities. Most of the

mentioned pharmacological studies were aimed on validating its traditional

uses. The different parts of B. racemosa have been employed for the treatment

of various ailments. Most of the pharmacological studies that have been

carried out on B. racemosa were conducted using uncharacterized crude

extracts. Thus, it is difficult to reproduce the results of these studies and point

the bioactive principles. Hence, there is a need of phytochemical

standardization and bioactivity-guided identification of bioactive metabolites.

Phytochemical research carried out on B. racemosa had led to the isolation of

some active principles. However, the vast traditional use and proven

pharmacological activities of B. racemosa indicates that an immense scope

still exists for its phytochemical exploration. The outcome of such

phytochemical study may further expand its existing therapeutic potential.

The outcome of the future research will provide a convincing support for the

future clinical use of B. racemosa in modern medicine.

CHAPTER 2

Ph.D. Thesis (2015), Integral University

2.2 Cordia dichotoma

2.2.1 Background

Cordia dichotoma Linn

with 2-5 cm long petiole, 6

pubescent or glabrous, margin usually subundulate

grows in Sri Lanka, India and other warmer countries (

commonly known as Indian cherry in English and Lasora in Hindi. Its

medicinal properties is known since long time and traditionally used to cure

several ailments. Its fruits are used as expectorant, astringent, cooling,

emollient, purgative and ant

hepatoprotective and several other activities have also been reported from the

plant [66,67]. Instant Joshanda Granules, polyherbal formulations, are

extensively used by the people in India for the treatment of

catarrh, common cold, respiratory distress, catarrh, flu like ailments, fevers of

which Cordia dichotoma

Figure 2.4: Parts of Cordia dichotoma

and fruits; and (C) twig with

2.2.2 Traditional Uses

Leaf paste of Cordia dichotoma

treatment of diarrhea. Its leaves and fruits

from leucorrhoea [69]. The fruit of the plant is used as purgative, diuretic,

antihelminthic, useful in dry cough, wound purification, for cure of mouth

2 LITERATURE REVIEW

Thesis (2015), Integral University

Cordia dichotoma Linn

Linn belonging to family Boraginaceae is a 3

5 cm long petiole, 6-13×4-9 cm leaf blade ovate to elliptic, sparsely

pubescent or glabrous, margin usually subundulate to undulate dentate which

grows in Sri Lanka, India and other warmer countries (Figure 2.4




emollient, purgative and anthelmintic [65]. Anti-inflammatory, analgesic,


. Instant Joshanda Granules, polyherbal formulations, are

extensively used by the people in India for the treatment of cough, sore throat,


Cordia dichotoma is the chief ingredient [68].

Cordia dichotoma. (A) Whole plant; (B) twig with leaves

wig with flowers.

Traditional Uses

Cordia dichotoma is given to the animal with water for the

diarrhea. Its leaves and fruits are given to the animal suffering

. The fruit of the plant is used as purgative, diuretic,


LITERATURE REVIEW

Page 27

belonging to family Boraginaceae is a 3-4 m tall tree

9 cm leaf blade ovate to elliptic, sparsely

to undulate dentate which

2.4) [64]. It is




inflammatory, analgesic,


. Instant Joshanda Granules, polyherbal formulations, are

cough, sore throat,


. (A) Whole plant; (B) twig with leaves

is given to the animal with water for the

are given to the animal suffering

. The fruit of the plant is used as purgative, diuretic,




ulcer, jaundice and increase male potency [70,71]. Leaves are used for

headaches and ulcers including decoction for sore throat [72]. Bark is used in

the treatment of ulcerative colitis and colic pain [70]. 100 g powder of stem

bark is taken with a glass of water to cure menstrual disorders by Korku tribe

of Amravati district of Maharashtra, India [73]. It is used as abortifacient in

traditional/folkloric medicine [74]. The pickled fruit is eaten as an appetizer in

certain areas of Taiwan. The indigestible seeds and sticky pulp of the fruit can

form a phytobezoar if ingested excessively [75].

2.2.3 Phytochemistry

The dry powdered seeds of Cordia dichotoma were found to contain

glycosides, alkaloids, carbohydrates, tannins and saponins [76]. Alkaloids,

flavonoids, amino acids and proteins are present in the fruits of Cordia

dichotoma [70,71]. Qualitative phytochemical tests, thin layer

chromatography and TLC-bioautography of the ethanolic extract of leaves

demonstrated the presence of common phytocompounds phenols, tannins and

glycosides as major active constituents [77]. The antinutrional factors such as

phytic acid (0.2±0.1%), tannin (1.386±0.0152%) and oxalic acid

(2.133±0.2081%) were found to be present in the powdered wild edible fruits

of the plant. Saponin was absent [78]. Cordia dichotoma bark is identified as

botanical source of Shleshmataka in Ayurvedic pharmacopoeia [79].

