6. RESULTS 6.1. Strychnos nux-vomica - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/4507/16/16_chapter 6.pdf · Strychnos nux-vomica 6.1.1 Pharmacognostic analysis 6.1.1.1

190

6. RESULTS

6.1. Strychnos nux-vomica

6.1.1 Pharmacognostic analysis

6.1.1.1 Micro-chemical analysis

In this study no quantification was made, qualitative changes in

the density and intensity of coloured patches were observed. The

transverse section of unprocessed seeds showed very high density,

intense violet and yellow coloured patches throughout the section.

Whereas the density and intensity of both colors were reduced further

after each step of detoxification.

The observations were compiled in table 6.1.1.1

Table 6.1.1.1.Effect of shodhana on major alkaloids of nux-vomica

seeds by micro-chemical tests

S.no. Sample Phytochemicals present

1. UNV Epidermis : Strychnine + brucine +

Endosperm inner portion:Strychnine ++++ Endosperm outer portion: Brucine ++++

2.

PNV1

Epidermis : Strychnine + brucine + Endosperm inner portion : Strychnine ++

Endosperm outer portion : Brucine ++

3

PNV 2

Epidermis : Strychnine + brucine +

Endosperm inner portion : Strychnine ++

Endosperm outer portion : Brucine ++

4

PNV 3

Endosperm inner portion : Strychnine +

Endosperm outer portion: Brucine +

5

PNV 4

Endosperm inner portion : Strychnine +

Endosperm outer portion : Brucine +

+ indicates less quantity, ++ indicates moderate quantity,

+++ indicates high quantity

191

6.1.1.2. Physico-chemical analysis

The pharmacognostic standardization of seeds of nux-

vomica before and after shodhana process showed that the loss on

drying was found to be 0.25 – 0.31 % w/w after complete

detoxification. This clearly indicates that the water content in the

detoxified powder is almost very less.

Total ash values ranges from 0.99 G% w/w to 1.66 % w/w

except in case of PNV1, which is 4.1 % w/w. The acid insoluble

ash values ranges from 0.05 % to 0.4% except PNV1, which is

0.67 % w/w.

The alcohol extractive and water extractive values were

increased after each step of detoxification.

Average of four repetitions was given and all these

parameters can be used for standardization purpose.

The results were summarized in table 6.1.1.2

192

Table 6.1.1.2

Effect of shodhana on physico-chemical properties of nux-vomica seeds

S.no Method (% w/w) UNV PNV1 PNV2 PNV3 PNV4 PNV5

1. Loss on drying

5.2±0.04 12.67±0.5 10.2±0.8 0.25±0.05 0.31±0.2 4.6±0.5

2. Water soluble

extractive

9.5±1.2 7.2±0.8 18.4±0.76 36.8±6.67 55.4±2.3 31.2±2.1

3. Ethanol(70%) soluble extractive

3.2±0.57

2.4±0.86

4.2±0.67

1.6±0.89

1.4±0.8

8.8±0.98

4. Total ash

1.33±0.6 4.1±.8 1.66±0.76 0.99±0.7 1.5±0.54 2.5±0.91

5. Acid insoluble ash

0.1±0.05 0.67±0.42 0.23±0.07 0.4±0.05 0.05±0.01 0.2±0.04

193

6.1.2 Phytochemical analysis

6.1.2.1 Preliminary phytochemicals analysis

It was observed from the preliminary phytochemical screening of

alcohol extract of shodhana processed and unprocessed nux-vomica

that unprocessed (UNV) extract contains alkaloids, glycosides,

flavanoids, tannins and proteins. Shodhana processed nux-vomica

PNV1 to PNV4 contains alkaloids, tannins, flavanoids and proteins.

But there was a decreased alkaloid content when compared with UNV.

The loganin glycosides were absent in completely shodhana processed

extracts.

The results were compiled in table 6.1.2.1

Table 6.1.2.1

Effect of shodhana on phytochemicals of nux-vomica seeds

Phytochemical constituents

UNV PNV1 PNV2 PNV3 PNV4

Alkaloids ++++ ++ + + +

Carbohydrates +++ + + + +

Flavanoids + + + + +

Glycosides +++ + - - -

Tannins ++ + + + +

Proteins ++ + +++ ++ ++

.

194

6.1.2.2 Phytochemical analysis of cow urine and cow milk before

and after shodhana process

It was observed that the alkaloids, loganic acid glycosides,

tannins and carbohydrates were found in cow urine after shodhana

process of nux-vomica seeds. It was found that there was a gradual

decrease in intensity of chemical reaction/color by processing with

cow urine from 1st day of treatment to 7th day of treatment.

In one more study it was observed that alkaloids and loganin

glycosides were found in cow milk after shodhana treatments (in both

methods i.e. swedana and seeds immersed in milk for 3 days without

heating).

It is found that there are no phytochemicals present in cow

urine and milk before they are used for detoxification process.

The results were compiled in table 6.1.2.2

Table 6.1.2.2

Phytochemical analysis of cow urine and cow milk before and

after shodhana process

Phytochemical constituents

Cow urine

before

shodhana

Cow urine

after

shodhana

Cow milk

before

shodhana

Cow milk

after

shodhana

Alkaloids _ ++ _ +

Carbohydrates _ + _ +

Flavanoids _ _ _ _

Glycosides _ ++ _ +

Tannins _ + _ _

Proteins _ + +++ ++

195

6.1.2.3. TLC analysis

It was confirmed by TLC studies that there was a gradual

decrease in strychnine and brucine contents in detoxified samples as

indicated by decreased intensity of colour of the spot.


Table 6.1.2.3

Identification of strychnine and brucine by TLC

before and after shodhana on seeds of nux-vomica

S.no. Samples Strychnine(Rf) Brucine(Rf)

1.

Strychnine 0.25 ___

Brucine ____

0.18

2. UNV

0.25 0.19

3. PNV1 0.26 0.18

4. PNV2 0.26 0.18

5. PNV3 0.25 0.18

6. PNV4 0.26 0.18

7. PNV5 0.24 0.18

196

6.1.2.4 Estimation of total alkaloids

It was observed by gravimetric analysis that there was a

gradual decrease in total alkaloid content in products after each

step of shodhana. The completely shodhana processed sample

contains about 1/4th of unprocessed drug. It was further found

that the PNV4 possessed least amount of total alkaloids.

The total alkaloid content in the products (namely PNV1,

PNV2, PNV3 & PNV4) intended to be used for screening

pharmacological activities was also calculated.

The results were summarized in table 6.1.2.4.1, 6.1.2.4.2

197

Table 6.1.2.4.1

Effect of shodhana on total alkaloidal content of nux-vomica seeds - gravimetric analysis

Table 6.1.2.4.2

Effect of shodhana on total alkaloid content of nux-vomica seeds-

total alkaloids in treatment dose

Samples

Total alkaloid

( % w/w)

Dose

(mg/kg)

Total alkaloid

present in dose (mg/kg)

UNV 2.74 52 1.42

PNV 1 1.47 520 7.64

PNV 2 1.13 520 5.87

PNV 3 0.74 520 3.84

PNV 4 0.60 520 3.12

PNV 5 0.30 520 1.56

S.no. Samples

Total alkaloid

Content

( % w/w)

1. UNV 2.74±0.08

2. PNV1 1.47±0.05

3. PNV2 1.13±0.025

4. PNV3 0.74±0.021

5. PNV4 0.6±0.013

198

6.1.2.5. Isolation & estimation of strychnine & brucine

The strychnine and brucine concentrations were decreased

gradually upon stepwise shodhana process. The fully detoxified

products contain lesser amounts of strychnine and brucine, when

compared to unprocessed and partially detoxified samples.

The content of strychnine and brucine in the dose of UNV,

PNV1, PNV2, PNV3 & PNV4 intended to be used for further

pharmacological screening was also determined.

The results were summarized in table 6.1.2.5.1, 6.1.2.5.2 &

6.1.2.5.3.

199

Table 6.1.2.5.1

Effect of shodhana on strychnine and brucine content of seeds of nux-vomica - spectroscopic analysis

Samples Strychnine

(% w/w)

Brucine

( % w/w)

UNV 1.5 0.85

PNV1 0.9 0.51

PNV2 0.5 0.45

PNV3 0.41 0.30

PNV4 0.3 0.24

Table 6.1.2.5.2

Effect of shodhana on strychnine content in treatment dose

Samples

Strychnine

(% w/w)

Dose

(mg/kg)

Strychnine present in the

dose (mg/kg)

UNV 1.5 52 0.78

PNV 1 0.90 520 4.68

PNV 2 0.50 520 2.60

PNV 3 0.41 520 2.13

PNV 4 0.30 520 1.56

PNV 5 0.30 520 0.78

Table 6.1.2.5.3

Effect of shodhana on brucine content in treatment dose

Samples Brucine (%w/w)

Dose (mg/kg)

Brucine present in the dose

(mg/kg)

UNV 0.85 52 0.42

PNV 1 0.51 520 2.65

PNV 2 0.45 520 2.34

PNV 3 0.30 520 1.56

PNV 4 0.24 520 1.25

PNV 5 0.12 520 0.625

200

6.1.2.6 Effect of shodhana on strychnine & brucine content of seeds of nux-vomica - HPTLC analysis

The distinct spots were seen in all the products (i.e. before and

after shodhana) at Rf value 0.26 and 0.18. These two Rf values were

in confirmation with the Rf values of standard strychnine & brucine

respectively and thereby confirmed the presence of these two principal

alkaloids in all the samples namely UNV, PNV1, PNV2, PNV3 & PNV4.

The HPTLC study of nux-vomica seeds after shodhana process

showed marked decrease in strychnine and brucine content when

compared with unprocessed seeds. The strychnine and brucine

content were decreased to about 1/3rd in shodhana processed i.e. PNV

3, when compared with unprocessed UNV. The strychnine content

was decreased to about 1/5th and brucine content was decreased

about 1/3rd in shodhana processed PNV4 when compared with

unprocessed UNV. It was also observed that four peaks were present

corresponding to four major compounds in unprocessed UNV.

Whereas there were only three peaks corresponds to three major

compounds were present after shodhana processed products, PNV 3

and PNV 4.

The results were summarized in table 6.1.2.6.1, 6.1.2.6.2 &

HPTLC chromatograms were depicted in fig. 6.1.2.6.1., 6.1.2.6.2,

6.1.2.6.3, 6.1.2.6.4 and 6.1.2.6.5.

