results and discussion of vitex negundo...
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CHAPTER-6 RESULTS AND DISCUSSION
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RESULTS AND DISCUSSION OF VITEX NEGUNDO LINN.
6.2.1 SOLUBILITY OF DIFFERENT EXTRACTS:
The solubility of each extracts of Vitex negundo Linn. is reported in the following table.
Table no. 54: Data showing the solubility of different extracts of Vitex negundo Linn.
Sr.
no. Name of the extracts
Solvents used for solubility for
screening of anti-tubercular activity
1 Aqueous extract Distilled water
2 Ethanol extract Dimethyl formamide
3 Petroleum ether extract Dimethyl formamide
4 Benzene extract Dimethyl formamide
5 Chloroform extract Dimethyl formamide
6 Acetone extract Distilled water
7 Successive ethanol extract Dimethyl formamide
8 Methanol extract Dimethyl sulphoxide
6.2.2 PERCENTAGE YIELD OF DIFFERENT EXTRACTS:
Extractions of leaves of Vitex negundo Linn. were performed. The aqueous extract
was obtained by maceration method. Ethanol extract is obtained by solvent extraction
method while petroleum ether, benzene, chloroform, acetone, ethanol, and methanol
extracts were obtained by successive solvent extraction method. The colour, consistency
and % yield of all the extracts is reported in the following table.
Table no. 55: Data showing colour, consistency and % yield of the extracts of Vitex
negundo Linn.
Sr.
no.
Name of the extracts
of Vitex negundo
Linn.
Colour and consistency of extract % yield
obtained for
the extracts
1 Aqueous extract Reddish black and semisolid 16.20
2 Ethanol extract Blackish and semisolid later crystalline 13.40
3 Petroleum ether
extract
Blackish and crystalline 5.74
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4 Benzene extract Blackish and slight crystalline 4.58
5 Chloroform extract Blackish and semisolid 4.04
6 Acetone extract Blackish and semisolid 3.34
7 Successive ethanol
extract
Blackish and crystalline 2.91
8 Methanol extract Brownish and semisolid 1.27
The maximum percentage of yield was obtained by maceration for aqueous
extract (16.20), while ethanol extract was having (13.40) obtained by soxhlet extraction
method. The lower percentage of yield obtained for petroleum ether (5.74), benzene
(4.58), chloroform (4.04), acetone (3.34), successive ethanol (2.91) and methanol extract
(1.27) which were obtained by successive solvent extraction method. The order of
percentage yield of different extracts were aqueous > ethanol > pet. ether > benzene >
chloroform >acetone >successive ethanol > methanol.
6.2.3 PHYTOCHEMICAL INVESTIGATION OF EXTRACTS:
All the extracts were subjected to preliminary organic qualitative analysis to identify
nature of compounds present by chemical tests(1).
Table no. 56: Data summarizes the phytochemicals of Vitex negundo Linn. leaves
extracts:
SR.
NO.
CHEMICAL
TEST AQU ETN
PET.
ETHER
CHL
ORO BEN ACE SE MET
1)
TEST FOR
CARBOHYDRAT
ES
a] Molisch test + + -- -- + + + +
1a Test for reducing
sugars
a] Fehling’s test + + -- -- + + + +
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b] Benedict’s test + + -- -- + + + +
1b Test for
monosaccharides
a] Barfoed’s test + -- -- -- -- -- -- --
1c Test for Pentose
Sugars
a] Bial’s orcinol test -- -- -- -- -- -- -- --
b] Aniline acetate -- -- -- -- -- -- -- --
1d Test for hexose
sugars
a] Selwinoff’s test -- -- -- -- -- -- -- --
b] Tollen’s
phlorglucinol test -- -- -- -- -- -- -- --
c] Cobalt chloride test -- -- -- -- -- -- -- --
1e Test for non-
reducing sugar
a] Fehling’s and
Benedict’s test -- -- -- -- -- + -- --
b] Hydrolyse test
solution -- + -- -- -- + -- +
1f Test for starch
a] Iodine test -- -- -- -- -- -- -- --
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b] Tannic acid test -- -- -- -- -- -- -- --
1g Test for gums -- + + -- -- -- -- +
1h Test for mucilage -- + + -- -- -- -- +
2) TEST FOR
PROTEINS
a] Biuret test + -- -- -- -- -- -- --
b] Million’s test + -- -- -- -- -- -- --
c] Xanthoprotein test + -- -- -- -- -- -- --
d] Test for protein
containing sulphur + -- -- -- -- -- -- --
e] Precipitation test
with
a] Absolute alcohol + -- -- -- -- -- -- --