Apigenin was isolated by column chromatography from methanol fraction of

crude methanol extract of C. dichotoma bark [80]. Arabinoglucan was

isolated from the fruits of Cordia dichotoma [81]. The phytoconstituents

arabinoglucan, D-glucose and L-arabinose are present in fruits; linolinic acid

in seed; and quercetin in leaves [82]. The structure and properties of the

newly identified natural cellulose fabrics from the branches of the Cordia

dichotoma indicated that they could be appropriate for blending and

processing by biodegradable polymers to make green composites [83]. The



mucilage isolated from the aqueous filtrate of macerated seeds, whose pulp

was previously removed, possess binding properties comparable to the starch.

An increase in mucilage concentration led to decrease in friability and

increase in disintegration time of the tablets [84].

2.2.4 Pharmacology

2.2.4.1 Analgesic, antibacterial and cytotoxic activities

The The crude ethanol extract of leaves of Cordia dichotoma produced

significant writhing inhibition in acetic acid induced writhing in mice at the

oral dose of 500 mg/kg b wt (p<0.001) which was comparable to that of the

standard drug diclofenac sodium at the dose of 25 mg/kg b wt. The extract

showed significant zone of inhibition against both Gram negative and Gram

positive bacteria Streptococcus aureus, Streptococcus pyogenes, Vibrio

cholerae, Streptococcus epidermis, Hafnia and Escherichia coli in disc

diffusion study which is compareable to that of Kanamycin (30 µg/ml). The

extract also showed potent toxicity against the brine shrimp Artemia salina

(LC50: 20 µg/ml and LC90:180 µg/ml) in brine shrimp lethality bioassay [66].

2.2.4.2 Anthelmentic activity

Five concentrations (10, 25, 50, 75 and 100 mg/ml) of ethanolic and aqueous

extracts prepared from pulp obtained after separation of seeds from fruits of

Cordia dichotoma by Soxhlet extraction were studied for anthelmentic

activity by using Eudrilus euginiae earthworms. Both ethanolic and aqueous

extracts showed paralysis and death of worms in concentration dependent

manner. Aqueous extract showed significant activity than ethanolic extract.

The extracts also showed the presence of alkaloid, glycosides, saponins,

flavonoid, triterpenoids, proteins, aminoacids and carbohydrates in

preliminary phytochemical investigations [65].



2.2.4.3 Antibacterial activity

Antibacterial activity of alcoholic and aqueous extracts of thirty four

medicinal plants including Cordia dichotoma were screened for potential

antibacterial activity against six bacterial strains belonging to

Enterobacteriaceae, viz. Escherichia coli, Enterobacter aerogenes, Klebsiella

pneumoniae, Salmonella typhimurium, Proteus mirabilis and Proteus vulgaris

by the agar well diffusion and disc diffusion methods. The ethanol extracts

were more active than aqueous extracts for all the plants studied. Aqueous

extract of Cordia dichotoma inhibited only to Proteus mirabilis, while

ethanolic extract inhibited to Klebsiella pneumonia and Proteus mirabilis

[85].

Antibacterial activity of Cordia dichotoma leaves extract studied by

two different methods i.e. well diffusion and disc diffusion methods against

procured strain of E. coli revealed that it possess antibacterial activity against

E. coli. The activity of extract is due to the flavonoid active constituent.

Phytochemical analysis showed positive result for tannins, protein and

flavonoids [86].

Antibacterial activity of petroleum ether, chloroform, methanol and

aqueous extracts of Cordia dichotoma ripened fruits against urinary tract

pathogens such as Pseudomonas aeruginosa, Escherichia coli, Klebsiella

pneumoniae, Proteus mirabilis, Proteus vulgaris and Staphylococcus aureus

using disc diffusion method was evaluated. Results of the study showed that

the methanol extract exhibited better antibacterial activity against the bacterial

strains as compared to other extracts [87].

2.2.4.4 Antioxidant activity or free radical scavenging activity

The methanolic extract of seeds and leaves of Cordia dichotoma

demonstrated positive antioxidant activity in a concentration dependent

manner during investigation of their free radical scavenging potential using in



vitro models viz. 1,1, diphenyl-2-picrylhydrazyl (DPPH) and hydrogen

peroxide model. The IC50 values for leaves were found 51.83 µg/ml and 55.73

µg/ml for DPPH and H2O2 models respectively. The IC50 values for seeds

were found 57.22 µg/ml and 48.28 µg/ml for DPPH and H2O2 models

respectively. In both the models, the methanolic extract of the leaves

exhibited low IC50 values as compared to the seeds’ extracts. Thus, this

activity was more pronounced in leaves as compared to seeds [88].

