chapter 2 review of literature 2. review of literature...

CHAPTER 2 REVIEW OF LITERATURE

HNGU, PATAN PhD THESIS 27

2. REVIEW OF LITERATURE

2.1 Berberis aristata

The Plant Berberis aristata DC belongs to family Berberidaceae, known as Indian

barberry in English and „Daruhaldi‟ in Hindi. It is an erect spinous shrub, often found

in small patches on the hilly slopes. This shrub is found growing wild in the sub-

Himalayan tract at altitude ranging from 1000-3000m. It also grows in the Nilgiri hills

in South1.

2.1.1 Taxonomical Classification1

Kingdom : Plantae

Phylum : Angiosperms, Eudicots

Order : Ranunculales

Family : Berberidaceae

Genus : Berberis

Species : aristata

2.1.2 Synonyms1

Sanskrit : Suvarnavarna

Bengali : Darhaldi

Gujarati : Daruhaldar

Hindi : Daruhaldi

Tamil : Mullukala

2.1.3 Pharmacognosy

I. Macroscopic2 : It is an erect spiny shrub ranging between 2-3m in height.

Wood hard and yellow, bark yellow to brown from outside and deep yellow

from inside, removable in longitudinal strips by hand; spines (which, in fact,

are modified leaves), three-branched and 1.5cm long. Leaves, in bunch of 5 to

8, phyllotaxy verticillate, lanceolate, simple spiny, toothed, leathery, sessile,

acuminate, with reticulate pinnate venation, 4.9cm long, 1.8cm broad, deep

green on the dorsal surface and light green on the ventral surface.



Fig. 2.1 Photograph of Berberis aristata (shrub, roots and powder)

II. Microscopic3: The transverse section of a young stem shows a wavy outline

and it consists of a single layer of epidermis comprising of cubical to radial

elongated type of cells covered by a thick cuticle. Some of the epidermal cells

are elongated to form short unicellular trichomes whose walls are very much

thickened so as to leave a narrow lumen within them. They are non – lignified.

This is followed by cortex divided into three zones. The outer one is a narrow

zone of layers of parenchymatous cells with relatively thick walls and less

number of intercellular spaces. They may appear to be tanniferous showing

deep coloration on their walls. The middle zone consists of 4-6 layers of

sclerenchymatous layers which are strongly lignified. The third zone of cortex

is only 1-2 layers wide and is comprised of thin walled parenchymatous cells.

The xylem fibres appear to be concentrated in the lower part of the individual

vascular bundle in V shape and the xylem vessels are mostly concentrated in

the basal and peripheral part of the bundle. The trachides are short.

III. Medicinal uses: Antimicrobial, Antiprotozoal and Antidiarrheal activity 4

2.1.4 Chemical constituents

Berberis aristata contains berberine, oxyberberine, berbamine, armoline, karachine,

palmatine, oxyacanthine and taxilamine5. It also contains protoberberine and bis

isoquinoline type of alkaloid. Root contains alkaloid like berbamine, berberine,

oxycanthine, epiberberine, palmatine, dehydrocaroline, jatrorhizine and

columbamine6,7

, Karachine8, dihydrokarachine, taximaline

9, oxyberberine,

aromoline10

. Four alkaloids, pakistanine, l-O-methylpakistanine, pseudoberberine



chloride were also isolated from Berberis aristata11,12

. A secobisbenzlisoquinoline or

simple isoquinoline alkaloid was isolated13

. The major alkaloid found in Berberis

aristata is bright yellow colored berberine (2.23%) followed by palamatine14

. It is

manufactured mostly from roots of Berberis aristata (5% in roots and 4.2% in stem

bark)15

.

Fig. 2.2 Structure of Berberine

2.1.5 Pharmacological Action

I. Anti-microbial activity: The antimicrobial activity of hydroalcoholic extracts

of B. aristata, were tested against eleven bacterial, Micrococcus luteus,

Bacillus subtilis, Berberis cereus, Enterobactor aerogenus, Escherichia coli,

Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa,

Staphylococcus aureus, Salmonella typhimurium, Streptococcus pneumoniae

and eight fungal strains. The extract showed very low IC50, minimum

inhibitory concentration (MIC), minimum microcidal concentration (MMC)

and minimum microbistatic concentration (MMS) indicating anti-microbial

activity16

.

II. Antidepressant activity: Berberine possesses the ability to inhibit

monoamine oxidase-A, an enzyme involved in the degradation of nor

epinephrine and serotonin (5-HT). Berberine exerted anti-depressant like

effect in various behavioral paradigms of despair possibly by modulating brain

biogenic amines. Further, nitric oxide pathway or sigma receptors are involved

in mediating its antidepressant like activity in mouse forced swim test17

.

III. Diabetes Mellitus: Testing in animal model indicated that treatment with

berberine led to healthier pancreatic tissue compared to controls. It is

suggested that the mechanism of action of berberine may be associated with

promoting regeneration and functional recovery of β-cells18,19

.



IV. Hepatoprotective activity: B. aristata leaves and fruits showed

hepatoprotection possibly through inhibitory action on hepatic drug

metabolizing enzymes. Pretreatment of animals with berberine prevented the

acetaminophen or CCl4 induced rise in serum levels of ALP, AST and ALT,

suggestive of hepatoprotection. Post-treatment with berberine reduced the

hepatic damage induced by acetaminophen, while CCL4 induced

hepatotoxicity was not modified, suggesting a selective curative effect against

acetaminophen18,19

.

V. Inotropic activity: The cardiac action of n-butanolic fraction of B. aristata

showed dose-dependent positive inotropic action with little effect on heart

rate20

.

2.1.6 Analytical methods for estimation of berberine

Joshi and Kanaki21

developed and validated a UV spectrophotometric method for the

quantitative determination of berberine in rasayan-churna. The method involved

measurement of absorbance of sample solutions at the absorption maxima 348nm for

berberine. The method was validated by determining linearity, precision and accuracy

as per the ICH guidelines obeying Beer‟s law in the concentration range of 2-20µg/ml

and found quite accurate with a percentage recovery of 99.21%.

Shah et al.22

developed TLC densitometric method using HPTLC for the

quantification of berberine in polyherbal formulation. Of the various solvent systems

tried, the one containing toluene: ethylacetate: methanol: formic acid (3:3:0.2:0.8)

gave best resolution of berberine (Rf = 0.2). The peak response was linear within the

concentration range of 8-64ng/spot with a correlation coefficient of 0.997.

Rojsanga et al.23

developed TLC densitometric method for the estimation of berberine

using precoated silica gel G F(254) plates as stationary phase and butanol:glacial

acetic acid: water (14:3:4) as a mobile phase. Detection and quantitation were

performed at the wavelength of 415nm over the linearity range of 240 – 840ng (R2

=

0.9982).