b] 5% HgCl2 + -- -- -- -- -- -- --
c] 5% CuSo4 + -- -- -- -- -- -- --
d]
5% lead acetate + -- -- -- -- -- -- --
e] 5% ammonium
sulphate + -- -- -- -- -- -- --
3) TEST FOR
AMINO ACIDS
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a] Ninhydrin test + -- -- -- -- -- -- --
4) TEST FOR FATS
AND OILS
a] Section test -- + + + -- + -- +
c] Solubility test -- + + + -- + -- +
d] Filter paper test -- + + + -- + -- +
5) TEST FOR
STEROIDS
a] Salkowski reaction -- + -- -- + -- + +
b] Libermann-
Burchard reaction -- + -- -- + -- + +
c] Libermann reaction -- + -- -- + -- + +
6) TEST FOR
VOLATILE OILS
a] Odour test + + -- -- -- -- -- --
b] Filter paper test + + -- -- -- -- -- --
c] Solubility test + + -- -- -- -- -- --
7) TEST FOR
GLYCOSIDES
a) Test for saponin
glycosides + + -- -- -- -- -- +
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b] Foam test + + -- -- -- -- -- +
c] Hemolytic test + + -- -- -- -- -- +
8) TEST FOR
FLAVONOIDS
a] Shinoda test + + + + -- + + +
b] Lead acetate
solution + + + + -- + + +
c] Sodium hydroxide
test + + + + -- + + +
9) TEST FOR
ALKALOIDS
a] Dragendorff’s test + + -- -- -- + -- +
b] Mayer’s test + + -- -- -- + -- +
c] Hager’s test + + -- -- -- + -- +
d] Wagner’s test + + -- -- -- + -- +
e] Murexide test + + -- -- -- + -- +
10)
TEST FOR
TANNINS &
PHENOLIC
COMPOUNDS
a] 5%Fecl3 + + + + -- + + +
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b] Lead acetate
solution + + + + -- + + +
c] Gelatin solution + + + + -- + + +
d] Bromine water + + + + -- + + +
e] Acetic acid
solution + + + + -- + + +
f] Potassium
dichromate + + + + -- + + +
g] Dil. iodine solution + + + + -- + + +
h] Dil. HNO3 + + + + -- + + +
i]
Dil. NH4OH &
Potassium
ferricyanide
solution
+ + + + -- + + +
j]
Dil. Potassium
permagnate
solution
+ + + + -- + + +
Abbreviations in table-----
AQU - aqueous extract ETN - ethanol extract
PET. ETHER - petroleum ether extract CHLORO - chloroform extract
BEN - benzene extract ACE - acetone extract
SE - successive ethanol extract MET - methanol extract
Preliminary phytochemical study was screened for the presence of
carbohydrates, proteins, glycosides, alkaloids, steroids, phenolic compounds, flavonoids,
tannins, volatile oils, gums, mucilage, sapponins, fats and oils. Phytochemical screening
of each different extracts of plant is summarized in the following table.
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Table no. 57: Data showing phytochemical investigation of different extracts of Vitex
negundo Linn. leaves.
Sr.
no.
Name of
the extracts
Phytochemicals investigated.
1 Aqueous Carbohydrates, proteins, alkaloids, phenolic compounds, tannins,
volatile oils., saponins, irridoid glycosides and flavonoids.
2 Ethanol Carbohydrates, fats, oils, polyphenolic compounds, tannins,
alkaloids, flavonoids, irridoid glycosides, steroids, volatile oils,
gums, mucilage and saponins.
3 Petroleum
ether
Steroids, gum, mucilage, fats and oils.
4 Chloroform Fats, oils, phenolic compounds, tannins and flavonoids.
5 Benzene Carbohydrates and steroids.
6 Acetone Carbohydrates, fats, oils, polyphenolic compounds, tannins,
alkaloids and flavonoids.
7 Successive
ethanol
Carbohydrates, polyphenolic compounds, tannins, steroids and
flavonoids
8 Methanol Carbohydrates, fats, oils, polyphenolic compounds, tannins,
alkaloids, flavonoids, glycosides, steroids, gums and mucilage.
Carbohydrate is present in all the extracts except pet. ether and chloroform extract
and protein is present in only aqueous extract. Aqueous, acetone, ethanol and methanol
extracts showed the presence of alkaloids while sapponins and vol. oils are present in
aqueous and ethanol extracts. Tannins and polyphenolic compounds are present in all
extracts except benzene and pet. ether extracts. Gums and mucilage are present in
methanol, pet. ether and ethanol extracts. All the extracts except aqueous and acetone
extract showed the presence of steroids. Aqueous, ethanol and methanol extracts showed
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the presence of glycosides. Maximum numbers of phytochemicals are present in the
aqueous, ethanol extracts followed by methanol extracts.