During nitroblue tetrazolium chloride (NBT) superoxide radical

scavenging assay, maximum antioxidant activity of ethanol and acetone

extracts of the fruits of the plant Cordia dichotoma was noticed to be 54.5 and

55.5% respectively. The inhibitory potential expressed in IC50 values of

acetone and ethanol extracts of C. dichotoma were found to be 131.0 and

149.0 µg/ml respectively [89].

The activity of peroxidase (POX), superoxide dismutase (SOD) and catalase

(CAT) was determined in the fruits. The SOD and CAT activities were

increased in mature fruits than ripened fruits, whereas POX activity was

found to be more in ripened fruits as compared to mature fruits [90].

C. dichotoma contains a considerable amount of phenols. The IC50

value was found to be 28 µg/ml for the methanolic extract and 36 for

butanolic extract in DPPH scavenging assay. The study revealed that the bark

has significant radical scavenging activity [79].

2.2.4.5 Antidiabetic activity

Marles and Farnsworth (1995) provided information on more than 1200

species of plants reported to have been used to treat diabetes and/or

investigated for antidiabetic activity, with a detailed review of representative

plants and some of great diversity of plant constituents with hypoglycemic

activity [91]. The aerial parts of Cordia dichotoma administered orally were

devoid of normal antidiabetic activity.



The dose of 500 and 1000 mg/kg of aqueous leaves extract

administered orally to alloxan induced and normoglycemic Wistar rats

showed a significant (p<0.5) decrease in blood glucose levels after 4, 8 and 24

h. The dose of 1000 mg/kg of the extract in normoglycemic rats significantly

(p<0.05) decreased the blood glucose levels at 8 and 24 h [92].

The methanol extract of fruits of Cordia dichotoma reduced the blood

glucose level in glucose loaded animal and alloxan induced diabetic animal

models when compared to diabetic control group and exerted significant

hypoglycemic and antidiabetic activities compared to standard drug

metformin. The extract also reduced the rate of body weight loss in normal

and alloxan induced diabetic animals [93].

2.2.4.6 Antimicrobial activity

Alcohol is found to be a better solvent for extraction of antimicrobially active

substances compared to water and hexane [94].

Ethanolic extract of the leaves showed antimicrobial activity only

against S. aureus (inhibition zone: 21-30 mm) and S. dysenteriae (inhibition

zone: 10-20 mm) in the study antimicrobial activity against certain drug-

resistant bacteria Salmonella paratyphi, Staphylococcus aureus, Escherichia

coli, Bacillus subtilis, Shigella dysenteriae and a yeast Candida albicans of

clinical origin. No correlation was observed between susceptibility of test

strains with the extract and antibiotic resistance behavior of the microbial

strains [77].

The acetone, chloroform, hexane, aqueous and ethanol extracts of the

C. dichotoma leaves and the chloroform, aqueous and acetone extracts of the

C. dichotoma stem bark did not show any antimicrobial activity against tested

strains of bacteria and fungi determined by macrobroth dilution method on the

concentration of 25 mg/ml to 0.10 mg/ml of the extracts. Only the hexane and

ethanol extracts were active against only certain fungal strains [95].



The acetone and ethanol extracts of Cordia dichotoma fruits exhibited

highest antimicrobial activity against Staphylococcus epidermidis,

Staphylococcus aureus, Escherichia coli, Candida albicans and Aspergillus

niger. Acetone extract showed maximum inhibitory zone of 19.1 mm whereas

no inhibitory effect was observed for the aqueous extract [89].

Petroleum ether, benzene, chloroform, acetone, methanol and aqueous

extracts of the plant were investigated for the antimicrobial efficacy by

modified Kirby-Bauer disc diffusion method against the clinical isolates of

oral cancer cases like the fungal pathogens Aspergillus fumigates, Candida

albicans and bacterial pathogens Escherichia coli, Staphylococcus aureus,

Staphylococcus epidermidis, Klebsiella pneumonia, Pseudomonas

aeruginosa, Proteus vulgaris, Proteus mirabilis. Only S. aureus was inhibited

significantly by the petroleum ether and acetone extracts (inhibition zone: 10

and 11.33 mm respectively; MIC: 250 and 125 µg/ml respectively) while only

S. epidermidis by aqueous extract (inhibition zone:10.33 mm; MIC: 125

µg/ml). Even, other isolates were not inhibited by the remaining extracts [96].