Andola et al.24

developed HPTLC method for the estimation of berberine using silica

gel G F254 as stationary phase and n – propanol:water:formic acid (90:8:0.4) as mobile

phase. The detection was performed at 366nm over the linearity range of 2–8ng (R2

=

0.996).



Pasrija et al.25

developed and validated HPLC method using C18 column as stationary

phase and mobile phase consisting of acetonitrile and potassium dihydrogen

phosphate buffer pH 2.5 in a gradient flow. Elution was monitored at 346nm. The

linear regression analysis data for the calibration plots showed R2

= 0.9942 in the

concentration range of 16380 – 30420µg/ml demonstrating developed method was

simple, sensitive, selective and precise.

Patel26

developed specific, sensitive and validated HPTLC method using Silica gel 60

F254 as stationary phase and n-butanol: glacial acetic acid: water (12:3:4) as mobile

phase. Densitometry scanning was performed at 350nm over a concentration range of

50-250ng/spot with R2 = 0.997.

Shigwan et al.27

developed RP-HPLC method using photodiode array detector and

isocratic mode consisting of 0.1% trifluroacetic acid: acetonitrile (60:40) at a flow

rate of 1 ml/min. Linearity range was 0.2µg/ml–150µg/ml. The limit of detection was

1ng and limit of quantification was 2ng.

Leona and Lombardi28

developed a surface-enhanced Raman scattering method for

the identification of berberine in microscopic textile samples by employing citrate

reduced Ag colloid. Using FT-Raman and FT-SERS relative intensity shifts were

compared and adsorption geometry of berberine on Ag nanoparticles was obtained.

2.2 Curcuma Longa

Drug consists of the dried rhizome of Curcuma longa Linn. (Syn. Curcuma domestica

Valeton) belonging to family Zingiberaceae. The plant is cultivated in almost all the

states of India, particularly in Chotanagpur, Bihar, Agartala, Pareshnath, Naga Hills,

Coimbatore, Cochin and Bengal29

.

2.2.1 Taxonomical classification

Kingdom : Plantae

Phylum : Angiosperm

Order : Commelinids

Family : Zingiberaceae

Genus : Curcuma

Species : longa



2.2.2 Synonyms29

Sanskrit : Haridra, Nisa

Bengali : Haldi

Gujarati : Halada

Hindi : Haldi

Malyalam : Manjal

Kannad : Arishina

Tamil : Manjal

Telugu : Pasupu

2.2.3 Pharmacognosy

I. Macroscopic29

: The central or primary rhizome ovate, oblong or round

(pyriform) or cylindrical to elongate, conical and varies from 3–8cm in length

and 2 or 3cm in diameter. The sessile lateral branches 7–11cm in length and 1-

1.5cm in diameter. Externally yellowish to yellowish–brown with root scars

and annulations, the latter forms the scars of leaf bases; internally orange–

yellow to yellow. Odour aromatic; taste warmly aromatic and bitter.

Fig.2.3 Photograph of Curcuma longa (herb, rhizomes and powder)

II. Microscopic30,31

: Cork composed of thin walled brown cells which in surface

view appear large and polygonal. Epidermis with thick-walled, cubical cells of

various dimensions. Cortex characterized by the presence of mostly thin

walled, rounded parenchyma cells; which are filled with gelatinized starch

grains and permeted with a bright yellow colouring matter. Covering



trichomes found scattered; unicellular, elongated and bluntly pointed. Vascular

bundles scattered and are of collateral type. Vessels mainly spirally thickened;

a few with reticulate and annular thickening. Cell of ground tissue contains

starch grains.

III. Medicinal uses32

: Anti-inflammatory, Stomachic, tonic.


Major constituents are curcuminoids (6%), the yellow coloring principles, of

which curcumin constitutes 50 % - 60%33,

Essential oil (2-7%) with high

content of bisabolane derivatives34

.

Fig. 2.4 Structure of Curcumin

Minors are desmethoxycurcumin, bisdesmethoxycurcumin, dihydrocurcumin35

;

common phytosterols, fatty acids36

and polysaccharides37

, viz., ukonan A, B, C & D.

2.2.5 Pharmacological action

I. Anti-inflammatory activity38,39

: Oral administration of C. longa significantly

reduced inflammatory swelling. The activity is attributed to its ability to

inhibit both biosynthesis of inflammatory prostaglandins from arachidonic

acid, and neutrophil function during inflammatory states. Curcuminoids also

inhibit LOX, COX, and Phospholipases, leukotrienes, prostaglandins,

thromboxane, nitric oxide elastase, hyaluronidase and interleukin12. They also

decrease prostaglandin formation and inhibit leukotriene biosynthesis via the

lipoxygenase pathway.

II. Antioxidant activity40

: In vitro study of curcumin on endothelial heme

oxygenase-1, an indiucible stress protein was conducted using bovine aortic

endothelial cells. Incubation with curcumin resulted in enhanced cellular

resistance to oxidative damage. Water and fat soluble extracts of turmeric and



its curcumin component exhibit strong antioxidant activity, comparable to

vitamins C and E.

III. Hepatoprotective activity: Studies have demonstrated hepatoprotective

properties of turmeric from a variety of hepatotoxic injuries, including CCl4,

galactosamine and acetaminophen. Hepatoprotective effect is a result of its

antioxidant properties and its ability to decrease the formation of

proinflammatory cytokines41,42

.

IV. Anticarcinogenic activity: Animal research demonstrates inhibition at all

three stages of carcinogenesis-initiation, promotion and progression. During

initiation and promotion, curcumin modulates transcription factors controlling

phase I and II detoxification of carcinogens, downregulates proinflammatory

cytokines, free radical-activated transcription factors and arachidonic acid

metabolism vicyclooxygenase and lipoxygenase pathways and scavenges free

radical43,44

.

V. Antidiabetic activity: A hexane extract (containing ar-turmerone), ethanolic

extract (containing ar-turmerone, curcumin, demethoxycurcumin and

bisdemethoxycurcumin) and ethanolic extract from the residue of the hexane

extraction (containing curcumin, demethoxycurcumin and

bisdemethoxycurcumin) were found to dose-dependently stimulate adipocyte

differentiation. The results indicate that turmeric ethanolic extract containing

both curcuminoids and sesquiterpenoids is more strongly hypoglycemic than

either curcuminoids or sesquiterpenoids45

.

VI. Antimicrobial activity: Turmeric extract and the essential oil of C. longa

inhibit the growth of a variety of bacteria, parasites and pathogenic fungi. A

study of chicks infected with the caecal parasite Eimera maxima demonstrated

that diets supplemented with turmeric resulted in a reduction in small

intestinal lesion scores improved weight gain. In another study guinea pigs

infected with either dermatophytes, pathogenic molds or yeast were treated

with topical application of turmeric oil resulting in inhibition of dermatophytes

and fungi46

.