6.2.4 TOXICITY STUDIES OF VITEX NEGUNDO LINN. LEAVES:
Acute oral toxicity and LD50 determination of the plant extract was done
according to OECD guidelines 423 for deciding the doses of the plant extract.
Table no. 58: Tabulation of response data (i.e. animals showing signs of toxicity
including nature, severity, duration of effects and mortality) and dose level for each
animal.
Extract of Vitex
negundo Linn.
No. of
animals used
Limit dose
(mg/kg)
Sign of
toxicity
Duration of
effect
Mortality
Ethanolic extract Five rats
(female)
2000 No No No
LD50 Data:
Test type: Limit test
Limit dose: 2000 mg/kg
Assumed LD50 : None.
No mortality was observed at limit dose 2000 mg/kg. As per OECD guidelines
LD50 value of ethanolic extract of Vitex negundo Linn. is greater than 2000 mg/kg of
body weight.
Acute oral toxicity:
Table no. 59: Acute oral toxicity results for rats:
Extract of Vitex negundo Linn. Dose (mg/kg) Short term result
Ethanolic extract 2000 00000
(0 = survived)
Results: Body weight changes: No change in body weight.
Acute oral toxicity was carried out by up and down regulation method for
ethanolic extract of Vitex negundo Linn. It is found that no mortality is observed for
ethanolic extract at limit dose of 2000 mg/kg.
6.2.5 PROPORTION METHOD:
M. tuberculosis H37Rv, the reference strain sensitive to all the anti-tuberculosis
agents, was used as the control strain. The proportion methods using L-J medium were
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performed as per established standard procedures and calculations for resistance were
also made as recommended in standard literature(2)
.
The number of colony forming units growing on the medium containing the drug
was compared with the number on the control bottle. The proportion of resistant cells in
the total viable population of the original inoculum was then calculated and expressed as
a percentage. The proportion of growth above which the isolate was labelled as resistant
was taken as 1%, as per the recommendation(2)
.
Table no. 60: Effects of 100 µg/ml of extracts of Vitex negundo Linn. by proportion
method:
Sr.
no.
Name of test drug/ extracts Concentration
of test drug
extract (µg/ml)
Mean number of colonies of
Mycobacterium tuberculosis
(Mean ± SEM)
1 Control -- 73 ±1.528
2 Aqueous extract 100 66.33 ± 1.764
3 Ethanol extract 100 18 ± 1.732
4 Successive ethanol extract 100 24 ± 2.082
5 Acetone extract 100 63.33 ± 1.202
6 Chloroform extract 100 24.66 ±1.453
7 Benzene extract 100 47 ± 1.155
8 Petroleum ether extract 100 24 ± 1.732
9 Methanol extract 100 31.66 ± 1.453
10 Rifampicin 40 0**
11 Isoniazide 0.2 0**
Values are expressed as mean+ SEM. Statistical analysis was performed using Dunnet’s
multiple comparison tests. *p<0.05 and **p<0.01 was taken as the criterion of
significance when compared to control where n=3.
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Fig. no. 41: Effects of 100 µg/ml extracts of Vitex negundo Linn. by proportion
method:
Aqueous, acetone, methanol and benzene extracts did not showed any anti-
tubercular activity at 100 µg/ml. Mean number of colonies of M. tuberculosis observed
by proportion method showed 73 in control bottle. Aqueous, acetone, methanol and
benzene extracts showed 66, 63, 31 and 47 mean number of colonies respectively, since
resistance value is greater than 1 % as compared to control. Hence M. tuberculosis is
resistant to all the above extracts.
Successive ethanol, petroleum ether and chloroform extracts all showed mean
number of M. tuberculosis colonies 24 while ethanol extract showed 18, since resistance
value is greater than 1 % as compared to control. Therefore M. tuberculosis is resistant to
these all extracts, which indicates all extracts did not showed any anti-tubercular activity
at 100 µg/ml.
Therefore it is concluded that all the extracts did not showed any anti-tubercular
activity by proportion method at 100 µg/ml. No growth of colonies was observed for
rifampicin and isoniazide.
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Table no. 61: Effect of 150 µg/ml extracts of Vitex negundo Linn. by proportion
method.
Sr.
no.
Name of the test drug/
extracts
Concentration
of test drug/
extract (µg/ml)
Mean number of colonies of
Mycobacterium tuberculosis
(Mean ± SEM)
1 Control -- 73 ±1.528
2 Aqueous extract 150 54.66 ± 2.186
3 Ethanol extract 150 0**
4 Successive ethanol extract 150 0**
5 Acetone extract 150 47.33 ± 1.202
6 Chloroform extract 150 14 ± 1.155
7 Benzene extract 150 26.66 ± 1.453
8 Petroleum ether extract 150 11± 1.155
9 Methanol extract 150 1.66 ±0.333*
10 Rifampicin 40 0**
11 Isoniazide 0.2 0**
Values are expressed as mean+ SEM. Statistical analysis was performed using Dunnet’s
multiple comparison tests. *p<0.05 and **p<0.01 was taken as the criterion of
significance when compared to control where n=3.