The methanol and butanol extracts of the bark showed remarkable zone

of inhibition of bacterial growth and fungal growth comparable with that of

standards drugs against the organisms tested Escherichia coli, Pseudomonas

aeruginosa, St. pyogenes, Staphylococcus aureus. Aspergillus niger,

A.clavatus, and Candida albicans. The activity of extracts increased linearly

with increase in extract concentration [97].

2.2.4.7 Antiulcer activity

Ethyl acetate, butanol and butanone fractions of ethanolic fruit extract

significantly decreased the volume of gastric secretion, free acidity, total

acidity and ulcer index with respect to control during the study of anti-ulcer

effect of fruit extracts (300 mg/kg b wt) in Wistar rats using pyloric ligation,

aspirin and indomethacin induced ulcer models [98].



The aqueous extract of fresh ripened fruits was found to be more

effective than alcoholic extract as compared to ranitidine in aspirin induced

gastric ulcer model (p<0.001) as well as in pylorus ligation model in rats [99].

2.2.4.8 Anti-inflammatory activity

The ethanol and aqueous extracts of Cordia dichotoma seeds at a dose of 250

mg/kg and 500 mg/kg orally showed significant activity compared with the

control and diclofenac sodium (10 mg/kg as standard) in dextran-induced paw

oedema and carrageenan-induced paw oedema models in rats on different

phases of acute inflammation [76].

Apigenin (5 mg/kg, po) showed significant healing and reduction in

inflammatory enzymes myeloperoxidase (from 360±0.2 U/ml due to acetic

acid induction to normal 222±22.5 U/ml due to treatment) and

malondialdehyde (from 9.98±1.5 nmol/ml to 2.11±1.5 nmol/ml) when

screened for ulcerative colitis induced by intrarectal administration of 150 µl,

5% acetic acid (pH 2.5) 3 cm from the anal margin. It was concluded that

apigenin from C. dichotoma bark may be responsible for the treatment of

ulcerative colitis [80].

The methanol fraction of methanolic extract of C. dichotoma bark

showed effective treatment of ulcerative colitis. It showed good healing and

lower pathological scores in treated animals. It significantly reduced MDA

and MPO levels in tissue and blood. It showed antioxidant potential and

contains a high level of phenolics [100].

2.2.4.9 Anti-cerebrovascular insufficiency activity

Long term cerebral hypoperfusion in rats resulted in propensity towards

listlessness and anxiety (elevated plus maze test and open field paradigm)

accompanied by deficits in memory and learning (Morri’s water maze test)

and tendency towards depression (Porsolt’s swim test). Gliosis, cellular

edema, astrocytosis and inflammatory changes were observed in forebrain.



Treatment by Cordia dichotoma (250 mg/kg po for 28 days) alleviated these

cognitive, behavioral and histopathological changes suggesting that Cordia

dichotoma may be useful in cerebrovascular insufficiency conditions [101].

2.2.4.10 Angiotensin-converting enzyme inhibitory activity

The ethanolic extract of bark showed high ability to inhibit the angiotensin

converting enzyme [102].

2.2.4.11 Diuretic activity

Petroleum ether, solvent ether and butanol fractions of alcoholic extract of the

fruits 300 mg/kg of body weight were tested for diuretic activity in rats for

total urine volume, urine concentration of Na+ and K

+ and showed increase in

cation excretion and urine volume [103].

2.2.4.12 Hepatoprotective activity

The methanolic extract of the leaves (300 mg/kg) significantly reduced the

ALT (p<0.001), AST (p<0.001) and TBR levels (p<0.01) and at 500 mg/kg

extract dose significantly reduced the AST ALT (p<0.001), (p<0.001), TBR

(p<0.01) and lipid peroxide levels (p<0.05) in male Wistar rats with liver

damage by carbon tetrachloride treatment [67].

2.2.4.13 Antifertility activity

The reversible nature of the developed phytopharmaceutical was studied by

performing pharmacological analyses followed by chronic toxicity studies.

The biochemical and histological estimations detected the reversible

contraceptive potential of the drug after withdrawal. The observations also

suggested that the developed phyto-pharmaceutical has potential antifertility

activity with safety aspects [104].

2.2.4.14 Wound healing activity

Petroleum ether, solvent ether, ethyl acetate, butanol and butanone successive

fractions of ethanolic extract of Cordia dichotoma fruits showed significant



(p<0.001) wound healing activity on excision, incision and dead space wound

models on Wistar albino rats [105].

2.2.5 Marketed Formulation

Instant Joshanda Granules, polyherbal formulations, are extensively used by

the people in India for the treatment of cough, sore throat, catarrh, common

cold, respiratory distress, catarrh, flu like ailments, fevers of which Cordia

dichotoma is the chief ingredient [68].



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