201

Table 6.1.2.6.1

Effect of shodhana on HPTLC chemical finger print of nux-vomica

seeds

S.no. Samples Strychnine

(Rf)

Brucine

(Rf)

1. Standard

markers 0.25 0.18

2. UNV 0.26 0.18

3. PNV3 0.26 0.18

4. PNV4

0.26 0.18

Table 6.1.2.6.2

Effect of shodhana on strychnine & brucine content in nux-vomica seeds - HPTLC analysis

S.no. Samples Strychnine(%g) Brucine(%g) No.of peaks

1. UNV 1.17 0.80 4

2. PNV3 0.38 0.28 3

3. PNV4 0.23 0.26 3

202

Fig 6.1.2.6.1 HPTLC chromatogram of strychnine marker

Fig 6.1.2.6.2 HPTLC chromatogram of brucine marker

Fig 6.1.2.6.3 HPTLC chromatogram of UNV

203

1-brucine, 2-strychnine

Fig 6.1.2.6.4

HPTLC chromatogram of PNV 3

1 – brucine, 2 -strychine

Fig 6.1.2.6.5 HPTLC chromatogram of PNV 4

1 – brucine, 3 –strychine

204

6.1.2.7. Effect of shodhana on phytochemicals of nux-vomica

seeds – LC-MS analysis

It was observed by LC-MS analysis of methanolic extracts of

shodhana processed and unprocessed seeds of nux-vomica that the

various phytochemicals were identified on the basis of their molecular

ion peaks. It was observed that higher concentrations of strychnine,

brucine and loganic acid were present in unprocessed sample (UNV).

LC-MS analysis of completely shodhana processed product,

PNV3 and PNV4 showed that the strychnine and brucine

concentrations were decreased and loganic acid glycoside was entirely

eliminated.

It was also observed that both shodhana processed and

unprocessed extracts showed the presence of various indole strychnos

alkaloids, glycosides etc. They were identified on the basis of their

molecular ion peaks. The intensity of molecular ion peak indicates the

quantity of particular constituent. There are other unidentified

molecular ion peaks were observed. Additionally some molecular ion

peaks of derivatives of nux-vomica constituents were also observed.

The results were summarized in table no. 6.1.2.7 & fig. no

6.1.2.7.1, 6.1.2.7.2, 6.1.2.7.3, 6.1.2.7.4, 6.1.2.7.5, 6.1.2.7.6,

6.1.2.7.7, 6.1.2.7.8, 6.1.2.7.9 & 6.1.2.7.10.

205

Table no 6.1.2.7

Effect of shodhana on phytochemicals of nux-vomica seeds - LC-MS analysis

UNV PNV1 PNV2 PNV3 PNV4

M+ M- M+ M- M+ M- M+ M- M+ M-

Strychnine m/z 335

Loganic acid m/z 375

Strychnine m/z 335

Brucine m/z 395

Strychnine m/z 335

Loganine m/z 389

Strychnine m/z 335

Mavacurine m/z 311

Strychnine m/z 335

Vomicine m/z 380

Brucine m/z 395

Brucine N-oxide m/z 411

Novacine m/z 425

Loganine m/z 389

Brucine m/z 395

Acetyl Loganine m/z 417

Brucine m/z 395

- Brucine m/z 395

-

Colubrine m/z 365

- Brucine N-

oxide m/z 411

- Novacine m/z 425

Loganic Acid

m/z 375

21,22- dihydroxy Strychnine m/z 369

- Novacine m/z 425

-

Vomicine m/z 381

-

21,22- dihydroxy strychnine m/z 369

- Colubrine m/z 365

- - - - -

Novacine m/z 425

- - - Mavacurin m/z 311

Strychnin -N-oxide derivative m/z 355

- - - -

- -

Dihydro strychnine derivative m/z 339

-

Dihydro strychnine

derivative m/z 339

-

Dihydroxy strychnine

derivative m/z 339

-

Dihydroxy strychnine derivative m/z 339

-

-

Strychnin-N-oxide

derivative m/z 353

Strychnin-N-oxide

derivative m/z 353

- - - Strychnin-N-

oxide m/z 355 -

Strychnine-N-oxide m/z 355

-

- - - - Brucine-N-

oxide derivative m/z 409

-

Brucine-N-oxide

derivative m/z 409

- Brucine-N-oxide

derivative m/z 409

-

206

Fig. 6.1.2.7.1

LC-MS analysis of unprocessed seeds of nux-vomica (UNV) M+ ion

peaks

Fig. 6.1.2.7.2

LC-MS analysis of unprocessed seeds of nux-vomica (UNV) M -

peaks

207

Fig. 6.1.2.7.3

LC-MS analysis of shodhana processed seeds of nux-vomica

(PNV1) M + peaks

Fig. 6.1.2.7.4


(PNV1) M - peaks

Fig. 6.1.2.7.5


(PNV2), M + peaks

208

Fig. 6.1.2.7.6

LC-MS analysis of shodhana processed nux-vomica seeds (PNV-2)

M - peaks

Fig. 6.1.2.7.7


(PNV3) M + peaks

Fig. 6.1.2.7.8


(PNV3) M - peaks

209

Fig. 6.1.2.7.9

LC-MS analysis of shodhana processed seeds of nux-vomica (PNV4) M + peaks

Fig. 6.1.2.7.10


(PNV4) M - peaks

210

6.1.3 Acute toxicity studies

Effect of shodhana on acute toxicity of nux-vomica seeds in mice

The LD50 value of UNV was found to be 260 mg/kg. But there

was a significant increase in LD50 of PNV1, PNV2, PNV3 and PNV4

(i.e. 10 times more) of UNV (2600 mg/kg).

In the present study 1/5th LD50 of all the products of nux-

vomica were selected for further study.

The results are compiled in tables 6.1.3.1 and 6.1.3.2 and

depicted in fig. 6.1.3.1 and 6.1.3.2.

Table 6.1.3.1

Effect of shodhana on acute toxicity of unprocessed seeds of nux-

vomica (UNV) in mice

Group Dose

mg/kg,po

Log dose Dead/

Total

% Dead Corrected

%

Probit

1 200 2.30 0/10 0 2.5* 3.04

2 225 2.35 1/10 10% 10 3.72

3 250 2.39 2/10 20% 20 4.16

4 275 2.43 6/10 60% 60 5.25

5 300 2.47 10/10 100% 97.5* 6.96

*corrected for 0% dead =100 (0.25/n) and 100% dead =100 (n-0.25/n)

Table 6.1.3.2

Effect of shodhana on acute toxicity of shodhana processed seeds of nux-vomica (PNV1-5) in mice

Group Dose

mg/kg,po

Log

dose

Dead/

Total % Dead

Corrected

% Probit

1 2000 3.30 0/10 0% 2.5* 3.04

2 2100 3.32 1/10 10% 10 3.72

3 2200 3.34 2/10 20% 20 4.16

4 2300 3.36 2/10 20% 20 4.32

5 2400 3.38 3/10 30% 30 4.48

6 2500 3.39 4/10 40% 40 4.75

7 2600 3.41 5/10 50% 50 5.00

8 2700 3.43 6/10 60% 60 5.25

9 2800 3.44 10/10 100% 97.5* 6.96

*corrected for 0% dead =100(0.25/n) and 100% dead =100(n -

0.25/n).

211

Fig. no 6.1.3.1

Effect of shodhana on acute toxicity of unprocessed seeds of nux-vomica (UNV) in mice

Fig 6.1.3.2

Effect of shodhana on acute toxicity of shodhana processed seeds

of nux-vomica (PNV1-5) in mice

212

6.1.4 Pharmacological study

6.1.4.1 Effect of shodhana on analgesic activity of nux-vomica seeds against acetic acid-induced writhings in mice

The shodhana processed samples suppressed the acetic acid

induced writhing response significantly. The % reduction of writhing

in the animals treated with UNV, PNV1, PNV2, PNV3, PNV4 and PNV5

were 25.92%, 45.48%, 48.14%, 55.35%, 60.08% and 65.23%

respectively. Where as aspirin (100 mg/kg) reduced the writhings to

the extent of 57.82%. The unprocessed sample UNV demonstrated

least protection against acetic acid induced writhing where as PNV3,

PNV4 and PNV5 reduced the acetic acid induced writhing significantly

higher than UNV and were comparable to that of standard drug

aspirin.

It was observed that each step of shodhana process enhanced

the analgesic potency of the nux-vomica seeds. It was also observed

that PNV 4, the product after complete detoxification (as per

traditional healer) possess maximum analgesic potency.

The results were summarized in table 6.1.4.1 & graphically

depicted in fig.6.1.4.1.

213

Table 6.1.4.1

Effect of shodhana on analgesic activity of nux-vomica seeds against acetic acid-induced writhings in mice

Treatment Dose Number of

writhing

%

Protection

Control Acetic acid (1%)

10ml/kg 81 ± 2.23

-

Aspirin 100 mg/kg 34.16 ± 1.72*** 57.82 UNV 52 mg/kg 60.5 ± 5.52* 25.92 PNV1 520mg/kg 44.16 ± 2.54*** 45.48 PNV2 520mg/kg 42 ± 1.94*** 48.14 PNV3 520mg/kg 36.16 ± 2.85*** 55.35 PNV4 520mg/kg 32.33 ± 2.37 *** 60.08 PNV5 520mg/kg 28.16±1.88*** 65.23

Values are mean ± SEM for 6 mice

*P <0.05, **P<0.01 and *** P<0.001 compared to control group

Fig 6.1.4.1

Effect of shodhana on analgesic activity of nux-vomica seeds

against acetic acid-induced writhings in mice

214

6.1.4.2. Effect of shodhana on anti-inflammatory activity of nux-

vomica seeds against carrageenan induced hind paw edema in rats

All the samples i.e. UNV (18.57%), PNV1 (32.96%), PNV2 (41.34%),

PNV3 (44.10%), PNV4 (46.50%) & PNV5 (70.40%) showed significant

anti-inflammatory property. It is observed that anti-inflammatory

potency increased after each step of shodhana. Maximum potency was

exhibited by PNV5 (the formulation containing shodhitha nux-vomica).

The anti-inflammatory potency of shodhitha nux-vomica i.e. PNV3,

PNV4 & PNV5 was comparable to that of standard during diclofenac

sodium (25mg/kg). It is evident from the results that the potency of

nux-vomica is enhanced by the shodhana process.

The results were summarized in table 6.1.4.2 and graphically

depicted in fig. 6.1.4.2.