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Fig. no. 42: Effects of 150 µg/ml extracts of Vitex negundo Linn. against M.
tuberculosis.
Aqueous, acetone, benzene and chloroform extracts did not showed anti-
tubercular activity at 150 µg/ml. Mean number of colonies of M. tuberculosis observed
was 73 by proportion method in control bottle. Aqueous, acetone and benzene extracts
showed 54, 47 and 26 mean number of colonies respectively; subsequently resistance
value is greater than 1 % as compared to control. Hence M. tuberculosis is resistant. to all
the above extracts.
one colony was observed for M. tuberculosis in methanol extract, subsequently
resistance value is greater than 1 % as compared to control. Hence M. tuberculosis is
resistant, which indicates the extract did not show any anti-tubercular activity at 150
µg/ml. But the colony count was very low as compared to control. Statistical analysis was
performed using Dunnet’s multiple comparison test for which probability values was
found to be (*p) <0.05. Therefore methanol extract possesses mild anti-tubercular
activity.
Therefore it is concluded that all the extracts except ethanol and successive
ethanol extract did not showed any anti-tubercular activity while methanol extract
possesses mild anti-tubercular activity at 150 µg/ml by proportion method. No growth of
M. tuberculosis colonies was observed in ethanol and successive ethanol extract at 150
µg/ml; hence the standard strain of M. tuberculosis is sensitive to ethanol and successive
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ethanol extract. Therefore it is concluded that extract possesses significant anti-tubercular
activity. No growth of colonies was observed for rifampicin and isoniazide.
Table no. 62: Effect of extracts of 200 µg/ml of Vitex negundo Linn. by proportion
method.
Sr.
no.
Name of the test drug/
extracts
Concentration
of test drug/
extracts (µg/ml)
Mean number of colonies of
Mycobacterium tuberculosis
(Mean ± SEM)
1 Control -- 73 ±1.528
2 Aqueous extract 200 36.66± 1.453
3 Ethanol extract 200 0**
4 Successive ethanol extract 200 0**
5 Acetone extract 200 29.33± 1.453
6 Chloroform extract 200 0**
7 Benzene extract 200 8 ± 0.5774
8 Petroleum ether extract 200 0**
9 Methanol extract 200 0**
10 Rifampicin 40 0**
11 Isoniazide 0.2 0**
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Fig. no. 43: Effects of 200 µg/ml extracts of Vitex negundo Linn. against M.
tuberculosis.
It was observed by proportion method that no. of colonies decreases as
concentration of extract increases. Results of anti-tubercular activity by proportion
method revealed that complete inhibition of growth of M. tuberculosis i.e. no growth of
colonies was observed at 150 µg/ml of ethanol, successive ethanol and at 200 µg/ml of
ethanol, successive ethanol, chloroform, pet. ether and only one colony observed on
methanol extract.
Therefore it is concluded that 150 & 200 µg/ml of ethanol and successive ethanol
extracts and 200 µg/ml of chloroform, pet. ether extract possesses most significant while
150 µg/ml of methanol extract possesses mild anti-tubercular activity. Rifampicin and
isoniazide at 40 µg/ml and 0.2 µg/ml respectively showed anti-tubercular activity.
Acetone, benzene and aqueous extracts of Vitex negundo Linn. did not showed any anti-
tubercular activity.
6.2.6: NITRATE REDUCTSE ASSAY:
In the present study, we have used NRA method as an alternative method for
evaluation of anti-tubercular activity, which is based on the ability of M. tuberculosis to
reduce nitrate to nitrite, the reduction detected by using specific reagents which produces
a colour change.
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All the extracts of Vitex negundo Linn. were tested against M. tuberculosis by the
nitrate reductase assay method to confirm the results of proportion method for anti-
tubercular activity. As 100 µg/ml conc. of all the extracts of Vitex negundo Linn. did not
showed any anti-tubercular activity by proportion method, therefore conc. of 150 and 200
µg/ml of different extracts of Vitex negundo Linn. were subjected for screening of anti-
tubercular activity by nitrate reductase assay method.
Table no. 63: Effect of 150 µg/ml extracts of Vitex negundo Linn. on M. tuberculosis
by nitrate reductase assay method:
Sr.
no.