VII. Cardiovascular diseases: Protective properties of the cardiovascular system

include lowering cholesterol and triglyceride levels, decreasing susceptibility



of low density lipoprotein to lipid peroxidation and inhibit platelet aggregation

was demonstrated by turmeric47

.

VIII. Neurological disorders: Studies in animal models of Alzheimer‟s disease

indicate a direct effect of curcumin in decreasing the amyloid pathology of

Alzheimer disease48

.

2.2.6 Analytical methods for Estimation of Curcumin

Sharma et al.49

developed and validated UV-visible spectrophotometric method for

the estimation of curcumin using methanol as solvent and detection wavelength of

421nm. The detector response was linear over the concentration range of 1-7µg/ml

with correlation coefficient 0.9995. The LOD and LOQ were 0.05 and 0.172µg/ml

respectively.

Gupta et al.50

developed a spectrofluorimetric method for the estimation of curcumin

by preparing a calibration curve in the concentration range of 1-10ng/ml using

methanol. The spectrofluorimetrically scanned solution shows excitation at 232nm

and emission at 614nm. The correlation coefficient obtained was 0.99.

Ashrafl et al.51

developed and validated HPTLC method for the estimation of

curcumin using toluene: chloroform: methanol (5:4:1, v/v/v) as mobile phase and

silica gel 60 F254 as stationary phase and detection wavelength of 430nm. Linearity

was observed in the concentration range of 200-1000ng/ml.

Gantait et al.52

developed a validated HPTLC method using silica gel 60 F254 as

stationary phase, dichloromethane: methanol (99:1) as mobile phase and 427nm as

detection wavelength. Linearity was observed in the concentration range of 0.8-

1.3µg/spot with correlation coefficient of 0.99395. LOD was 49ng and LOQ was

148ng/spot.

Soni et al.53

carried out HPLC separation of curcumin using Cyber Lab C18 column

(250 x 4 mm, 5µ) as stationary phase and mobile phase comprising of acetonitrile and

0.1% orthophosphoric acid solution in water in the ratio of 60:40 (v/v) at flow rate of

0.5ml/min and detection wavelength of 425nm.

Nagappan et al.54

developed a liquid chromatography method for the simultaneous

determination of curcumin and piperine in food products using C18 column (250 x

4.6mm) by isocratic elution with 50mM potassium dihydrogen orthophosphate (pH



3.5) : acetonitrile (40:60) and detection at 424nm and 340nm using photodiode array

detector for curcumin and piperine respectively. The calibration plot was linear over

the range 100-3200ng/ml and 200-700ng/ml respectively with a correlation of 0.999.

Wichitnithad et al.55

developed and validated isocratic HPLC method for the

simultaneous determination of curcuminoids in commercial turmeric extracts using

Alltima C18 column with isocratic elution of acetonitrile and 2% V/V acetic acid

(40:60, V/V) at a flow rate of 2.0 ml/min and UV detection at 425nm.

Moorthi and Kathiresan56

developed and validated RP-HPLC-PDA method for

simultaneous estimation of curcumin and silibinin in nano-formulation using C18

column with an isocratic elution of mobile phase composed of a degassed mixture of

0.1% orthophosphoric acid and acetonitrile (50:50V/V) at a flow rate of 1.0ml/min.

2.3 Embelia ribes

Vidang consists of dried mature fruits of Embelia ribes Burn large scandent shrub

with long slender, flexible branches; distributed throughout hilly parts of India upto

1600m57

.


Kingdom : Plantae

Phylum : Angiosperms

Order : Ericales

Family : Myrsinaceae

Genus : Embelia

Species : ribes

2.3.2 Synonyms57

Sanskrit : Jantughna, Krimghna, Vella, Krimhara, Krmiripu

Bengali : Vidang

Gujarati : Vavding, Vayavadang, Vavading

Hindi : Vayavidanga, Bhabhiranga, Baberang

Malyalam : Vizhalari, Vizalari

Kannad : Vayuvidanga, Vayuvilanga

Tamil : Vayuvilangam, Vayuvidangam

Telugu : Vayuvidangalu



2.3.3 Pharmacognosy

I. Macroscopic57

: Fruit brownish black, globular, 2-4mm in diameter, warty

surface with a beak like projection at apex, often short, thin pedicel and

persistant calyx with usually 3 or 5 sepals present; pericarp brittle enclosing a

single seed covered by a thin membrane; entire seed, reddish and covered with

yellowish spots (chitra tandula); odor, slightly aromatic; taste, astringent.

Fig. 2.5 Photograph of Embelia ribes (herb, fruits and powder)

II. Microscopic57

: Transverse section of fruit shows epicarp consisting of single

raw of tabular cells of epidermis, usually obliterated, in surface view cells

rounded with wrinkled cuticle; mesocarp consists of a number of layers of

reddish-brown colored cells and numerous fibro vascular bundles and rarely a

few prismatic crystals of calcium oxalate; inner part of mesocarp and

endodermis composed of stone cells; endodermis consisting of single layered,

thick walled, large, palisade-like stone cells; speed coat composed of 2-3

layered reddish-brown colored cells; endosperm cells irregular in shape, thick-

walled, containing fixed oil and proteinous masses; embryo small when

present otherwise most of the seeds sterile.

III. Medicinal uses: Anthelmintic, Pain, staomachic.


Embelin is the principle chemical compound reported from the seeds (4.33%)58

.



Fig. 2.6 Structure of Embelin

It is water insoluble but forms a water soluble, violet colored complex, in alkaline

medium59

. The other constituents isolated from the seeds are quercitol, tannin,

christembine and volatile oil60

. It also contains chemical constituents like embolic

acid, volatile oil, fixed oil, resin, tannin, christembine (alkaloid)61

. Phenolic acids like

caffeic acid, vanillic acid, chronogenic acid, cinnamic acid, o-cumaric acid62

.

Phytochemical investigation of the seeds revealed 3 new compounds identified as 3-

(4”-hydroxyoctadecanyloxy)-p-quinonyl-5-methylene-8-(10-pentanyloxy)-p-quinine

(embelinol), n-pentacosanyl-n-nonadeca-71-en-9

1-alpha-ol-1

1-oate (embeliaribyl

ester), 1,2,4,5-tetrahydroxy3-undecanyl benzene (embeliol) and a known compound

embelin63

.


I. Hepatoprotective: Ethanol extract of E. ribes administrated orally reported a

dose dependant fall in the serum SGPT levels as compared to elevated levels

in the mice whose liver cell damage is induced by paracetamol64

.

II. Anthelmintic activity: E. ribes seed oil when administered at different doses

reported death of worms (Pheretima posthuma). But response of worms to

different doses altered in the time of paralysis parameter. Increase in dose

reported a decreased time of paralysis, and the values are significant when

compared with standard piperazine citrate65

.