215

Table 6.1.4.2 Effect of shodhana on anti-inflammatory activity of nux-vomica seeds against carrageenan

induced hind paw edema in rats

Treatment

Dose

(mg/kg)

Mean paw-size in mm

% Inhibition

% Inhibition

1h 2h 4h 6h 1h 2h 4h 6h

Control - 0.52±0.056 0.81±0.09 0.83±0.077 0.71±0.05 - - - -

Diclofenac

Sodium

25 0.47±0.061 0.40±0.083** 0.35±0.090** 0.31±0.064** 5.0 50.98 57.98 55.86

UNV 52 0.49±0.02ns 0.76±0.07ns 0.65±0.08ns 0.58±0.03ns 1.8 5.1 21.9 18.57

PNV1 520 0.46±0.10 0.76±0.07 0.53±0.08* 0.48±0.02* 8.0 6.12 36.0 32.96

PNV2 520 0.47±0.03 0.70±0.117 0.52 ±0.12* 0.42±0.09** 5.0 13.23 36.85 41.34

PNV3 520 0.35±0.03 0.65±0.086 0.53±0.04** 0.4±0.03** 28.3 19.3 38.05 44.1

PNV4 520 0.43±0.05 0.62±0.038 0.50±0.030** 0.38±0.02** 13.4 23.4 39.01 46.5

PNV5 520 0.45±0.07 0.65±0.051 0.37 ±0.04** 0.21±0.07** 9.6 20.3 54.9 70.4

All values represent mean ± SEM, n=6 in each group, *P <0.05 and **P<0.01 compared to control group

216

Anti-inflammatory activity- 2h of the treatment

00.10.20.30.40.50.60.70.80.9

1

Control Diclofenac

sodium

UNV PNV1 PNV2 PNV3 PNV4 PNV5

Treatment

Mean

paw

siz

e (

mm

)

**

ns ns nsns

nsns


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Control Diclofenac

sodium


Treatment

Mean

paw

siz

e (

mm

)

*

nsns

nsns

nsns

Fig.6.1.4.2

Effect of shodhana on anti-inflammatory activity of nux-vomica

seeds against carrageenan induced hind paw edema in rats

217

Anti-inflammatory activity-6h of the treatment

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Control Diclofenac

sodium


Treatment

Mean

paw

siz

e (

mm

)

ns

**

* ****

**

**


0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

Control Diclofenac

sodium


Treatment

Mean

paw

siz

e i

n m

m

** **

****

**

ns

218

6.1.4.3 Effect of shodhana on anti-ulcer activity of nux-vomica

A. Ethanol induced gastric ulcers in rats

Oral administration of ethanol produced severe ulceration. The

detoxified (Shodhana) samples PNV1 to PNV5 (520 mg/kg) and

standard drug lansoprazole (8 mg/kg p.o) significantly reduced the

ulcer index and sevearity of ulceration in ethanol induced ulcer model.

Out of all these processed samples, the completely processed products

PNV3, PNV4 and formulation PNV5 showed maximum inhibition of

gastric lesions against ethanol induced gastric mucosal damage when

compared with the positive control group. The gastro-protective effect

of detoxified samples showed significant protection when compared

with unprocessed sample. The extent of gastro-protective activity of

PNV4 and PNV5 were more than standard drug.

Pretreatement with PNV1to PNV5, significantly increased tissue

GSH and decreased tissue LPO in ethanol induced ulcer model in rats.

The gastroprotective effect of nux-vomica is significantly enhanced by

shodhana process.

The results were summarized in table 6.1.4.3.1 and graphically

depicted in fig. 6.1.4.3.1, 6.1.4.3.2 & 6.1.4.3.3

219

Table 6.1.4.3.1

Effect of shodhana on anti-ulcer activity of nux-vomica seeds against ethanol induced gastric ulcers in rats

Treatment Ulcer index % Protection

GSH

% Protection

LPO % Protection

Control _ _ 0.65±0.82 _ 0.112±0.080 _

Control (99.5%Ethanol)

4.66 0.84 ___ 0.183 0.01 __ 0.358 0.012 __

Lansoprazole(8mg/kg)

1.41 0.30*** 69.74 0.418 0.046*** 56.22 0.141 0.004*** 60.61

UNV (52mg/kg) 2.91 0.66ns 37.55 0.245 0.024ns 25.30 0.296 0.028ns 17.31

PNV 1 (520mg/kg) 1.66 0.40*** 64.37 0.31 0.028** 40.96

0.240 0.027** 32.96

PNV 2 (520mg/kg) 1.5 0.31*** 67.81 0.3450.042*** 46.95 0.226 0.021*** 36.87

PNV 3 (520mg/kg) 1.45 0.30*** 68.88 0.45 0.063*** 59.33 0.210 0.014*** 41.34

PNV 4 (520mg/kg) 1.16 0.27*** 75.10 0.537 0.010*** 65.92

0.167 0.012*** 53.35

PNV 5 (520mg/kg) 1.33 0.30***

71.45 0.492 0.025*** 62.80 0.195 0.019*** 45.53

All values represent mean ± SEM, n=6 in each group, ns = non significant,**P <0.01, ***P <0.001 V/s control group

220

0

0.1

0.2

0.3

0.4

0.5

0.6

Cont

rol

Lans

opraz

ole

UNV

PNV1

PNV2

PNV3

PNV4

PNV5

Treatment

GS

H

***

ns

*****

******

***

0

1

2

3

4

5

6

Cont

rol

Lans

opraz

ole

UNV

PNV1

PNV2

PNV3

PNV4

PNV5

Treatment

Ulc

er

ind

ex

***

ns

****** ***

*** ***

Fig 6.1.4.3.1

Effect of shodhana on anti-ulcer activity of nux-vomica seeds against ethanol induced gastric ulcers in rats

Fig 6.1.4.3.2

Effect of shodhana on anti-ulcer activity of nux-vomica seeds on

tissue GSH against ethanol induced gastric ulcers in rats

221

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

Cont

rol

Lans

opraz

ole

UNV

PNV1

PNV2

PNV3

PNV4

PNV5

Treatment

LP

O

***

ns

******

***

******

Fig 6.1.4.3.3


tissue LPO against ethanol induced gastric ulcers in rats

222

B. Pylorus ligation induced ulcer in albino rats

Pyloric ligation produced gastric ulcers, increased the free &

total acidity and reduced the pH of gastric content. Pretreatment with

UNV, processed PNV1 to PNV4 and formulation PNV5 significantly

reduced the ulcer index, tissue lipid peroxidation and enhanced the

tissue GSH levels. The anti-secretory property of shodhana processed

samples in pylorus ligation model was evident from its significant

reduction in gastric volume, free acidity, total acidity, ulcer index and

significantly increased gastric pH. The gastro-protective property

produced by processed samples was significantly higher than the

UNV. In this model also, the gastro-protection offered by the processed

samples was comparable to that of the standard lansoprazole.

The results were summarized in table no. 6.1.4.3.2 and

6.1.4.3.3 & graphically depicted in fig. no. 6.1.4.3.4, 6.1.4.3.5,

6.1.4.3.6, 6.1.4.3.7, 6.1.4.3.8, 6.1.4.3.9 and 6.1.4.3.10.

223

Table 6.1.4.3.2

Effect of shodhana on anti-ulcer activity nux-vomica seeds against pylorus ligation induced ulcer in rats

Treatment Ulcer index %Protection

GSH

%Protection

LPO

%Protection

Control

5.5 0.50

---

0.180.02

----

0.27 0.006

----

Lansoprazole (8mg/kg)

0.580.30*** 89.45

0.370.02***

51.35

0.093 0.007***

65.55

UNV

(52mg/kg) 2.66 0.55*** 51.63 0.300.004*** 40.00 0.145 0.008*** 46.29

PNV1 (520mg/kg)

1.660.10*** 69.81 0.320.006*** 43.75 0.120.002*** 55.55

PNV2

(520mg/kg) 1.00.18*** 81.81 0.350.005*** 49.43 0.10.007*** 62.96

PNV3 (520mg/kg)

0.660.10*** 88.0 0.400.005*** 55.99 0.093 0.006*** 65.55

PNV4

(520mg/kg) 0.5830.30*** 89.4 0.440.006*** 59.73 0.0870.001*** 67.77

PNV5 (520mg/kg)

0.660.24*** 88.0 0.420.003*** 57.14 0.0930.002*** 65.55

All values represent mean ± SEM, n=6 in each group. ns = non significant,*P<0.05,**P <0.01, ***P <0.001 V/s control group.

224

0

1

2

3

4

5

6

7

Cont

rol

Lans

opraz

ole

UNV

PNV1

PNV2

PNV3

PNV4

PNV5

Treatment

Ulc

er

ind

ex

***

***

******

*** *** ***

Fig. 6.1.4.3.4

Effect of shodhana on anti-ulcer activity of nux-vomica seeds against pylorus ligation induced ulcer in albino rats

Fig. 6.1.4.3.5


tissue GSH against pylorus legation induced ulcer in albino rats

225

0

0.05

0.1

0.15

0.2

0.25

0.3

Cont

rol

Lans

opraz

ole

UNV

PNV1

PNV2

PNV3

PNV4

PNV5

Treatment

LP

O

***

***

*** *** ******

***

Fig. 6.1.4.3.6

Effect of shodhana on anti-ulcer activity nux-vomica seeds on tissue LPO against pylorus legation induced ulcer in rats

226

Table 6.1.4.3.3

Effect of shodhana on anti-ulcer activity of nux-vomica seeds on gastric secretion following pylorus ligation induced ulcer in rats

Treatment

Dose

Gastric

volume(ml)

pH

Free Acidity (Eq/I)

Total Acidity (Eq/I)

Control -- 8.61 0.99 2.56 0.19 97.82 6.97 108.1 6.07

Lansoprazole 8mg/kg 3.73 0.54*** 6.75 0.29*** 37.62 6.28*** 50.57.6***

UNV 52mg/kg 5.71 0.23** 3.28 0.26ns 64.59 6.96** 82.57 7.3ns

PNV1 520mg/kg 4.30.21** 4.50.65* 53.658.12* 73.658.1*

PNV2 520mg/kg 4.10.78*** 4.90.8*** 48.67.9*** 65.79.3***

PNV3 520mg/kg 3.90.36*** 4.950.56*** 46.58.2*** 56.78.5***

PNV4 520mg/kg 3.610.3*** 5.210.53*** 43.84.2*** 52.095***

PNV5 520mg/kg 3.8 0.33*** 5.25 0.4*** 38.65.5*** 49.08.3***

All values represent mean ± SEM, n=6 in each group.

ns = non significant *P<0.05, **P <0.01, ***P <0.001 V/s control group.