Name of the test drug/
extracts
Concentration
of test
drug/extracts
(µg/ml)
Growth of Mycobacterium
tuberculosis indicated by
reddish/violet colour
I II III
1. Control without drug -- + + +
2. Rifampicin 40 -- -- --
3. Isoniazide 0.2 -- -- --
4. Aqueous extract 150 + + +
5. Ethanol extract 150 -- -- --
6. Petroleum ether extract 150 + + +
7. Benzene extract 150 + + +
8. Acetone extract 150 + + +
9. Chloroform extract 150 + + +
10. Successive ethanol extract 150 -- -- --
11. Methanol extract 150 -- -- --
-- indicates no change in colour means no growth of M. tuberculosis.
+ indicates development of reddish violet colour means growth of M. tuberculosis.
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In all control bottles reddish/violet colouration was observed on the surface of the
slants indicative of a positive NRA and growth of standard strain of M. tuberculosis on
addition of specific reagent. Reddish/violet colouration was also observed on the surface
of the slants indicative of a positive NRA and growth of standard strain of M. tuberculosis
on addition of specific reagent in aqueous, acetone and benzene extract containing bottles
of Vitex negundo Linn.
No reddish/violet coloration was observed on the surface of the slants for
rifampicin and INH and absence of colouration interpreted as negative NRA. No
reddish/violet coloration was observed on the surface of the slants in ethanol, successive
ethanol and methanol extract containing bottles of Vitex negundo Linn. and absence of
colouration interpreted as negative NRA. Therefore it is concluded that aqueous, pet.
ether, acetone, chloroform and benzene extracts at 150µg/ml did not showed any anti-
tubercular activity while ethanol, successive ethanol and methanol extracts possesses anti-
tubercular activity by nitrate reductase assay method. The results were obtained after
seven days of inoculation in all the control and extract containing bottles.
Table no. 64: Effect of 200 µg/ml extracts of Vitex negundo Linn. on M. tuberculosis
by nitrate reductase assay method:
Sr.
no.
Name of the test drug/
extracts
Concentration
of test drug/
extracts
(µg/ml)
Growth of Mycobacterium
tuberculosis indicated by
reddish/violet colour
I II III
1 Control without drug -- + + +
2 Rifampicin 40 -- -- --
3 Isoniazide 0.2 -- -- --
4 Aqueous extract 200 + + +
5 Ethanol extract 200 -- -- --
6 Petroleum ether extract 200 -- -- --
7 Benzene extract 200 + + +
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8 Acetone extract 200 + + +
9 Chloroform extract 200 -- -- --
10 Successive ethanol extract 200 -- -- --
11 Methanol extract 200 -- -- --
-- indicates no change in colour means no growth of M. tuberculosis.
+ indicates development of reddish violet colour means growth of M. tuberculosis.
A] Control bottle B] Extract sample bottle
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C] Isoniazide bottle D] Rifampicin bottle
Fig. no. 44: Observations of growth of M. tuberculosis indicated by colour in
different bottles by NRA method. (Control bottles A showed pink colour indicating growth of M.
tuberculosis. Others B, C and D all showed blue colour (remains same) indicating no growth of M.
tuberculosis in extract sample, isoniazide and rifampicin bottles respectively).
In all control bottles reddish/violet coloration was observed on the surface of the
slants indicative of a positive NRA and growth of standard strain of M. tuberculosis on
addition of specific reagent. Reddish/violet coloration was also observed on the surface of
the slants indicative of a positive NRA and growth of standard strain of M. tuberculosis
on addition of specific reagent in all extracts except ethanol, successive ethanol, pet.
ether, methanol and chloroform extract containing bottles of Vitex negundo Linn.
No reddish/violet coloration was observed on the surface of the slants for
rifampicin and INH and absence of colouration interpreted as negative NRA. No
reddish/violet coloration was observed on the surface of the slants in ethanol, successive
ethanol, pet. ether, methanol and chloroform extract containing bottles of Vitex negundo
Linn. and absence of colouration interpreted as negative NRA.
Therefore it is concluded that aqueous, acetone and benzene extracts at 200 µg/ml
did not showed any anti-tubercular activity while ethanol, successive ethanol, pet. ether,
methanol and chloroform extracts possesses anti-tubercular activity by nitrate reductase
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assay method. The results were obtained after seven days of inoculation in all the control
and extract containing bottles.
The results of susceptibility testing of all the extracts by the proportion method
and NRA method for Vitex negundo Linn. were compared with control and it was
observed that the results of NRA for all extracts for this standard strain of M. tuberculosis
(MIC 200 µg/ml) was similar to that by the proportion method, since both showed that M.
tuberculosis was sensitive to these all extracts. Susceptibility testing of all extracts by
these two methods in the M. tuberculosis was in 100 % agreement.
Therefore it is concluded in the present study that the NRA is rapid and superior
to L-J proportion method, which is time-consuming. NRA has emerged as the method of
choice for the susceptibility testing of M. tuberculosis organism and is important to study
the drug resistance pattern.