III. Anti-bacterial activity: E. ribes reported zone of inhibition when compared

to the standard drug nitrofurazone against test organism Bacillus subtilis.

Embelia ribes did not produce any inhibitory/antimicrobial activity against

Pseudomonos aeruginosa, Staphylococcus aureus and Escheresia coli66

.



IV. Antioxidant activity: Aqueous extract of E. ribes administered orally

significantly decreased the levels of pancreatic superoxide dismutase, catalase,

glutathione and pancreatic β-cells in the streptozotocin induced diabetic rats.67

.

V. Antifungal activity: Antifungal activity was studied by lest method NCCLS

(The national committee for clinical laboratory standard M27-A2 protocol).

NCCLS method revealed that methanol extract of E. ribes and embelin had

lowest MIC50 range of 120mg/L against Candida albican (MTCC no. 183) and

among four Candida species tested embelin had reported MIC 50 values below

700mg/L68

.

2.3.6 Analytical methods for Estimation of Embelin

Chauhan et al.69

carried out TLC identification and spectrophotometric estimation of

embelin in E. ribes by measuring the absorbance of three different concentrations of

embelin at 285nm against chloroform as blank. Correlation coefficient was 0.999.

Sudani et al.70

developed and validated HPTLC method for quantitation of embelin

using TLC aluminum plate precoated with silica gel 60 F254 as stationary phase and

chloroform: ethylacetate: formic acid (5:4:1) as mobile phase. Densitometric analysis

was carried out in the absorbance mode at 291nm showing good correlation

coefficient 0.9986.

Kukkar et al.71

developed a validated HPTLC method for the estimation of embelin

and strychnine in krimimudgara rasa by using silica gel 60 F254 as stationary phase

and mobile phase composed of n-propanol: n-butanol: 4N ammonia (7:1:2). Detection

was carried out at 333nm with a linearity range of 400-2400ng/spot and correlation

coefficient of 0.9966.

Shelar et al.72

developed a HPLC method for quantitative estimation of embelin in E.

ribes by using isocratic mode consisting of 0.1% trifluroacetic acid in water and

methanol (88:12) at a flow rate of 1.0ml/min and 290nm. The plot was found to be

linear over the range of 5.0 – 75.0µg/ml with a relative standard deviation of 0.61-

0.96%. LOD and LOQ were 20ng and 50ng respectively.

Gopinath et al.73

developed RP-HPLC method for simultaneous estimation of

plumbagin and embelin by using mobile phase comprising of acetonitrile: 50mM

phosphate buffer (45:55) pH 3.5, flow rate 1ml/min and detection at 290nm.



Sudani et al.74

carried out quantitative and chromatographic fingerprint analysis of E.

ribes churna formulations by HPLC method using C18 column with a mobile phase of

Methanol: Phosphate buffer pH 3.0 (90:10), flow rate 1.4ml/min and detection

wavelength 291nm. Method showed good regression with correlation coefficient

0.9988.

2.4 Piper nigrum

Drug consists of dried ripe fruits of Piper nigrum Linn. (Syn. P. trioicum Roxb. P.

baccatum DC. and Muldera multinervis Miq.). Plant is mostly cultivated in the hot

and moist parts of India from konkan southwards (especially N.kanara and Kerala)75

.


Kingdom : Plantae

Phylum : Angiosperms, Magnoliids

Order : Piperales

Family : Piperaceae

Genus : Piper

Species : nigrum

2.4.2 Synonyms75

Sanskrit : Maricha

Bengali : Golmorich, Kalimirch

Gujarati : Kalamari, Kalomirich

Hindi : Golmirch, Kalimirch

Malyalam : Kurumulaku

Kannad : Karemensu

Tamil : Milagu

Telugu : Miriyalu, Maichamu

2.4.3 Pharmacognosy

I. Macroscopic76,77

: Fruits are globular or oblong, 4-6mm in diameter.

Externally blackish brown, with raised reticulated wrinkles. One seeded seeds

white and hollow. Odour aromatic; taste aromatic and strongly pungent.

II. Microscopic78

: Epicarp consisting of an outer layer of tangentially elongated

cells having dark brown to blackish contents. Non-glandular trichomes are



seen on the surface of the epicarp. Stone cells are isodiametric to radially

elongate which are present just beneath the epicarp and also found in the

endocarp.

Fig. 2.7 Photograph of piper nigrum (herb, fruits and powder)

The parenchymatous mesocarp possesses oil globules, simple and compound starch

grains. Inner zone of perisperm cells are radially elongated which embeds largely

oleoresins besides starch and protein substance. Tracheids are pitted; some of them

show helical thickenings on their secondary wall.

III. Medicinal uses: Fruits are used as anticonvulsant and bioavailability enhancer79

2.4.4 Chemical constituents80

The drug contains volatile oil (1 % - 2.5 %), alkaloids/ amides (5 % - 9 %) and a

resin81

. Major constituent is pungent alkaloid, piperine (2 % - 5 %)82

.

Fig. 2.8 Structure of Piperine

Minors are number of alkaloids/ amides83

,84

eg. pipericine, piperettine, piperanine,

guineensine, sarmentine, pipericide; propenylphenols viz., eugenol, myristicine,

safrole; mono and sesquiterpenes eg. 1, 8 cineole, p-cymene, carvone, b-bisabolene.




I. Antioxidant activity: Regenerated tissues of P. nigrum like callus, in vitro

shoots, roots, in vitro plantlets, peppercorn and acclimated plantlets possesses

antioxidant activity which is probably due to the presence of flavonoids and

phenolic contents. P.nigrum also prevent the intestine induced oxidative stress,

inhibit lipid peroxidation, arresting different radicals such as hydroxyl and

super oxides radicals, decrease induced lung carcinogenesis and inhibit human

lipoxygenase85

.

II. Antidiarrhoeal activity: It has been reported the effect of piperine in mice

small intestine fluid accumulation activated by castor oil. In such induced

situation piperine sequentially prevent the small intestine fluid accumulation86

.

III. Antimutagenic and antitumor activity: It has been reported that when

Drosophill melanogaster was exposed to mutation through promutagen ethyl

carbamate, in such induced situation the P. nigrum is effective to reduce

mutational events. P. nigrum and its active derivatives especially peppercorn

extract has been reported to inhibit tumors formation in experimental

models87

.

IV. Antihypertensive activity: Piperine in, in vitro study on rabbit heart causes a

partial reduction of force, contraction of tissues and blood flow in coronary

vessels. In rabbit aortic ring, piperine partially inhibited phenylephrine and

inhibited high K+, pre-contractions due to blockade Ca

2+ channel. In Ca

2+ free

medium, piperine in low doses exhibited vasoconstrictor effect88

.