227

0

2

4

6

8

10

12

Cont

rol

Lans

opraz

ole

UNV

PNV1

PNV2

PNV3

PNV4

PNV5

Treatment

Gastr

ic v

olu

me (

ml)

****** **

**

*** *** ***

0

1

2

3

4

5

6

7

8

Cont

rol

Lans

opraz

ole

UNV

PNV1

PNV2

PNV3

PNV4

PNV5

Treatment

Gastr

ic p

H

***

ns

**** *** *** ***

Fig.6.1.4.3.7

Effect of shodhana on anti-ulcer activity of nux-vomica seeds on gastric volume following pylorus ligation induced ulcer in albino

rats

Fig.6.1.4.3.8


gastric pH following pylorus ligation induced ulcer in albino rats

228

0

20

40

60

80

100

120

Cont

rol

Lans

opraz

ole

UNV

PNV1

PNV2

PNV3

PNV4

PNV5

Treatment

Fre

e a

cid

ity(m

Eq

/l/1

00g

)

***

****

*** ****** ***

0

20

40

60

80

100

120

Cont

rol

Lans

opraz

ole

UNV

PNV1

PNV2

PNV3

PNV4

PNV5

Treatment

To

tal

acid

ity (

mE

q/l

/100g

)

***

ns

****

****** ***

Fig.6.1.4.3.9

Effect of shodhana on anti-ulcer activity of nux-vomica seeds on free acidity following pylorus ligation induced ulcer in rats

Fig.6.1.4.3.10


total acidity following pylorus ligation induced ulcer in rats

229

6.1.4.4. Effect of shodhana on hepatoprotective activity of nux-

vomica seeds against CCl4 induced hepatic damage in rats

In the control group (+ve control) CCl4 significantly enhanced

the biochemical markers of liver injury like SGPT, SGOT, ALP,

bilirubin, and cholesterol. Pre-treatment with UNV (52 mg/kg), PNV3

and PNV4 (520 mg/kg) significantly reduced the elevated levels of all

the above mentioned biochemical markers. In addition, CCl4 depleted

the tissue GSH and enhanced the tissue lipid peroxidation. Pre-

treatment with UNV, PNV3 and PNV4 prevented the depletion of tissue

GSH levels and reduced lipid peroxidation. Pre-treatment with PNV3

and PNV4 significantly reduced the elevated levels of biochemical

markers to near normal levels. Pre-treatment with study products

prevented the depletion of tissue GSH and reduced LPO significantly.

There was a significant protection exhibited by pretreatment with

PNV4 when compared with PNV3. Evidently, the hepatoprotective

effect of PNV3 and PNV 4 were near to that of standard silymarin.

Both PNV3 and PNV4 produced higher hepato-protection than

unprocessed UNV group.

The results were summarized in tables 6.1.4.4.1 and 6.1.4.4.2

& graphically depicted in fig. 6.1.4.4.1, 6.1.4.4.2, 6.1.4.4.3, 6.1.4.4.4,

6.1.4.4.5, 6.1.4.4.6, 6.1.4.4.7 and 6.1.4.4.8

230

Table 6.1.4.4.1

Effect of shodhana on hepatoprotective activity of nux-vomica seeds against CCl4 induced hepatic damage in

rats

Values are mean ± SEM for six rats

***P <0.001 V/s CCl4 group, figures in parentheses are the percentage of protection

Treatment

SGOT

(U/L)

SGPT

(U/L) ALP(U/L)

Total

bilirubin (mg/dl)

Direct

bilirubin (mg/dl)

Cholesterol

(mg/dl)

Control

(saline 0.5ml po,

5days)

53.53 ± 2.39 56.38 ± 1.43 117.4 ± 3.13 0.88 ± 0.07 0.38 ± 0.06 113.33 ± 4.85

CCl4 (2ml/kg sc) 305 ± 3.84 321.01 ± 6.63 279.46 ± 11.07 4.52 ± 0.27 1.48 ± 0.16 174.98 ± 2.63

Silymarin (100mg/kg,po) 5 days

+ CCl4

70.58 ± 3.51***

(76.85)

66.28 ± 2.62***

(79.35)

90.88 ± 2.37***

(67.48)

1.06± 0.05***

(76.54)

0.255± 0.01***

((82.77)

118.81± 4.03***

(32.10)

UNV (52mg/kg,po) 5 days + CCl4

106.58±6.14*** (65.24)

112.83±8.3*** (65.10)

110.45±7.63*** (60.06)

1.21± 0.06*** (73.23)

0.32 ± 0.02*** ((78.37)

150.33± 3.98*** ((14.08)

PNV3 (520mg/kg, po)

5 days + CCl4

99.53 ± 6.62***

(67.36)

101.6 ± 6.67***

(68.53 )

102.31± 7.83***

(63.39)

1.21± 0.07***

(73.23)

0.29 ± 0.02***

((80.40)

126.88± 3.62***

(27.64)

PNV4 (520mg/kg, po) 5

days + CCl4

69.61 ± 3.62***

(77.17)

67.9 ± 3.63***

(78.84)

91.98 ± 1.67***

(67.08)

1.14± 0.12***

(74.77)

0.27 ± 0.02***

((81.75)

122.03± 2.80***

((30.26)

231

Fig. 6.1.4.4.1

Effect of shodhana on hepatoprotective activity of nux-vomica seeds on SGOT against CCl4 induced hepatic damage in rats

Fig. 6.1.4.4.2

Effect of shodhana on hepatoprotective activity of nux-vomica

seeds on SGPT against CCl4 induced hepatic damage in albino rats

232

Fig. 6.1.4.4.3

Effect of shodhana on hepatoprotective activity nux-vomica seeds on ALP against CCl4 induced hepatic damage in rats

Fig. 6.1.4.4.4


seeds on total bilirubin against CCl4 induced hepatic damage in

rats

233

Fig. 6.1.4.4.5


seeds on direct bilirubin against CCl4 induced hepatic damage in

rats

Fig. 6.1.4.4.6


seeds on cholesterol against CCl4 induced hepatic damage in rats

234

Table 6.1.4.4.2

Effect of shodhana on hepatoprotective activity of nux-vomica seeds in liver tissue GSH & LPO in CCl4 induced hepatic damage in rats

Treatment

GSH

% Protection

LPO

% Protection

Control

(saline 0.5ml po,

5 days)

0.266 ± 0.012

___

0.0813 ± 0.005

____

CCl4 (2ml/kg,sc)

0.0695 ± 0.0132

___ 0.236 ± 0.021

____

Silymarin (100mg/kg,po) 5 days + CCl4

0.25 ± 0.0256***

72.2

0.0821 ±0.004***

65.21

UNV (52mg/kg,po)

5 days + CCl4

0.169 ± 0.017***

58.87

0.12 ± 0.015***

48.30

PNV3 (520mg/kg,po)

5 days + CCl4

0.209 ± 0.007***

66.74

0.090 ± 0.01***

61.61

PNV4 (520mg/kg,po) 5 days + CCl4

0.246 ± 0.015***

71.74

0.083± 0.004***

64.83

Values are mean ± SEM for 6 rats ***P <0.001 V/s CCl4 group

235

Fig 6.1.4.4.7


seeds in liver tissue GSH levels in CCl4 induced hepatic damage in

rats

Fig. 6.1.4.4.8


seeds in liver tissue LPO levels in CCl4 induced hepatic damage in rats

236

Histopathological observations of liver

Group I –Control group: Liver section showed normal lobular

architecture and normal hepatic cells with a well preserved cytoplasm

and well-defined nuclei with nucleolus.

Group II - CCl4 treated group: Liver section showed centrilobular

necrosis, some cells showed loss of nucleus and nucleoli. Liver

sinusoids were congested and infiltration by inflammatory cells.

Group III - Sylimarin treated group: Liver section showed some cells

with loss of nucleus, but there was well defined cytoplasm. Occasional

Kupffer cell proliferation was seen. Hepatocytes regeneration was

observed.

Group IV - UNV treated group: Liver section showed normal lobular

architecture with some cells showing loss of nucleus, occasional areas

of kupffer cells proliferation with mild dilatation along with fatty

degeneration. Hepatocytes regeneration was observed.

Group V - PNV3 treated group: Liver section showed normal lobular

architecture with mild dilatation and mild fatty degeneration.

Hepatocytes regeneration was observed.

Group VI - PNV-4 treated group: Liver section showed normal lobular

architecture with mild hepatocyte dilatation and mild fatty

degeneration. Hepatocytes regeneration was observed. Therefore it

may be inferred that hepatoprotective property of nux-vomica is

enhanced by shodhana process. The histopathalogical reports are

summarized in plate no.6.1.4.4.

237

Plate no. 6.1.4.4

Photographs showing histopathology of rat liver

C. Liver histopathalogy of

sylimarin treated rat

A. Liver histopathalogy of

control rat

B. Liver histopathology of

CCl4 treated rat

238

D.Liver histopathology of unprocessed nux-vomica (UNV) treated rat

E. Liver histopathology of shodhana processed

nux-vomica (PNV3) treated rat

F. Liver histopathology of shodhana processed

nux-vomica (PNV4) treated rat

239

6.1.4.5 Effect of shodhana on nephroprotective activity nux-

vomica seeds against cisplatin induced nephrotoxicity in albino rats

In the control group, cisplatin significantly increased the

biochemical markers of kidney injury like BUN (89.18 mg/dl),

creatinine (3.36 mg/dl), kidney weight (1.16 G % w/w) and the body

weight was decreased (-7.41 G % w/w). Treatment with UNV (52

mg/kg), PNV3 and PNV4 (520 mg/kg) reversed the cisplatin challenge

i.e. prevented the elevation of all the biomarkers of kidney damage

(BUN: 42.05 mg/dl, 32.31 mg/dl, 24.84 mg/dl respectively,

creatinine: 1.74 mg/dl, 1.01 mg/dl, 0.99 mg/dl respectively and

kidney weight: 0.92 G % w/w, 0.79 G % w/w, 0.79 G % w/w

respectively and prevented the loss in body weight: -3.45 G % w/w,

7.96 G % w/w, 6.59 G % w/w respectively). Even, the treatment with

shodhana processed samples like PNV3 (7.96 G % w/w) and PNV4

(6.59 G % w/w) enhanced the body weight.