6.2.7 BACT/ALERT3D SYSTEM:
All the extracts of Vitex negundo Linn. were subjected for screening anti-
tubercular activity by BacT/Alert3D system to obtain confirmatory results as it is more
sensitive compared to first two methods and also comparison for the results by all these
three methods was also possible.
Source of inoculam for BacT/Alert method:
When seed bottle flagged positive by BacT/Alert then Gram staining was
performed according to standard procedure of seed bottle for the presence of gram – ve or
gram + ve organisms.
Observation: No presence of any gram – ve or gram + ve organisms.
Result: The seed bottle did not containing any gram – ve or gram + ve organisms.
Hence it was confirmed that there was no any contamination in seed bottle. When
seed bottle flagged positive by BacT/Alert and AFB smear positive it was not incubated
for more than 48 hrs. A “seed” bottle has utilized as source of inoculam for further
screening of anti-tubercular activity of test extract samples.
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Table no. 65: Effect of 150 µg/ml extracts of Vitex negundo Linn. on M. tuberculosis
by BacT/Alert 3D system.
Sr.
no.
Name of the test drug/
extracts
Concentration
of test drug/
extracts (µg/ml)
No. of days required for
growth of Mycobacterium
tuberculosis (Mean + SEM)
1 Proportional control -- 10.3833 ± 0.01333
2 Direct control -- 6.25 ± 0.02309
3 Ethanol extract 150 19.8566 ± 0.02309**
4 Petroleum ether extract 150 8.3966 ± 0.02309
5 Benzene extract 150 7.25 ± 0. 02309
6 Chloroform extract 150 8.4233 ± 0. 01333
7 Acetone extract 150 7.3033 ± 0. 0133
8 Successive ethanol extract 150 10.4660 ± 0. 01333*
9 Methanol extract 150 9.2833 ± 0.01764
10 Aqueous extract 150 5.2333 ± 0.01667
11 Rifampicin 1 24.5933 ± 0.01333**
12 Isoniazide 0.1 19.2166 ± 0.01667**
Values are expressed as mean+ SEM. Statistical analysis was performed using Dunnet’s
test. *p<0.05 and **p<0.01 was taken as the criterion of significance when compared to
proportional control where n = 3.
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Fig. no. 45: Effect of 150 µg/ml extracts of Vitex negundo Linn. on M. tuberculosis by
BacT/Alert 3 D system.
From BacT/Alert method, it was observed that the growth of M. tuberculosis was
found after 10.38 days in the proportional control bottle and after 6.25 days in direct
control bottle after inoculation. Aqueous, acetone, benzene, pet. ether, chloroform and
methanol extracts 150 µg/ml used as test bottles, the growth of M. tuberculosis was found
in all above extracts during period 5.23-9.28 days after inoculation, since the growth of
M. tuberculosis compared to the proportional control bottle, which is earlier than
proportional control bottle, hence it indicates these extracts did not possesses any anti-
tubercular activity. The standard rifampicin and INH bottles showed M. tuberculosis
growth at 24 and 19.5 days respectively after inoculation compared to the proportional
control bottle, since growth was observed later than the proportional control bottle, which
indicates anti-tubercular activity.
From this result, it is concluded that all these extracts except ethanol and
successive ethanol did not showed anti-tubercular activity at 150 µg/ml of Vitex negundo
Linn. by BacT/Alert 3D system which was similar as shown by proportion method,
nitrate reductase assay method except for the methanol extract. Susceptibility testing of
all extracts except methanol extract by these three methods in the M. tuberculosis was in
100 % agreement.
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Methanol extract possesses anti-tubercular activity by proportion and NRA
method while same extract did not possess anti-tubercular activity by BacT/Alert 3 D
system. The methanol extract, in which the results of proportion and NRA method were
not corroborated by BacT/Alert 3 D system. Therefore the discordant types of results
were observed in -between these methods for methanol extract.
Table no. 66: Effect of 200 µg/ml extracts of Vitex negundo Linn. on M. tuberculosis
by BacT/Alert 3 D system.
Sr.
no.
Name of the test drug/
extracts
Concentration
of test drug/
extracts (µg/ml)
No. of days required for
growth of Mycobacterium
tuberculosis (Mean + SEM)
1 Proportional control -- 10.3833 ± 0.01333
2 Direct control -- 6.25 ± 0.02309
3 Ethanol extract 200 23.7766 ± 0.02309**
4 Petroleum ether extract 200 21.8966 ± 0.02309**
5 Benzene extract 200 9.41 ± 0. 02309
6 Chloroform extract 200 14.3833 ± 0. 01333*
7 Acetone extract 200 8.25 ± 0. 02309
8 Successive ethanol extract 200 14.5933 ± 0. 01333*
9 Methanol extract 200 11.4833 ± 0.01667*
10 Aqueous extract 200 6.2166 ± 0.01667
11 Rifampicin 1 24.5933 ± 0.01333**
12 Isoniazide 0.1 19.2166 ± 0.01667**
Values are expressed as mean+ SEM. Statistical analysis was performed using Dunnet’s
test. *p<0.05 and **p<0.01 was taken as the criterion of significance when compared to
proportional control where n=3.