V. Hepatoprotective activity: It has been reported that when experimental mice

with D-galactosamine induced liver toxicity were exposed to dose dependent

piperine, it inhibited increase in serum GPT and GOT levels and suggested

that this inhibitory effect depended on the reduced sensitivity of hepatocytes to

tumor necrosis factor85

.

VI. Digestive action: Piperine increases the production of saliva and gastric

secretion. Ingestion of peppercorn increases the production and activation of

salivary amylase89

.



2.4.6 Analytical methods for Estimation of Piperine

Gupta and Jain90

developed a UV-spectrophotometric method for the estimation of

piperine in pippli churna by preparing calibration curve in methanol and measuring

absorbance at 342.5nm. Piperine obeys Beer Lambert law in concentration range 10-

50µg/ml with correlation coefficient of R2

= 0.9961.

Patel and Vyas91

developed a validated spectrofluorimetric method for estimation of

piperine by preparing standard curve at excitation and emission wavelengths of

339nm and 450nm respectively. The linearity was found to be in the range of 10-

60ng/ml. The correlation coefficients were 0.9958 indicating good linearity between

fluorescence intensity and concentration.

Parameswaran and Koshti92

developed an HPTLC method for quantitative estimation

of piperine and diosgenin in ayurvedic formulation. These compounds were

chromatographed on precoated silica gel G60254 in the mobile phase comprising of

toluene: hexane: ethylacetate (6.8:0.2:3). The calibration plot was linear in the range

of 0.2-1µg/spot for piperine and 1.0-3.0µg/spot for diosgenin. The correlation

coefficient was 0.9979 and 0.9915 for piperine and diosgenin respectively.

Tapadiya et al.93

developed HPTLC method for quantitative estimation of piperine.

The stationary phase was precoated silica gel G60254. The mobile phase was toluene

and methanol (80:10). The detection was carried out at 332nm. The Rf value was

found to be 0.49±0.01. The linearity curve found to be linear in between 10-

45ng/spot.

Upadhyay et al.94

developed and validated RP-HPLC method for estimation of

piperine. This method was carried out by using (250x4 mm, 5 µ), C18 column as

stationary phase and mobile phase consisting of acetonitrile: water: acetic acid

(60:39.5:0.5). The flow rate was 1.0ml/min with UV detection at 340nm, run time

10min and injection volume 20µl.

Santosh et al.95

developed RP-HPLC method for determination of piperine using

methanol and water as mobile phase. The detection and quantification was performed

at 345nm. Lineariry of detector response was between 0.005% to 0.1%. The

correlation coefficient was 0.998.



2.5 Glycyrrhiza glabra

Drug consist of dried, unpeeled, stolon and root of Glycyrrhiza glabra Linn, a tall

perennial herb upto 2m high found cultivated in Europe, Persia, Afghanistan and to a

little extent in some parts of India96

.


Kingdom : Plantae

Phylum : Angiosperms, Eudicots,Rosids

Order : Fabales

Family : Fabaceae

Genus : Glycyrrhiza

Species : glabra

2.5.2 Synonyms 96

Sanskrit : Yastimadhuka, Yastika, Madhuka

Bengali : Yashtimadhu

Gujarati : Jethimadha, Jethimard, Jethimadh

Hindi : Mulethi, Mulathi

Malyalam : Irattimadhuram

Kannad : Madhuka, Atimadhurav

Tamil : Athimadhuram

Telugu : Atimadhuramu

2.5.3 Pharmacognosy

I. Macroscopic97

: Root nearly cylindrical, up to 2cm in diameter. Outer surface

yellowish brown or dark brown, externally longitudinally wrinkled with

patches of cork. Fracture, coarsely fibrous in bark and splintery in wood.

Odor, characteristic; taste sweetish.



Fig. 2.9 Photograph of Glycyrrhiza glabra (herb, rhizomes and powder)

II. Microscopic98

: Cork consisting of several layers of orange-brown, thin-walled

cells. Cortex relatively narrow. The secondary phloem is a broad band.

Pholem fibres abundant, occur in groups, unlignified or slightly lignified,

surrounded by a calcium oxalate prism sheath. Secondary xylem distinctly

radiate with medullary rays. 3-5 cells wide. Vessels thick, yellow, pitted,

reticulately thickened walls. Xylem fibres also in bundles. Roots without pith

at the centre.

III. Medicinal uses: Anti inflammatory, Anti-ulcer.


Major constituent of glycyrrhiza is triterpenoid saponin glycyrrhizin (2-9%)99,100

a

mixture of potassium and calcium salts of glycyrrhizinic acid101

.

Fig. 2.10 Structure of Glycyrrhyetenic acid



Minors, include other triterpenoid saponins viz. glabranin A & B, glycyrrhetol,

glabrolide, isoglabrolide102

; isoflavones viz. formononetin, glabrone, neoliquiritin,

hispaglabridin A & B; Coumarins viz. herniarin, umbeliferone; triterpene sterols viz.

onocerin, beta-amyrin, stigmasterol103.


I. Chronic Hepatitis: In two clinical trials, Stronger Neo-Minophagen C has

been shown to significantly lower aspartate transaminase, alanine

transaminase and gammaglutamyltransferase concentrations, while

simultaneously ameliorating histologic evidence of necrosis and inflammatory

lesions in the liver104,105.

II. Antitussive and Antidemulcent Activity: The extract of the powdered drug

in water was found to be effective in the treatment of sore throat cough

bronchial catarr. It is antitussive and expectorant loosening tracheal mucus

secretion. The demulcent action is attributed to glycyrrhizin106

.

III. Peptic Ulcer: Oral administration of aqueous extract of licorice in rats

reduced gastric secretions and inhibited the formation of gastric ulcers induced

by pyloric ligation, aspirin, and ibuprofen. The mechanism of antiulcer

activity involves acceleration of mucin excretion through increasing the

synthesis of glycoprotein at the gastric mucosa, prolonging the life of the

epithelial cells, and antipepsin activity 101

.

IV. Antilipidemic activity: Biological study of the effect of licorice roots extract

on serum lipid profile, liver enzymes and kidney function tests in albino mice

was studied. Results showed that it decreases total cholesterol, triglyceride,

low density lipoprotein, high density lipoprotein demonstrating antilipidemic

activity107

.

2.5.6 Analytical methods for Estimation of Glycrrhetenic acid

Roshan et al.108

developed UV-spectrophotometric method for the estimation of

glycyrrhetenic acid in pratisyayghna kwath by preparing calibration curve of

glycyrrhetenic acid in methanol at 204nm. The method obeys Beers law in

concentration ranges 10-50µg/ml.



Song et al.109

developed UV spectrometry method for determination of total

glycyrrhetenic acid in glycyrrhizae radix by second derivative spectrometry using ion-

pair extraction technique. Glycyrrhizin obtained was hydrolyzed into glycyrrhetenic

acid in 2NHCl and methanol (1:1) and extracted from aqueous phase in the form of an

ion-pair complex with tetrapentylammoniumbromide (TPA) as a counter ion.