In addition, cisplatin depleted the levels of kidney tissue GSH and

enhanced the tissue lipid peroxidation. Treatment with UNV, PNV3

and PNV4 prevented the depletion of tissue GSH levels and there was

reduction in lipid peroxidation. Both the processed samples PNV3 and

PNV4 showed significantly higher protective property than

unprocessed UNV treated group. Treatment with processed seeds

PNV3 and PNV4 showed recovery in kidney damage when compared

with unprocessed seeds UNV. The results were summarized in table

6.1.4.5.1, 6.1.4.5.2 and graphically depicted in fig.6.1.4.5.1, 6.1.4.5.2,

6.1.4.5.3, 6.1.4.5.4, 6.1.4.5.5 and 6.1.4.5.6.

240

Histopathology observations of kidney

Group I: Normal control (-ve control) showed structure of kidney with

normal glomeruli, proximal and distal tubules and with normal

interstitial cells and blood vessels.

Suggestive: normal kidney architecture

Group II: Cisplatin treated group (+ control) showed structure of

kidney with glomerular congestion. Interstitium showed infiltration

with inflammatory cells. This group animal showed severe interstitial

congestion.

Suggestion: Severe interstitial congestion

Group III: Treatment with UNV showed structure of kidney with

maintained glomeruli, proximal and distal tubules. There was less

interstitial congestion as compared to cisplatin treated group.

Suggestion: Recovery of interstitial congestion

Group IV: Treatment with PNV-3 showed structure of kidney with

maintained glomeruli, proximal and distal tubules. There was no

interstitial congestion.

Suggestion: Interstitial congestion is like normal.

Group V: Treatment with PNV-4 showed structure of kidney with

maintained glomeruli, proximal and distal tubules. There was no

interstitial congestion.

Suggestion: Interstitial congestion is like normal.

The histopathology of kidney photographs are depicted in plate no.

6.1.4.5

241

Table 6.1.4.5.1

Effect of shodhana on nephroprotective activity of nux-vomica seeds on biochemical & physical parameters against cisplatin induced nephrotoxicity in rats

Treatment

BUN (mg/dl)

Serum

Creatinine

(mg/dl)

Change in

body weight

(G % w/w)

Kidney weight (G % w/w)

Control

(saline 0.5ml po, 6 days) 21.59±1.13 0.95± 0.05 8.06±0.31 0.786± 0.02

Cisplatin 89.18±2.07 3.36± 0.41 -7.41±0.91 1.16± 0.04

UNV (52mg/kg,po 6 days) +

Cisplatin 42.05±2.60*** 1.74± 0.04*** -3.45±1.89 0.92± 0.04***

PNV3 (520mg/kg,po 6 days) +

Cisplatin 32.31±2.28*** 1.01±0.12***

7.96±2.69***

0.79± 0.03***

PNV4 (520mg/kg, po 6 days)+

Cisplatin 24.84±1.19*** 0.99±0.08*** 6.59±2.79*** 0.79± 0.01***

Values are mean ± SEM for 6 rat, ***P <0.001 V/s control group

242

Fig. 6.1.4.5.1

Effect of shodhana on nephroprotective activity of nux-vomica

seeds on BUN against cisplatin induced nephrotoxicity in rats

Fig. 6.1.4.5.2

Effect of shodhana on nephroprotective activity of nux-vomica seeds on creatinine against cisplatin induced nephrotoxicity in

rats

243

Fig. 6.1.4.5.3


seeds on % change in body weight against cisplatin induced

nephrotoxicity in rats

Fig. 6.1.4.5.4


seeds on % change in kidney weight against cisplatin induced nephrotoxicity in rats

244

Table 6.1.4.5.2

Effect of shodhana on nephroprotective activity of nux-vomica seeds on in-vivo GSH & LPO in cisplatin

induced nephrotoxicity in rats

Treatment

GSH

% increase

LPO

% protection

Control(saline 0.5 ml p.o.,

6days) 0.298±0.0153 ____ 0.128±0.0008 _____

Cisplatin(6mg/kg, iv)

0.122±0.006

___

0.324±0.022

__

UNV (52mg/kg, po 6 days)+ cisplatin

0.241±0.02***

49.37

0.215±0.024**

33.64

PNV3(520mg/kg, po 6 days) + cisplatin

0.275±0.01***

55.63

0.172±0.0165***

46.13

PNV4 (520mg/kg, po

6 days)+ cisplatin

0.29±0.0199***

58.05

0.159±0.012***

50.92


ns = non significant *P<0.05, **P <0.01 and ***P <0.001 V/s control group.

245

Fig. 6.1.4.5.5


seeds in kidney tissue GSH levels against cisplatin induced

nephrotoxicity in rats

Fig. 6.1.4.5.6


seeds on tissue LPO against cisplatin induced nephrotoxicity in rats

246

Plate no 6.1.4.5. Photographs showing histopathology of kidney

A. Normal rat kidney histopathalogy

B. Cisplatin treated rat kidney histopathalogy

C. UNV treated rat kidney histopathalogy

D.PNV3 treated rat kidney hisytopathalogy

247

E.PNV4 treated rat kidney histopathalogy

248

6.2. Commiphora mukul (guggul)

6.2.1. Pharmacognostic analysis

6.2.1.1 Physico-chemical analysis

The pharmacognostic analysis of oleo-gum resin of guggul

before and after shodhana process showed that loss on drying of

unprocessed guggul GUG-1 is 4.8 G% w/w. The loss on drying of

GUG-2 and GUG-3 were 6.85 G% w/w and 5.8 G% w/w

respectively. This clearly indicates that the water content in the

shodhana processed guggul by cow urine treatment contains more

moisture than processed guggul.

Total ash values of unprocessed guggul GUG-1 was 8.42 G%

w/w and acid insoluble ash value is 4.91 G% w/w. The total ash

value of GUG-2 and GUG-3 were 7.92 G% w/w and 11.4 G% w/w

respectively. The acid insoluble ash values of GUG-2 and GUG-3

was 4.12 G% w/w and 4.69 G% w/w respectively.

The water extractive values were decreased after each

process of detoxification except in GUG-3. The alcohol extractive

values were decreased after each process of detoxification.

The data of the average of four times repetitions was given

hereunder and all these parameters can be used for standardization

purposes. The results were summarized in the table 6.2.1.1

249

Table 6.2.1.1

Effect of shodhana on physico-chemical properties of guggul

S.no.

Method ( G% w/w)

GUG 1

GUG 2

GUG 3

1. Loss on drying 4.8±0.05 6.85±0.04 5.8±0.20

2. Water soluble extractive 14.56±0.60 7.2±0.04 18.4±0.07

3.

Alcohol(70%) extractive

18.42±0.09

2.43±0.06

4.2±0.067

4. Petroleum ether(60-80ºC)

extractive 10.12±0.07 7.81±0.06 8.56±0.12

5. Total ash 8.42±0.075 7.92±0.076 11.4±0.72

6.

Acid insoluble ash

4.91±0.025

4.12±0.057

4.69±0.061

7. Water soluble ash

3.13±0.022 2.6±0.021 4.85±0.045

8. Foreign organ matter

5.11±0.1 1.1±0.5 0.8±0.12

250


6.2.2.1. Effect of shodhana on phytochemicals of guggul

It was observed from the phytochemical screening of ethyl

acetate extracts of guggul that steroids, carbohydrates, flavanoids

were present in unprocessed guggul extract GUG1. Cow milk

processed guggul GUG2 contains steroids, proteins, carbohydrates

and flavanoids. Similarly thriphala aqueous extract treated guggul

GUG-3 contains steroids, tannins, proteins and carbohydrates.


Table 6.2.2.1

Effect of shodhana on phytochemicals of guggul

Phytochemical

constituents GUG1 GUG2 GUG3

Steroids ++++ +++ +++

Carbohydrates +++ ++ ++

Flavanoids + + +

Tannins _ _ +++

Proteins _ +++ _

251

6.2.2.2 Phytochemical analysis of shodhana processed materials

after guggul shodhana

It was observed from the phytochemical analysis of cow urine after

shodhana with guggul that it contains steroids, carbohydrates and

flavanoids. It was observed from cow milk after shodhana that it

contains steroids, carbohydrates and flavanoids. Similarly steroids,

tannins, flavanoids and carbohydrates were present in aqueous

extract of triphala after shodhana treatment.

The results were summarized in table 6.2.2.2.

Table 6.2.2.2

Phytochemical analysis of shodhana processed materials after

guggul shodhana

Types of

phytochemical

constituents

Cow milk

Triphala

aqueous

extract

Steroids + +

Carbohydrates + +

Flavanoids + +

Tannins _ +++

252

6.2.2.3 TLC analysis of guggul

The standard samples of guggulsterone E and

guggulusterone Z showed bluish violet spots at Rf values 0.38 and

0.46 respectively. Similarly GUG1, GUG2, GUG3 and GUG4 also

showed bluish violet coloured spots at identical Rf values and

thereby confirms the presence of both the guggulsterones. The

presence of both the guggulusterones was confirmed by TLC.


Table 6.2.2.3

TLC analysis of guggul before and after shodhana

S.no. Samples Guggulsterone E (Rf)

Guggulsterone Z (Rf)

1. Guggulsterone E 0.38 _

2. Guggulsterone Z _ 0.46

3. GUG1 0.38 0.46

4. GUG2 0.39 0.46

5. GUG3 0.38 0.46

253

6.2.2.4 Estimation of guggul lipid by gravimetry

The guggul lipid contains guggulsterone E, guggulsterone Z

and other guggulsterones I - VI. Therefore the estimation of guggul

lipid is the indirect measure of guggulusterone content. Gravimetric

estimation of guggul revealed that, content of guggul lipids are

reduced upon shodhana with triphala kashaya. Contrary to this,

guggul lipid content was increased upon shodhana with cow milk.

The results were summarized in a table 6.2.2.4

Table 6.2.2.4

Effect of shodhana on guggul lipid content in guggul -

gravimetric analysis

S. no.

Samples Guggul Lipid

( G% w/w)

1 GUG 1 5.0

2 GUG 2 6.5

3

GUG 3

3.2

254

6.2.2.5 Effect of shodhana on guggulsterone E &

guggulsterone Z content of guggul – HPLC analysis

It was observed that guggulsterone Z concentration was

increased in GUG-2 and decreased in GUG-3 when compared with

GUG-1. But the guggulsterone E concentrations were decreased in

both processed guggul GUG-2 and GUG-3. It was furthered

observed that four major peaks were present in GUG-1 in which two

peaks were identified as guggulsterone E and guggulsterone Z. One

smaller peak at retention time 5.42 min was observed. However in

GUG-2, 4 peaks were observed corresponding to 4 major compounds

of which two compounds was identified as guggulsterone E & Z and

third and fourth peak were similar to GUG-1. But, the peak at

retention time 5.40 was also seen with enhanced intensity. However,

this peak could not be identified. But in GUG-3 another prominent

peak, which was not present in GUG-1 and GUG-2 was seen at

retention time 2.30 min. The intensity of this peak was more than

the other peaks. The constituent responsible for this peak was not

identified. This peak may be due to the addition of another

phytochemical during detoxification with triphala extract.

