6. results 6.1. strychnos nux-vomica -...
TRANSCRIPT
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.
The results were summarized in table 6.1.2.3
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
LC-MS analysis of shodhana processed seeds of nux-vomica
(PNV1) M - peaks
Fig. 6.1.2.7.5
LC-MS analysis of shodhana processed seeds of nux-vomica
(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
LC-MS analysis of shodhana processed seeds of nux-vomica
(PNV3) M + peaks
Fig. 6.1.2.7.8
LC-MS analysis of shodhana processed seeds of nux-vomica
(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
LC-MS analysis of shodhana processed seeds of nux-vomica
(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
Anti-inflammatory activity- 1h of the treatment
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Control Diclofenac
sodium
UNV PNV1 PNV2 PNV3 PNV4 PNV5
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
UNV PNV1 PNV2 PNV3 PNV4 PNV5
Treatment
Mean
paw
siz
e (
mm
)
ns
**
* ****
**
**
Anti-inflammatory activity- 4h of the treatment
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
Control Diclofenac
sodium
UNV PNV1 PNV2 PNV3 PNV4 PNV5
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
Effect of shodhana on anti-ulcer activity of nux-vomica seeds on
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
Effect of shodhana on anti-ulcer activity of nux-vomica seeds on
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
Effect of shodhana on anti-ulcer activity of nux-vomica seeds on
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
Effect of shodhana on anti-ulcer activity of nux-vomica seeds on
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
Effect of shodhana on hepatoprotective activity of nux-vomica
seeds on total bilirubin against CCl4 induced hepatic damage in
rats
233
Fig. 6.1.4.4.5
Effect of shodhana on hepatoprotective activity of nux-vomica
seeds on direct bilirubin against CCl4 induced hepatic damage in
rats
Fig. 6.1.4.4.6
Effect of shodhana on hepatoprotective activity of nux-vomica
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
Effect of shodhana on hepatoprotective activity of nux-vomica
seeds in liver tissue GSH levels in CCl4 induced hepatic damage in
rats
Fig. 6.1.4.4.8
Effect of shodhana on hepatoprotective activity of nux-vomica
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
Effect of shodhana on nephroprotective activity of nux-vomica
seeds on % change in body weight against cisplatin induced
nephrotoxicity in rats
Fig. 6.1.4.5.4
Effect of shodhana on nephroprotective activity of nux-vomica
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
Values are mean ± SEM for six rats
ns = non significant *P<0.05, **P <0.01 and ***P <0.001 V/s control group.
245
Fig. 6.1.4.5.5
Effect of shodhana on nephroprotective activity of nux-vomica
seeds in kidney tissue GSH levels against cisplatin induced
nephrotoxicity in rats
Fig. 6.1.4.5.6
Effect of shodhana on nephroprotective activity of nux-vomica
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 Phytochemical analysis
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.
The results were summarized in table 6.2.2.1
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.
The results were summarized in table 6.2.2.3.
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
HPLC chromatogram of GUG-2
257
Fig. 6.2.2.5.4
HPLC chromatogram of GUG-3
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.
The results were summarized in table 6.2.4.2 and graphically
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
Effect of shodhana on anti-hyperlipidemic activity of guggul on
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
Effect of shodhana on anti-hyperlipidemic activity of guggul on
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
Values are mean ± SEM for six rats
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
Control Diclofenac GUG-1 GUG-2 GUG-3
Treatment
Mean
paw
siz
e i
n m
m
**** **
ns
275
Anti-inflammatory activity- 4h of the treatment
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Control Diclofenac GUG-1 GUG-2 GUG-3
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
Control Diclofenac GUG-1 GUG-2 GUG-3
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.
The results were summarized in table 6.2.4.4 and graphically
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
against ethanol induced gastric ulcers in rats
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 Phytochemical analysis
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.
The results were summarized in table 6.3.2.1
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.
The results were summarized in table 6.3.2.2
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.
The results were summarized in table 6.3.2.3.
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.
The results were summarized in table 6.3.2.4
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).
The results were summarized in table 6.3.4.1 and graphically
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
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 DM-1 DM-2 DM-3
Treatment
Mean
paw
siz
e i
n m
m
ns
nsns
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
Control Diclofenac DM-1 DM-2 DM-3
Treatment
Mean
paw
siz
e i
n m
m
**
ns
**
ns
293
Anti-inflammatory activity- 4 h of the treatment
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Control Diclofenac DM-1 DM-2 DM-3
Treatment
Mean
paw
siz
e i
n m
m
**
* ****
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
Control Diclofenac DM-1 DM-2 DM-3
Treatment
Mean
paw
siz
e i
n m
m
****
**
**
294
6.3.4.2. Effect of shodhana on anti-ulcer activity of Datura seeds
against ethanol induced gastric ulcers in rats
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.
The results were summarized in table 6.3.4.2 and graphically
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