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Figure no. 46: Effect of 200 µg/ml extracts of Vitex negundo Linn. on M. tuberculosis
by BacT/Alert 3D system.
Fig. no. 47: Negative and positive bottles of BacT/Alert technology indicating no
growth and growth of M. tuberculosis respectively.
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Ethanol, successive ethanol, chloroform, methanol and pet. ether extracts of
Vitex negundo Linn. at 200 µg/ml observed the growth of M. tuberculosis after 23.77,
14.59, 14.38, 11.48 and 21.89 days respectively which was later than proportional control
bottle (after 10.38 days) after inoculation. Growth of M. tuberculosis identified by yellow
colour at the bottom of bottle and indicated +ve signal which was also confirmed by acid
fast staining.
Other extracts of aqueous, acetone, benzene and methanol of plant did not
showed any anti-tubercular activity since the growth of M. tuberculosis in each test bottle
was earlier than the proportional control bottle.
Therefore the results of BacT/Alert 3 D system concluded that successive
ethanol, methanol and chloroform extracts of Vitex negundo Linn. at 200 µg/ml showed
significant and ethanolic, pet. ether extracts showed highly significant anti-tubercular
activity while other extracts of Vitex negundo Linn did not showed any anti-tubercular
activity at 200 µg/ml by BacT/Alert 3D system.
• Acid fast staining: Acid fast staining was performed according to standard
procedure for proportional control bottles, direct control bottles, and each test
extract bottles for the presence of Mycobacterium tuberculosis.
Observation: Bright red bacilli were observed.
Result: In proportional control bottles, direct control bottles and each test extract bottles
observed the growth of Mycobacterium tuberculosis which is identified by acid fast
staining method. Therefore it is concluded that growth of Mycobacterium tuberculosis in
all above bottles which also indicates no contamination to these bottles.
The NRA result of ethanol, successive ethanol and chloroform extracts for this
standard strain of M. tuberculosis (MIC 200 µg/ml) was similar to that by the BacT/Alert
method, since both showed that M. tuberculosis was sensitive to these all three extracts.
The NRA result of aqueous, pet. ether, acetone, benzene, methanol and chloroform
extracts for this standard strain of M. tuberculosis (MIC 200 µg/ml) was similar to that by
the BacT/Alert method, since both showed that M. tuberculosis was resistant to these all
extracts. A good agreement was found between NRA and BacT/Alert method of drug
susceptibility testing of M. tuberculosis in our study. Therefore it is concluded that 100 %
agreement between results of these extracts of Vitex negundo Linn. by both these
methods. These finding showed that the NRA and BacT/Alert method is superior to the
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224
proportion method in terms of accuracy, simplicity of performance and the rapidity of
results.
6.2.8 ANTI-TUBERCULAR ACTIVITY OF FRACTIONS OF ETHANOLIC
EXTRACTS:
Screening of anti-tubercular activity of pet. ether, chloroform and methanol
fractions of ethanolic extract were performed by nitrate reductase assay method and
BacT/Alert method.
In all control bottles and methanol fraction reddish/violet coloration was observed
on the surface of the slants indicative of a positive NRA. No reddish/violet coloration was
observed on the surface of the slants for rifampicin, INH, pet. ether fraction and
chloroform fraction of ethanolic extract, absence of colouration interpreted as negative
NRA. The results were obtained after seven days of inoculation in all the control and test
sample containing bottles. Therefore results revealed that 150 µg/ml of pet. ether,
chloroform fractions showed anti-tubercular activity by NRA method.
In pet. ether fraction and chloroform fraction of ethanolic extract of Vitex
negundo Linn. at 150 µg/ml observed the growth of M. tuberculosis later than
proportional control bottle (after 10.38 days) after inoculation. Growth of M. tuberculosis
identified by yellow colour at the bottom of bottle and indicated +ve signal which was
also confirmed by acid fast staining. Therefore Pet. ether fraction and chloroform fraction
of ethanolic extract showed anti-tubercular activity since the growth of M. tuberculosis in
each test bottle was later than the proportional control bottle by BacT/Alert method.
Therefore it is observed that from NRA method and BacT/Alert method pet. ether
and chloroform fraction of ethanolic extract of Vitex negundo Linn. showed anti-
tubercular activity at 150 µg/ml.