The results were recorded in table 6.2.2.5 and graphically

depicted in fig. 6.2.2.5.1, 6.2.2.5.2, 6.2.2.5.3, 6.2.2.5.4.

255

Table 6.2.2.5

Effect of shodhana on guggulsterone E & guggulsterone Z content of guggul - HPLC analysis

Sample Guggulsterone E (%w/w) GuggulsteroneZ (%w/w) No. of peaks

GUG 1 0.13 0.16 4

GUG 2 0.09 0.26 4

GUG 3 0.09 0.10 4

Fig. 6.2.2.5.1

HPLC chromatogram of chemical markers guggulsterone E &

guggulsterone Z

256

Fig. 6.2.2.5.2

HPLC chromatogram of GUG-1

Fig 6.2.2.5.3


257

Fig. 6.2.2.5.4


258

6.2.2.6 Effect of shodhana on phytochemicals of guggul - LC-MS

analysis

The LC-MS data of methanolic extracts of GUG1, GUG2 & GUG3

showed the presence of various phytoconstituents. An attempt was

made to identify the phytoconstituents on the basis of their molecular

ion peaks.

It is surprising to note that the peaks of galloyl glucose & stearic

acid are seen in GUG3. This may be due to the addition of these two

constituents from thriphala kashaya during swedhana.

The results were summarized in table 6.2.2.6 and LC-MS

spectra depicted in fig. 6.2.2.6.1 to 6.2.2.6.6.

Table no. 6.2.2.6 Effect of shodhana on phytochemicals of guggul - LC-MS analysis

GUG-1 GUG-2 GUG-3

M+ M- M+ M- M+ M-

Guggulsterone E/

Guggulsterone Z

m/z 309

- Guggulsterone E/

Guggulsterone Z

m/z 309

- Guggulsterone E/

Guggulsterone Z

m/z 309

-

Myricyl alcohol

m/z 438

- Myricyl alcohol

m/z 438

- Myricyl alcohol

m/z 438

-

Cembrene A

m/z 273

- Cembrene A

m/z 273

- Cembrene A

m/z 273

-

Sesamin

m/z 355

- Sesamin

m/z 355

- Sesamin

m/z 355

-

- - - - Galloyl glucose m/z 334

-

- - - - Stearic acid

m/z 286

-

259

Fig. 6.2.2.6.1

LC-MS analysis of unprocessed guggul (GUG-1): M+ ion peaks

Fig. 6.2.2.6.2

LC-MS analysis of unprocessed guggul (GUG-1): M- ion peaks

Fig. 6.2.2.6.3

LC-MS analysis of shodhana processed guggul (GUG-2): M+ ion

peaks

260

Fig. 6.2.2.6.4

LC-MS analysis of shodhana processed guggul (GUG-2): M- ion peaks

Fig. 6.2.2.6.5

LC-MS analysis of shodhana processed guggul (GUG-3): M+ ion

peaks

Fig. 6.2.2.6.6

LC-MS study of shodhana processed guggul (GUG-3): M- ion peaks

261

6.2.3. Acute toxicity study

There was no mortality, after treatment with 2000 mg/kg of all the

three products GUG1, GUG2 & GUG3 indicating that shodhana did

not influence the acute toxicity of guggul. Hence, 2500 mg/kg can be

considered as the cut off dose. Therefore 1/10th dose (250 mg/kg) was

selected for further pharmacological studies.

6.2.4 Pharmacological studies

6.2.4.1 Effect of shodhana on cardioprotective activity of guggul against isoproterenol (ISO) induced myocardial infarction in

albino rats

ISO treated rats exhibited (p<0.001) higher levels of marker

enzymes of serum myocardial injury such as AST (401.16±7.28 U/L),

ALT (138±2.28 U/L), LDH (3250±84.77 U/L), CPK (1217.66±24.15

IU/L) compared to that of control rats. Pretreatment with GUG-1,

GUG-2 and GUG-3 for 14 days and ISO (200 mg/kg s.c for 2 days)

administration showed significant reduction in enzyme levels.

However, GUG2 & GUG3 were found to be more potent than GUG1.

The GUG-1 reduced elevated enzyme levels when compared with

isopretenol treated group.

Pretreatment with GUG1, GUG2 & GUG3 prevented the

depletion of tissue GSH & reduced tissue LPO in isopretenol induced

cardiac injury in rats.

The results were summarized in table 6.2.4.1.1 & 6.2.4.1.2 &

graphically depicted in fig. 6.2.4.1.1, 6.2.4.1.2, 6.2.4.1.3, 6.2.4.1.4,

6.2.4.1.5, & 6.2.4.1.6.

262

Table 6.2.4.1.1

Effect of shodhana on cardioprotective activity of guggul on biochemical

parameters against isoproterenol induced myocardial infarction in albino rats

Treatment AST (U/L) ALT(U/L) LDH(U/L) CPK(IU/L)

Group-I-control 315±2.51# 92.83±3.9# 2297.5±54.69# 1001.5±26.19#

Group-II

Control-ISO ( 200mg/kg) 401.16±7.28 138±2.28 3250±84.77 1217.66±24.15

Group-III-GUG-1 + ISO

(250mg/kg, 200mg/kg) 338.6±4.4* 121.5±3.0ns

2458.16±

79.58*** 1071±28.19**

Group-IV-GUG-2 + ISO

(250mg/kg, 200 mg/kg) 316±6.22*** 113.6±4.79** 2409±125.2***

1023.66±

25.09***

Group-V-GUG-3 + ISO

(250mg/kg, 200 mg/kg) 319.16±2.84*** 116.5±7.73** 2321±82.04***

1011.5±

29.38***

Values are mean ± SEM for six rats # P<0.001 V/s control

ns = non significant, *P <0.05, **P<0.01, ***P<0.001 V/s ISO control group.

263

Fig. 6.2.4.1.1

Effect of shodhana on cardioprotective activity of guggul on AST parameter against isoproterenol induced myocardial infarction in

rats

Fig. 6.2.4.1.2

Effect of shodhana on cardioprotective activity of guggul on ALT

parameter against isoproterenol induced myocardial infarction in

rats

264

Fig. 6.2.4.1.3

Effect of shodhana on cardioprotective activity of guggul on LDH parameter against isoproterenol induced myocardial infarction in

rats

Fig. 6.2.4.1.4

Effect of shodhana on cardioprotective activity of guggul on CPK parameter against isoproterenol induced myocardial infarction in

rats

265

Table 6.2.4.1.2

Effect of shodhana on cardioprotective activity of guggul on tissue GSH & LPO in isoproterenol (ISO) induced

cardiotoxicity in rats

Treatment

GSH

%

increase

LPO

%

Protection

Group-I -Control 0.360±0.045 _ 0.110±0.045 _

Group-II-control-ISO

( 200mg/kg)

0.182±0.018

_

0.192±0.080

_

Group-III GUG-1

+ ISO (250mg/kg, 200mg/kg)

0.282±0.050***

54.94

0.128±0.02***

33.33

Group-IV GUG-2

+ ISO (250mg/kg,

200 mg/kg)

0.354±0.092***

94.50

0.120±0.050***

37.50

Group-V GUG-3 +

ISO (250mg/kg,

200 mg/kg)

0.346±0.010***

90.10

0.126±0.010***

34.37

Values are the mean S.E.M. of 6 rats/ treatment. Significance ***P<0.001 compared to ISO treatment group

266

Fig. 6.2.4.1.5

Effect of shodhana on cardioprotective activity of guggul on tissue GSH levels in isoproterenol induced cardio toxicity in rats

Fig. 6.2.4.1.6

Effect of shodhana on cardioprotective activity of guggul on

tissue LPO levels in isoproterenol induced cardio toxicity in rats

267

6.2.4.2. Effect of shodhana on anti-hyperlipidemic activity of

guggul in Triton WR-1339 induced hyperlipidemia in rats

Pretreatment with GUG1, GUG2 & GUG3 prevented the

elevation in lipid profile upon triton challenge in rats.

The antihyperlipidemic potency of GUG3 was significantly higher

than GUG2 & GUG1. It indicating that shodhana with thriphala

kashaya is more beneficial when it is intended to be used for treating

hyperlipidemia.


depicted in fig. 6.2.4.2.1 to 6.2.4.2.6

268

Table.6.2.4.2

Effect of Shodhana on anti-hyperlipidemic activity of guggul on lipid profile in tritox WR-1339 induced hyperlipidemia in rats

Group Cholesterol Triglycerides Phospholipids HDL LDL VLDL

Group-I Control 56.2±1.28 58.4±4.17 144.6±5.6 27.6±2.06 18.32±2.34 10.28± 0.44

Group-II Triton 400mg/kg

161±19.53ª 147.6±14.9ª 197.9±12.5ª 17.9±0.74ª 119± 18.56ª 24.06 ±1.15ª

Group-III

Triton+GUG-1,

400mg/kg, 250mg/kg

77.6±6.08*** 72.4±8.75*** 156.4±8.5*** 29.2±1.65*** 29.68± 3.00*** 18.72± 2.13*

Group-IV

Triton+GUG-2, 400mg/kg,

250mg/kg

57.4±4.85*** 61.8±6.12*** 151.8±6.5*** 28.4±0.92*** 16.8 ±4.46*** 12.2 ±1.53***

Group-V

Triton+GUG-3, 400mg/kg,

250mg/kg

44.8±1.46*** 60.8±4.45*** 147.7±7.4*** 27.4±1.20*** 6.2± 1.15*** 11.2±1.46***

Group-VI Triton+fenofibrate

400mg/kg,

65mg/kg

56.3±1.38*** 70.6±3.98*** 159.5±5.8*** 28.3±0.58*** 18.7± 1.11*** 9.3± 0.30***

Values are the mean S.E.M. of 6 rats/treatment, significance ***P<0.001 compared to triton treated group