Maximum Z value was obtained when 1000 fold or greater molar ratio of TPA was

used at pH 11. Reaction time, temperature and ionic strength did not affect ion-pair

formation. Dichloromethane was an effective extraction solvent of the ion-pair

complex. The linearity was obtained in the range 4-120µg/ml.

Trivedi and Santani110

developed HPTLC method for simultaneous estimation of

withaferin-A and 18-β-glycyrrhetenic acid using toluene: ethyl acetate: glacial acetic

acid: chloroform (5:5:1:2) as mobile phase and silica gel 60 F254 as stationary phase.

Scanning wavelength of 223nm and 254nm for withaferin A and 18-β-glycyrrhetenic

acid with a slit dimension of 8.0x0.40mm, scanning speed of 20mm/s and data

resolution of 100µm/step was employed.

Trivedi and Mishra111

developed a simple and rapid HPTLC method for simultaneous

estimation of glycyrrhetenic acid and piperine using precoated silica gel G60254 as

stationary phase and toluene: glacial acetic acid:ethylacetate (12.5:7.5:0.5) as mobile

phase. The detection was carried out at 260nm.

Patil et al.112

developed a validated UV spectrophotometric method for estimation of

glycyrrhetenic acid. The method was performed at 254nm using phosphate buffer pH

6.8: Methanol (70:30) with a correlation coefficient of 0.999.

Potawale et al.113

developed HPTLC method for densitometric analysis of 18β-

glycyrrhetenic acid and β-sitosterol in polyherbal drug formulation. The stationary

phase was precoated silica gel G60254 and mobile phase was toluene: methanol: ethyl

acetate (14.3:1.9:3.8). The detection was carried out at 429nm with correlation

coefficient of 0.999.

2.6 Mucuna Pruriens

Drug consists of dried mature seed of Mucuna Prurita Hook., Syn. M. pruriens Baker.

(Fam.Fabaceae); a slender extensive climbing plant found almost all over the

country114

.




Kingdom : Plantae


Order : Fabales

Family : Fabaceae

Genus : Mucuna

Species : pruriens

2.6.2 Synonyms114

Sanskrit : Kapikacchu, Markati, Kandura

Gujarati : Kavach, Kaucha

Hindi : Kewanch, Kaucha

Malyalam : Naikurana

Kannad : Nasugunne, Nasugunnee

Tamil : Poonaikkali

2.6.3 Pharmacognosy

I. Macroscopic114

: Seed ovoid, slightly laterally compressed, with a persistant

oblomg, funicular hilum, dark brown with spots; usually 1.2 – 1.8cm long, 0.8

– 1.2cm wide, hard, smooth to touch, not easily breakable; odour not distinct;

taste, sweetish-bitter.

Fig. 2.11 Photograph of Mucuna pruriens (herb, seeds and powder)

II. Microscopic114

: Mature seed shows a thin seed – coat and two hard

cotyledons; outer testa consists of single layered palisade-like cells; inner testa



composed of 2 or 3 layers, outer layer of tangentially elongated, ovoid, thin –

walled cells; tegmen composed of a wide zone of oval to elliptical, somewhat

compressed, thin-walled, parenchymatous cells; some cells contain starch

grains; cotyledons composed of polygonal, angular, thin-walled, compact

parenchymatous cells, containing aleurone and starch grains; starch grains

small, simple, rounded to oval measuring 6-41μ in diameter but not over 45μ

in diameter, a few vascular bundles with vessels showing reticulate thickening

or pitted present.

III. Medicinal uses: Aphrodisiac


Seeds are known to produce the unusual non-protein amino acid 3-

(3,4dihydroxyphenyl)-l-alanine (L-Dopa), a potent neurotransmitter precursor that is,

at least in part, believed to be responsible for the toxicity of mucuna seeds115

. Beside

this it also contain some other amino acids, glutathione, lecithin, gallic acid and beta-

sitosterol. It has unidentified bases like mucunine, mucunadine, prurienine,

prurieninine. Other bases isolated from the pods, seeds, leaves and roots include

indole-3-alkylamines-N, N-dimethyltryptamine. Leaves also gave 6-methoxyharman.

Serotonin is present only in pods116

. The seeds also contain oils including palmitic,

stearic, oleic and linoleic acids117

.

Fig. 2.12 Structure of L-Dopa


I. Antioxidant activity: The free radical scavenging activity of various extracts

of whole plant of M. pruriens by different in-vitro methods was evaluated by

hydroxyl radical scavenging activity, DPPH scavenging activity, nitric oxide

radical scavenging activity with reference standard ascorbate and total phenol



content respectively. Results indicated higher amount of phenolic contents and

marked antioxidant activity against ascorbate.118

II. Antimicrobial activity: Methanol extract of M. pruriens was determined by

disc diffusion method with various Gram positive and Gram negative micro-

organisms. Methanol extract of M. pruriens showed broad spectrum

antimicrobial activity against various microorganisms‟ species like

Staphylococcus aureus, Bacillus pumillus, Escherichia coli and Vibrae

cholera119

.

III. Aphrodisiac activity: The role of M. pruriens was studied on 60 subjects who

were undergoing infertility screening and were found to be suffering from

psychological stress, assessed on the basis of a questionnaire and elevated

serum cortisol levels. Infertile subjects were administered with M. pruriens

seed powder orally. The results demonstrated decreased sperm count and

motility in subjects who were under psychological stress120

.

IV. Anti-inflammatory activity: The anti-inflammatory activity of aerial parts of

M. pruriens was studied using carrageenin induced rat paw edema model and

cotton pellet implattion method in rats. The extracts significantly reduced

carragenin induced paw edema in rats. In cotton pellet implantation model, the

extract showed a significant reduction in the weight of cotton pellet in test

animal compared to control121

.

2.6.6 Analytical methods for Estimation of L-Dopa

Yukesoy 122

developed derivative spectrophotometry for simultaneous determination

of levodopa and benserazide. The method is based on the use of UV derivative

spectrophotometric measurements the first derivative at 271.6nm and the second

derivative at 239.4nm for levodopa in 0.1N HCl and second derivative at 219.4nm for

benserazide in 0.1NHCl. The mean percentage recovery of the drugs by standard

addition method was 100.6% for levodopa and 100.2% for benserazide, respectively.

Hussein et al.123

developed spectrophotometric method for determination of Levodopa

using 4-aminoantipyrine (4-AAP) as a chromogenic reagent. The method is based on

oxidative coupling reaction of L-dopa with 4-AAP in the presence of sodium

hydroxide as alkaline media to form a red water soluble dye product, that has



maximum absorption at 519nm. Linearity was observed in the range of 0.2-30µg/ml

with correlation coefficient of 0.999.