269

Fig 6.2.4.2.1

Effect of shodhana on anti-hyperlipidemic activity of guggul on

cholesterol in Triton WR-1339 induced hyperlipidemia in rats

Fig 6.2.4.2.2


triglycerides in Triton WR-1339 induced hyperlipidemia in rats

270

Fig 6.2.4.2.3

Effect of shodhana on anti-hyperlipidemic acivity of guggul on

phospholipids in Triton WR-1339 induced hyperlipidemia in rats

Fig 6.2.4.2.4

Effect of shodhana on anti-hyperlipidemic activity of guggul on HDL in Triton WR-1339 induced hyperlipidemia in rats

271

Fig 6.2.4.2.5

Effect of shodhana on anti-hyperlipidemic activity of guggul on LDL in Triton WR-1339 induced hyperlipidemia in rats

Fig 6.2.4.2.6


VLDL in Triton WR-1339 induced hyperlipidemia in rats

272

6.2.4.3. Effect of shodhana on anti-inflammatory activity of

guggul in carrageenan induced paw edema in rats

The shodhana (detoxified) processed guggul GUG2 and GUG3

showed higher anti-inflammatory effect when compared with

unprocessed guggul GUG1. Unprocessed guggul GUG1 did not

showed significant anti-inflammation activity. GUG2 and GUG3 did

not showed anti-inflammatory activity at 1st h but showed significant

anti-inflammatory activity at 2nd, 4th and 6th h after carrageenan

challenge. Diclofenac sodium (25 mg/kg) was used as standard in the

present study. The shodhana processed samples showed significant

anti-inflammatory activity which is comparable to that of standard

drug. GUG3 showed maximum anti-inflammatory activity (60.47%

inhibition) at 6th h.

The results were summarized in table 6.2.4.3 and fig. 6.2.4.3.

273

Table 6.2.4.3

Effect of shodhana on anti-inflammatory activity of guggul in carrageenan induced paw edema in rats

Treatment

Mean paw-size in mm

% Inhibition

Dose

(mg/kg)

1h 2h 4h 6h 1h 2h 4h 6h

Control - 0.52±0.056 0.81±0.09 0.83±0.077 0.71±0.05 - - - -

Diclofenac Sodium

25 0.47±0.06 ns 0.40±0.083** 0.35±0.090** 0.31±0.064** 5.0 50.98 57.98 55.86

GUG 1 250 0.51±0.04ns 0.66±0.04ns 0.65±0.039ns 0.70±0.04ns 1.9 18.38 21.96 1.1

GUG 2 250 0.42±0.04 ns 0.54±0.04** 0.48±0.04** 0.33±0.02** 19.2 37.7 42.37 53.5

GUG 3 250 0.48±0.07 ns 0.50±0.05** 0.48±0.04** 0.28±0.04** 3.8 33.7 42.37 60.47


ns = non significant, *P <0.05, **P<0.01 V/s the control

274

Fig. 6.2.4.3

Effect of shodhana on anti-inflammatory activity of guggul in

carrageenan induced paw edema in rats

Anti-inflammatory activity-1 h of the treatment

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Control Diclofenac GUG-1 GUG-2 GUG-3

Treatment

Mean

paw

siz

e (

mm

)

ns ns

ns

ns

Anti-inflammatory activity -2 h of the treatment

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1


Treatment

Mean

paw

siz

e i

n m

m

**** **

ns

275


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1


Treatment

Mean

paw

siz

e s

ize i

n m

m

**

ns

** **

Anti-inflammatory activity- 6 h of the treatment

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8


Treatment

Mean

paw

siz

e i

n m

m

**

ns

** **

276

6.2.4.4. Effect of shodhana on anti-ulcer activity of guggul

against ethanol induced gastric ulcers in rats

Pretreatment with GUG1, GUG2 and GUG3 significantly reduced

the gastric mucosal damage due to ethanol challenge. Both the

detoxified samples produced higher antiulcer effect than GUG1.

Further pretreatment with GUG1, GUG2 & GUG3 also reduced the

lipid peroxidation & prevented the depletion of tissue GSH. However,

GUG3, (i.e. detoxified with thriphala kashaya) was found to be the

most potent one. The antiulcer potency of GUG3 was comparable with

that of lansaprazole. Therefore it may be inferred that shodhana of

guggul with thriphala kashaya is more beneficial, when it is intended

to be used for healing ulcers.


depicted in fig. 6.2.4.4.1, 6.2.4.4.2 & 6.2.4.4.3

277

Table 6.2.4.4

Effect of shodhana on anti-ulcer activity of guggul against ethanol induced gastric ulcers in rats

Treatment Ulcer index %

Protection GSH

%

protection LPO

%

protection

Control

(99.5%Ethanol) 7.16 0.5

__

0.265 0.01

__ 0.250.007

__

Lansoprazole

(8mg/kg) 0.4160.25*** 94.18 0.8210.022*** 67.72 0.09 0.002*** 64.0

GUG1 (250mg/kg)

3.00.33*** 58.10 0.4480.01*** 40.84 0.24 0.013ns 04.0

GUG2

(250mg/kg) 1.330.38*** 81.42 0.55±0.22*** 51.81 0.165±0.006*** 34.0

GUG3

(250mg/kg) 0.083±0.083*** 98.84 0.801±0.03*** 66.9 0.0920.027*** 63.2

All values are mean ± SEM for six rats, ***P <0.001 V/s control

278

Fig. 6.2.4.4.1

Effect of shodhana on anti-ulcer activity of guggul against ethanol

induced ulcer index in rats

0

1

2

3

4

5

6

7

8

9

Control Lansaprazole GUG-1 GUG-2 GUG-3

Treatment

Ulc

er

ind

ex

***

***

***

***

Fig. 6.2.4.4.2

Effect of shodhana on anti-ulcer activity of guggul on tissue GSH


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Control Lansaprazole GUG-1 GUG-2 GUG-3

Treatement

GS

H

***

******

ns

279

Fig. 6.2.4.4.3

Effect of shodhana on anti-ulcer activity of guggul on tissue LPO against ethanol induced gastric ulcers in rats

0

0.05

0.1

0.15

0.2

0.25

0.3

Control Lansoprazole GUG-1 GUG-2 GUG-3

Treatment

LP

O

***

***

***

ns

280

6.3 Datura metel var.fastuosa

6.3.1 Pharmacognostic study

6.3.1.1. Effect of shodhana on physico-chemical parameters of datura seeds

It was observed from the loss on drying method that there was

increase in moisture content after shodhana process of datura seeds

DM2 and DM3 when compared with unprocessed seeds DM1. There

was increase in alcohol soluble extractive values after shodhana

process of the Datura seeds DM2 and DM3 than DM1. Water soluble

extractive value was increased in DM2 than DM1 except DM3.

Petroleum ether extractive values were decreased after shodhana

process in datura seeds. Total ash content was increased after

shodhana process. Acid insoluble ash content was increased after

detoxification. The water soluble ash content was also increased after

detoxification when compared with unprocessed datura seeds.

The average of four repetitions was compiled in the result

table 6.3.1.1

281

Table 6.3.1.1

Effect of shodhana on physico-chemical parameters of datura seeds

S.no.

Method (%G w/w)

DM1

DM2

DM3

1.

Loss on drying

3.12±0.02

7.15±0.08

4.2±0.34

2.

Watersoluble extractive

13±0.060

18.2±0.14

11.4±0.15

3.

Ethanol(70%)extractive

2.42±0.09

8.5±0.10

3.05±0.05

4. Petroleum ether (60-

80ºC) extractive 15.96±0.15 12.09±0.10 14.8±0.51

5. Total ash 3.02±0.05 4.51±0.08 5.54±0.07

6.

Acid insoluble ash

0.91±0.02

2.12±0.023

3.15±0.061

7.

Water soluble ash

1.35±0.08

2.1±0.056

1.87±0.06

282


6.3.2.1 Effect of shodhana on phytochemicals of datura seeds

It was observed from the preliminary phytochemical screening of

70% ethanol extracts of Datura metel var. fastuosa seeds (DM1) that

the plant contains tropane alkaloids, steroids, carbohydrates and

proteins. Whereas cow urine treated seeds (DM2) contains lesser

amounts of tropane alkaloids along with steroids, carbohydrates and

proteins. Cow urine and cow milk swedana processed (DM3) contains

lesser amounts of tropane alkaloids along with steroids, proteins and

carbohydrates.


Table 6.3.2.1

Effect of shodhana on phytochemicals of datura seeds

Phytochemicals DM1 DM2 DM3

Steroids + + +

Carbohydrates + + +

Alkaloids ++++ ++ ++

Proteins + + +++

283

6.3.2.2 Phytochemical analysis of cow urine and cow milk after

datura seeds shodhana

Preliminary phytochemical analysis showed presence of tropane

alkaloids, steroids and carbohydrates, proteins showed in cow urine

and cow milk after shodhana of datura seeds.


Table 6.3.2.2

Phytochemical analysis of cow urine and cow milk after datura

seeds shodhana

Phytochemicals Cow urine Cow milk

Steroids + +

Carbohydrates + +

Alkaloids ++ +

Proteins + +

284

6.3.2.3 TLC analysis

A single spot was observed on TLC plate at Rf value 0.65, 0.66

& 0.67 in DM1, DM2 & DM3 respectively and this sample was

identified as atropine.


Table 6.3.2.3

Identification of atropine in datura seeds by TLC

Sl.No. Samples Atropine (Rf)

1. Atropine 0.65

2. DM1 0.65

3. DM2 0.66

4. DM3 0.67

285

6.3.2.4. Effect of shodhan on total alkaloid content of datura

seeds

Total alkaloidal content before shodhana i.e. in DM1 was

0.210±0.81 G% w/w. The alkaloidal content after shodhana in DM2 &

DM3 was 0.190± 0.96 G% w/w and 0.172±0.54 G% w/w respectively.

The results clearly indicate that shodhana treatment reduces

the total alkaloidal content of datura seeds.


Table 6.3.2.4

Effect of shodhana on total alkaloid content of datura seeds

S.No. Sample Total alkaloid

content (G % w/w)

1. DM1 0.210±0.81

2. DM2 0.190±0.96

3. DM3 0.172±0.54

286

6.3.2.5. Effect of shodhana on phytochemicals of datura seeds -

LC-MS analysis

LC-MS analysis of all the three products DM1, DM2 and DM3

showed several molecular ion peaks. Phytoconstituents present in

them were identified on the basis of molecular ion peaks.