Patil et al.124

developed spectrophotometric method for determination of levodopa

with ninhydrin. This method is based on reaction of ninhydrin with primary amine

present in the levodopa in the presence of dimethyl formamide. This reaction

produces purple colour product which absorbs maximally at about 624nm. Beers law

was obeyed in the range of 10-50µg/ml with correlation coefficient value 0.9940.

Sundaram and Gurumoorthi125

developed validated HPTLC method for quantitative

estimation of l-dopa using TLC aluminum plates precoated with silica gel as the

stationary phase in twin trough glass chamber saturated with n-butanol: glacial acetic

acid: water (4:1:1) as mobile phase. The Rf value of l-Dopa was found to be 0.39.

Linearity was found to be in the concentration range of 100-1200ng/spot.

Raina and Khatri126

developed HPTLC method using precoated silica gel 60 GF254 as

stationary phase and n-butanol: acetic acid: water (4:1:1) as mobile phase.

Quantification was done at 280nm. Linearity was found in the concentration range of

100-1000ng/spot with correlation coefficient value of 0.9980.

Shivananda et al127

developed HPLC method for the estimation of L-dopa. In this

method L-dopa from M. pruriens seeds was extracted using 0.1 M orthophosphoric

acid. The extracted L-dopa was analyzed in HPLC using sodium orthophosphate

buffer (pH 2.8) as mobile phase.

2.7 Tribullus terristris

Drug consists of dried ripe, entire fruits of Tribullus terristris Linn. The plant is

distributed throughout India up to 3500m128

.


Kingdom : Plantae


Order : Zygophyllales

Family : Zygophyllaceae

Genus : Tribulus

Species : terrestris



2.7.2 Synonyms128

Sanskrit : Gokshura

Bengali : Gokshura, Gokhri

Gujarati : Betagokhru, Mithagokhru, Nanagokharu

Hindi : Chota-gokhru, Gokhru

Malyalam : Neringil, Nerinnil

Kannad : Sannaneggilu, Negalu

Tamil : Nerunji

2.7.3 Pharmacognosy

I. Macroscopic129,130

: Fruit pedicellate, globose, possessing five woody wedge

shaped cocci, covered with two pairs of short stiff spines, one pair larger than

the other. Tips of spines almost meet in pairs together forming pentagonal

frame work around the fruit. Outer surface of the schizocarp is rough. Odour

faintly aromatic and slightly acrid in taste.

Fig. 2.13 Photograph of Tribullus terristris (herb, fruits and powder)

II. Microscopic131

: The pericarp is differentiated into epicarp, mesocarp and

endocarp. Outer surface of the epicarp is surrounded by non-glandular trichomes.

The parenchymatous mesocarp is 6-10 layers thick which embeds calcium oxalate

crystals. The sclerenchymatous endocarp is 3-4 layers thick and the cells are

compact containing prismatic crystals of calcium oxalate. Fruits are penta locular.



Vessels have simple pits and some vessels show helical thickenings. Fibres are

lignified, linear long with tapered ends.

III. Medicinal uses: Diuretic, antiurolithiatic


Major constituents are steroidal saponins132

e.g. terrestrosins A, B, C, D and E,

desgalactotigonin, F-gitonin, desglucolanatigonin, gitonin etc.; hydrolysed products

include diosgenin, hecogenin and neotigogenin etc133

.

Fig. 2.14 Structure of diosgenin

Minors are uncharacterized alkaloids134

; phytosterols e.g. β-sitosterol, stigmasterol126

;

a cinnamic amide derivative – terrestiamide and 7 – methylhydroindanone135

.


I. Diuretic activity: The diuretic properties of T. terristris are due to large quantities

of nitrates and essential oil present in its fruits and seeds. The diuretic activity can

also be attributed to the presence of potassium salts in high concentration. The

aqueous extract of T. terristris elicited a positive diuresis, which was slightly more

than that of furosemide. Sodium and chloride concentrations in the diuretic

activity helped in the propulsion of stones along the urinary tract136

.

II. Aphrodisiac activity: T. terristris extract exhibited a pro-erectile effect on rabbit

corpus cavernosum smooth muscle ex vivo after oral treatment. A dose dependent

improvement in sexual behaviour was observed with the lyophilized aqueous

extract treatment which was more prominent on chronic administration. A

significant increase in serum testosterone levels too was observed. These findings

confirm the traditional use of T. terristris as a sexual enhancer in the management

of sexual dysfunction in males137

.



III. Antiurolithiatic activity: An ethanol extract of T. terristris fruits was tested in

urolithiasis induced by glass bead implantation in albino rats. It exhibited

significant dose-dependent protection against deposition of calculogenic material

around the glass bead, leukocytosis and elevation in serum urea levels.

Subsequent fractionation of the ethanol extract led to decrease in activity138

.

IV. Immunomodulatory activity: Saponins isolated from the fruits of T. terristris

demonstrated dose-dependent increase in phagocytosis, indicating stimulation of

nonspecific immune response. An alcohol extract of the whole plant of T.

terristris exhibited a significant dose-dependent increase in humoral antibody titre

and delayed type hypersensitivity response, indicating increased specific immune

response139

.

V. Hepatoprotective activity: The T. terristris extract showed a remarkable

hepatoprotective activity against acetaminophen-induced hepatotoxicity in

Oreochromis mossambicus fish. The elevated biochemical parameters and

decreased level of reduced glutathione enzymes were normalized by treatment

with T. terristris extract for acetaminophen-induced toxicity in freshwater fish140

.

VI. Analgesic activity: Analgesic activities of T. terristris were studied in male mice

using formalin and tail flick test. The study indicated that the methanol extract of

T. terristris produced analgesic activity. This effect may be mediated centrally or

peripherally141

.

VII. Anthelmintic activity: The methanol extract of T. terristris was found to be more

effective than the petroleum ether, chloroform and water extracts for in vitro

anthelmintic activity on the nematode Caenorhabditis elegans. Further

bioactivity-guided fractionation confirmed tribulosin and β-sitosterol-d-glucoside

to be the active components with ED50 of 76.25 and 82.5 μg/ml respectively142

.

2.7.6 Analytical methods for Estimation of Diosgenin

Trivedi et al.143

developed a validated quantitative TLC method for estimation of

diosgenin in various plant samples, extract and market formulation. The spots were

visualized by spraying with modified anisaldehyde sulfuric acid reagent. The method

was validated according to ICH guidelines. Increased detection sensitivity was

observed with linearity from 98-588ng/spot and a correlation coefficient R2

= 0.988.



Parameswaran and Koshti144

developed HPTLC method for quantitative estimation of

piperine and diosgenin using precoated silica gel G60254 as stationary phase and

toluene: hexane: ethyl acetate (6.8:0.2:3) as mobile phase. The calibration plot was

linear in the range of 0.2-1µg/spot for piperine and 1.0-3.0µg/spot for diosgenin. The

correlation coefficient was 0.9979 and 0.9915 for piperine and diosgenin respectively.