The constituents present in all the three products are summerised

in table no. 6.3.2.5 fig. no. 6.3.2.5.1 to 6.3.2.5.6

Further it is observed that the intensity of molecular ion peak of

hyoscyamine in DM2 and DM3 was higher than DM1. Therefore it is

inferred concentration of hyoscyamine is increased upon shodhana

treatment. It was also observed that withameteline B and calystegine

were absent in DM3. But their concentration was reduced in DM2 as

indicated by the reduction in the intensity of molecular ion peak.

287

Table no. 6.3.2.5

Effect of shodhana on phytochemicals of datura seeds - LC-MS analysis

DM-1 DM-2 DM-3

M+ M- M+ M- M+ M-

Hyoscyamine

m/z 290

Linoleic acid

m/z 279

Hyoscyamine

m/z 290

Linoleic acid

m/z 279

Hyoscyamine

m/z 290

Linoleic acid

m/z 279

Megastigmine

m/z 245

Oleic acid

m/z 281

Megastigmine

m/z 245

Oleic acid

m/z 281

Megastigmine

m/z 245

Oleic acid

m/z 281

Datura metelin A m/z 602

Meteloidine m/z 255

Datura meteline A m/z 602

Meteloidine m/z 255

Datura meteline A m/z 602

-

Datura metelin E

m/z 553 _

Daturanolone

Calystegin C1 Withameteline -

Withameteline

m/z 408 - - - - -

Withameteline B m/z 437

- - - - -

Calystegine C1

m/z 193 - - - - -

288

Fig 6.3.2.5.1

LC-MS analysis of unprocessed seeds of datura DM-1: M+ ion

peaks

Fig 6.3.2.5.2

LC-MS analysis of unprocessed seeds of datura DM-1: M- ion

peaks

Fig 6.3.2.5.3

LC-MS analysis of shodhana processed seeds of datura DM-2: M+

ion peaks

289

Fig 6.3.2.5.4

LC-MS analysis of shodhana processed seeds of datura DM-2: M-

ion peaks

Fig 6.3.2.5.5

LC-MS analysis of shodhana processed seeds of datura DM-3: M+

ion peaks

Fig 6.3.2.5.6

LC-MS analysis of shodhana processed seeds of datura DM-3: M-

ion peaks

290

6.3.3. Acute toxicity study

There was no mortality, after treatment with 2000 mg/kg of all the

three products DM1, DM2 & DM3 indicating that shodhana did not

influence the acute toxicity of datura. Hence, 2500 mg/kg can be

considered as the cut off dose. Therefore 1/10th dose (250 mg/kg) was

selected for further pharmacological studies.

6.3.4 Pharmacological studies

6.3.4.1. Effect of shodhana on anti-inflammatory activity of

Datura seeds against carrageenan induced hind paw edema in rats

The % reduction in oedema after pretreatment with DM1, DM2 &

DM3 was 27.9%, 39.0% & 50.0% respectively at 4th h after

carrageenan challenge. The results are indicating that DM3 possess

significantly higher anti-inflammatory potency than DM1 & DM3. The

anti-inflammatory potency of DM3 was comparable with that of

standard diclofenac sodium (25 mg/kg).


depicted in fig. 6.3.4.1

291

Table 6.3.4.1

Effect of shodhana on anti-inflammatory activity of Datura seeds against carrageenan induced hind paw edema

in rats

Treatment

Mean paw-size in mm

% Inhibition

Dose

(mg/kg)

1h 2h 4h 6h 1h 2h 4h 6h

Control - 0.52±0.056 0.81±0.09 0.83±0.077 0.71±0.05 - - - -

Diclofenac

Sodium

25 0.47±0.061ns 0.40±0.083** 0.35±0.090** 0.31±0.064** 5.0 50.98 57.98 55.86

DM 1 250 0.57±0.04ns 0.80±0.15ns 0.6±0.005* 0.41±0.04** -9.6 1.9 27.9 41.89

DM 2 250 0.51±0.04ns 0.72±0.07ns 0.5±0.08** 0.21±0.05** 1.9 9.1 39.0 69.83

DM 3 250 0.50±0.07ns 0.58±0.117* 0.48±0.08** 0.3±0.04** 3.8 28.5 50.0 58.1

All values represent mean ± SEM for six rats

ns = non significant, *P <0.05, **P<0.01 V/s control group.

292

Fig. 6.3.4.1

Effect of shodhana on anti-inflammatory activity of Datura seeds against carrageenan induced hind paw edema in rats


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Control Diclofenac DM-1 DM-2 DM-3

Treatment

Mean

paw

siz

e i

n m

m

ns

nsns

ns


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1


Treatment

Mean

paw

siz

e i

n m

m

**

ns

**

ns

293


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1


Treatment

Mean

paw

siz

e i

n m

m

**

* ****


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8


Treatment

Mean

paw

siz

e i

n m

m

****

**

**

294

6.3.4.2. Effect of shodhana on anti-ulcer activity of Datura seeds


Pretreatment with DM1, DM2 and DM3 significantly reduced the

gastric mucosal damage due to ethanol challenge. Both the detoxified

samples produced higher antiulcer effect than DM1. Further

pretreatment with DM1, DM2 & DM3 also reduced the lipid

peroxidation & prevented the depletion of tissue GSH. However, DM3

was found to be most potent. The antiulcer potency of DM3 was

comparable with that of lansaprazole.Therefore it may be inferred that

shodhana of datura seeds with cow urine then swedana with cow milk

is more beneficial when it is intended to be used for healing ulcers.


depicted in fig. 6.3.4.2.1 and 6.3.4.2.2

295

Table 6.3.4.2

Effect of shodhana on anti-ulcer activity of Datura seeds against ethanol induced gastric ulcers in rats

Treatment Ulcer index

% Protection

GSH % increase

LPO

% Protection

Control (99.5%Ethanol) 7.16 0.54

___ 0.265 0.01

__ 0.259 0.007

__

Lansoprazole(8mg/kg) 0.416 0.23*** 94.18 0.8210.022*** 67.7 0.090 0.002*** 65.25

DM 1 (250mg/kg) 2.58 0.56*** 63.96 0.508 0.027*** 47.8 0.215 0.005** 16.98

DM 2 (250mg/kg) 2.25 0.33*** 68.57 0.63 0.02*** 57.9 0.172 0.009*** 33.59

DM 3 (250mg/kg) 0.083 0.083*** 98.84 0.7850.016*** 66.2 0.087 0.019*** 66.40

All values represent mean ± SEM for six rats, ns = non significant, *P <0.05, **P<0.01***P<0.001, V/s control group

296

Fig. 6.3.4.2.1

Effect of shodhana on ulcer index of Datura seeds against ethanol induced gastric ulcers in rats

Fig. 6.3.4.2.2

Effect of shodhana on tissue GSH of Datura seeds against ethanol

induced gastric ulcers in rats

297

Fig. 6.3.4.2.3

Effect of shodhana on tissue LPO of Datura seeds against ethanol induced gastric ulcers in rats

The overall research work was explained by the following

conclusion flow charts.

298

Strychnos nux-vomica

Shodhana processed seeds Un-processed seeds

Pharmacognostic study Acute toxicity Pharmacological screening

Physicochemical analysis

Phytochemical analysis Organprotective activity in-vitro anti-oxidant activity Analgesic activity

Total alkaloid analysis Shodhana processed Gastric ulcer screening Tissue GSH & LPO estimation Acetic acid induced writhing model

TLC analysis seeds less toxic Ethanol induced ulcer Ethanol & pylorus ligation ulcer Anti-inflammatory activity

Spectroscopical analysis Pylorus ligation model Hepatotoxicity CCl4 induced Carrageenan induced inflammation

HPTLC analysis Hepatoprotective activity Nephrotoxicity-cisplatin induced

LC-MS analysis CCl4 induced livertoxicity

Nephroprotective activity possess in-vitro possess significant analgesic

cisplatin induced nephrotoxicity anti-oxidant activity & anti-inflammatory activities

Reduced total alkaloids, strychnine

& brucine, elimination of loganic acid Possess significant organ protective activity

Detoxified seeds of nux-vomica showed less toxic & enhansed therapeutic efficacy

Fig.no.6.4.1 Schematic flow chart of seeds of nux-vomica before & after shodhana with conclusions

299

Commiphora mukul (guggul)

Shodhana processed guggul unprocessed guggul

Pharmacognostic study Acute toxicity & dose fixation Pharmacological screening

Physico-chemial analysis non-toxic at 2000mg/kg Anti-hyperlipidemic study

Phytochemical analysis Cut off dose 2500 mg/kg (triton induced hyperlipidemic rats)

Thinlayer chromatographic analysis 1/10th i.e. 250 mg/kg dose selected Anti-inflammatory activity

High performance liquid chromatographic (carrageenan induced inflammation in rats)

(HPLC) analysis Anti-ulcer activity study

LC-MS analysis (Ethanol induced gastric ulcers in rats)

Cardioprotective activity

not possessing toxicity before shodhana (isoproterenol induced cadiotoxity)

Tissue anti-oxidant activity study

Heart tissue GSH, LPO estimation

Stomach tissue GSH & LPO estimation

Guggulsterone Z enhanced in GUG2, possess significant pharmacological actions

Galloylglucose included in GUG3

Detoxified products of guggul showed more therapeutic efficacy

Fig. no. 6.4.2 Schematic flow chart of guggul before & after shodhana with conclusions

300

Datura metel var. fastuosa seeds

Shodhana processed seeds Un-processed seeds

Pharmacognostic study Acute toxicity & dose fixation Pharmacological screening

Physico-chemical analysis Non-toxic at 2000mg/kg Anti-inflammatory study

Phytochemical analysis

Thin layer chromatography analysis Cut off dose 2500 mg/kg Carrageenan induced rat paw edema

Total alkaloids analysis 1/10th i.e. 250 mg/kg dose selected Anti-ulcer study

LC-MS analysis Ethanol induced gastric ulcers

Decreased total alkaloids & not possessing toxicity before shodhana Tissue anti-oxidant activity

Increased hyoscyamine Stomach tissue GSH & LPO

Possess significant pharmacological actions

Fig no. 6.4.3 Schematic flow chart of seeds of Datura metel var.fastuosa before and after shodhana processes with conclusions

All the detoxified products of seeds of Datura metel var.fastuosa showed more therapeutic efficacy

Documents

6. RESULTS 6.1. Strychnos nux-vomica - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/4507/16/16_chapter 6.pdf · Strychnos nux-vomica 6.1.1 Pharmacognostic analysis 6.1.1.1