Blunden et al.145

carried out densitometric analysis of diosgenin. TLC was employed

to separate diosgenin from other plant constituents present in dioscorea tuber extract.

The diosgenin was rendered visible by antimony trichloride and the colored spot

estimated by scanning with a photoelectric densitometer attached to an integrating

logarithmic recorder. Diosgenin could be estimated with an experimental error of

0.1% then applied in concentration between 10 and 75mcg. Assay results obtained for

diosgenin by this method were slightly lower than those obtained by conventional

infrared spectrophotometry.

Amir et al.146

carried out HPTLC analysis of diosgenin using TLC aluminum plates

precoated with silica gel 60F-254 as stationary phase and petroleum ether:

isopropanol (12:1) as mobile phase. Detection was carried out at 366nm after spraying

with methanolic sulphuric acid. The linear regression analysis data for the calibration

plots showed good linear relationship with R2

= 0.995 in the concentration range of

100-1000ng/spot.

Warke et al.147

developed a validated RP-HPLC method for estimation of diosgenin

using RP C18 ODS hypersil column (150x4.6 mm) as stationary phase and acetonitrile:

water in the ratio of 90:10 V/V as mobile phase at a flow rate of 1ml/min with UV

detection of 203nm. Diosgenin was well resolved on the stationary phase and the

retention time was 10.5min. The calibration curve was linear R2

= 0.997 in the

concentration range of 2-10µg/ml.

2.8 Withania somnifera

Drug consists of dried roots of Withania somnifera (Linn) Dunal (syn. Physalis

somnifera Linn. P. flexuosa Linn. P. arborescence DC.) The plant is widely

distributed in North-Western India, Bombay, Gujarat, Rajashthan, Madhya Pradesh,

Uttar Pradesh, Punjab plains and extends to the mountain regions of Himachal

Pradesh and Jammu148

.




Kingdom : Plantae


Order : Solanales

Family : Solanaceae

Genus : Withania

Species : somnifera

2.8.2 Synonyms148

Sanskrit : Ashvagandha, Ashvakandika, Vajigandha

Bengali : Ashvagandha

Gujarati : Ghodakun, Asan, Asoda

Hindi : Asgandh

Malyalam : Ammukuram

Kannad : Angarberu, Asvagandhi

Tamil : Pennerugadda, Panneru, Pulivendram, Vajigandha

2.8.3 Pharmacognosy

I. Macroscopic148

: Roots 10-20cm long and 6-12mm in diameter, with a few (2

to 3) lateral roots of slightly smaller size; straight, unbranched. Outer surface is

buff to grey-yellow with longitudinal wrinkles and in the center soft, solid mass

with scattered pores. Odor characteristic; taste bitter and acrid.

Fig. 2.15 Photograph of Withania somnifera (herb, roots and powder)



II. Microscopic149,150

: The cork cells isodiametric and nonlignified. Intercellular

spaces are present in the phloem parenchyma while it is absent in xylem

parenchyma. Fibres absent in phloem and present in xylem. Simple, reniform

and oval starch grains found in the parenchyma of the cortex and vascular

region.

III. Medicinal uses: Adaptogen


Majority of the constituents are Withanolides, steroidal lactones with ergostane

skeleton include withanone151

, withaferin A152

, withanolides I, II, III, A, D , E, F, G,

H, I, J, K, L, M, WS-I, and S153

withasomidienone154

, withanolide C155

and alkaloids

(total 0.2%) e.g. cusohygrine, anahygrine, tropine, pseudotropine, anaferine,

isopellatierine, 3-tropyltigloate.

Fig. 2.16 Structure of Withaferin A


I. Anti-inflammatory activity: Withaferin A exhibits anti-inflammatory activity

effective as hydrocortisone sodium succinate. It was found to suppress

effectively arthritic syndrome without any toxic effect. It showed this property

in many animal models of inflammations like carrageenan-induced

inflammation, cotton pellet granuloma and adjuvant-induced arthritis156

.

II. Anti stress/Adaptogenic activity: Antistressor effect of asgand was

investigated in rats using cold water swimming test. The drug treated animals

showed better stress tolerance157

. A withanolide free aqueous fraction isolated

from the roots of W. somnifera exhibited anti-stress activity in a dose

dependent manner in mice. Asgand has been evaluated for its adaptogenic

activity. Administration of asgand with other drugs in experimental animals



exposed to a variety of biological, physical and chemical stressors was found

to offer protection against these stressors206

.

III. Musculotropic activity: The total alkaloids of asgand showed relaxant and

antispasmodic effects against several spasmogens on intestinal, uterine,

bronchial, tracheal and blood vascular muscles. The pattern of smooth muscle

activity of the alkaloids was similar to that of papaverine which suggested a

direct musculotropic action158

.

IV. Hepatoprotective activity: Withaferin A showed significantly protective

effect against CCl4-induced hepatotoxicity in rats as effective as

hydrocortisone159

.

V. Macrophage-activating Effect: The chemotactic activity of macrophages and

production of interleukin-1 (IL-1) and tumor necrosis factor (TNF) were

significantly reduced in mice treated with the carcinogen ochratoxin A (OTA).

Administration of asgand with other drugs was found to be significantly

inhibit OTA-induced suppression of macrophage chemotaxis and production

of IL-1 and TNF-α by macrophages160

.

2.8.6 Analytical methods for Estimation of Withaferin A

Gauttam and Kalia161

developed HPTLC method for simultaneous estimation of

vicine, trigonelline and withaferin A using silica gel 60 F-254 aluminium backed TLC

plates of 0.2mm layer thickness as stationary phase pre-derivatized with 0.02M

sodium acetate and n-butanol:acetic acid: water (5:1:5) as mobile phase, scanned at

wavelength of 235nm. The linearity was in the range of 100-600ng/band with

correlation coefficient of 0.999.

Rajendra et al.162

carried out HPLC estimation of withaferin – A and boswellic acid

using Lichosorb C18 RP column (250x4.6mm, 5µm particle size) as stationary phase

and acetonitrile: methanol: orthophosphoric acid (55:45:1) as mobile phase at flow

rate of 1.2ml/min and monitored at 224nm. The gel shows the presence of about 95-

105% of the amount of withaferin A in them against 100% of expected value.

Chaurasiya et al.163

developed RP-HPLC method for the simultaneous analysis of nine

withanolides using a linear binary gradient solvent system comprising methanol and

water containing 0.1% acetic acid. Both photodiode array and evaporative light

scattering detection were used to profile the extract compositions and to quantify the



withanolides therein. The method has been validated with respect to various

parameters of performance with correlation coefficient of 0.950.



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chapter 2 review of literature 2. review of literature...

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