6.2.9: RESISTANCE RATIO METHOD:
From the above observations and the results, it is concluded that the standard
strain of M. tuberculosis was sensitive to ethanolic extract by proportion method, nitrate
reductase assay method and BacT/Alert method. Pet .ether and chloroform fractions of
ethanolic extract was sensitive to standard strain of M. tuberculosis by nitrate reductase
assay method and BacT/Alert method.
Therefore the ethanolic extract and pet. ether and chloroform fractions of
ethanolic extract were screened for anti-tubercular activity by resistance ratio method.
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The resistance ratio (RR) method utilizes the ratio of the minimum inhibitory
concentration (MIC) for the patient’s strain to the MIC of the drug-susceptible reference
strain, M. tuberculosis H37RV, both tested in the same experiment. Growth is defined as
the presence of twenty or more colonies after four weeks of incubation and MIC is
defined as the lowest drug concentration in the presence of which the number of colonies
is less than 20. The result was expressed as a resistance ratio by comparison with a
control culture as follows.
Eighteen numbers of colonies of M. tuberculosis H37RV were observed for
ethanolic extract of Vitex negundo Linn. at 100 µg/ml while sixteen number of colonies
were observed for patient strain at 150 µg/ml. Therefore MIC for standard strain and
patient strain is 100 µg/ml and 150 µg/ml respectively. The resistance ratio is found to be
1.5 which indicates M. tuberculosis is sensitive to the ethanolic extract, since the value of
resistance ratio (RR) is less than two. Therefore ethanolic extract is sensitive to M.
tuberculosis.
Fourteen and twelve numbers of colonies of M. tuberculosis H37RV were
observed for pet. ether and chloroform fractions of ethanolic extract of Vitex negundo
Linn. at 100 µg/ml while twelve and ten number of colonies were observed for patient
strain at 100 µg/ml. Therefore MIC for standard strain and patient strain is 100 µg/ml and
100 µg/ml respectively. The resistance ratio is found to be 1 which indicates M.
tuberculosis is sensitive to the pet. ether and chloroform fractions of ethanolic extract,
since the value of resistance ratio (RR) is less than two. Therefore it is concluded that pet.
ether extract and chloroform fractions of ethanolic extract is sensitive to M. tuberculosis.
Table no. 67: Effect of all extracts of Vitex negundo Linn. on M. tuberculosis by
different methods used for screening of anti-tubercular activity.
METHOD AQU ETN PET.ET ACE BEN S. ETN CHN MET
Proportion method
150 µg/ml R S R R R S R S
200 µg/ml R S S R R S S S
NRA method
150 µg/ml R S R R R S R S
200 µg/ml R S S R R S S S
BacT/Alertmethod
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150 µg/ml R S R R R S R R
200 µg/ml R S S R R S S S
Where R = resistant and S = sensitive
Fig. no. 48: Effect of all extracts on M. tuberculosis by different methods used for
screening of anti-tubercular activity.
From the results obtained by all four methods it is observed that M. tuberculosis
H37RV is sensitive to ethanolic extract of Ricinus communis Linn. On correlation
analysis using Mc Nemar Chi-square test, no significant difference between the four
methods is observed (p>0.05). Hence 100 % agreements is observed in between
proportion, NRA, BacT/Alert and resistance ratio method for ethanolic extract of Vitex
negundo Linn.
6.2.10: ANTITUBERCULAR ACTIVITY OF ISOLATED COMPOUNDS:
A total of fourteen compounds collected from different column chromatography
of pet. ether and chloroform fractions of ethanolic extract of Vitex negundo Linn. and
anti-tubercular activity was performed for all fourteen compounds by nitrate reductase
assay which showed in following table.
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Table no. 68: Effects of isolated compounds on M. tuberculosis by nitrate reductase
assay method.
Sr.
no.
Name of the isolated
compounds
Concentration
(µg/ml)
Growth of Mycobacterium
tuberculosis indicated by
reddish/violet colouration
I II III
1 Control without drug -- + + +
2 Rifampicin 40 -- -- --
3 Isoniazide 0.2 -- -- --
4 Compound HEA-1 50 + + +
100 + + +
5 Compound HEA-2 50 + + +
100 -- -- --
6 Compound CM-20 50 + + +
100 -- -- --
7 Compound CM-24 50 + + +
100 -- -- --
8 Compound PE-9 50 + + +
100 + + +
9 Compound PE-10 50 + + +
100 + + +
10 Compound PE-19 50 + + +
100 + + +
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11 Compound PE-34 50 -- -- --
100 -- -- --
12 Compound PEA-22 50 + + +
100 + + +
13 Compound PEA-29 50 + + +
100 + + +
14 Compound PEA-48 50 + + +
100 + + +
-- indicates no change in colour means no growth of M. tuberculosis
+ indicates development of reddish violet colour means growth of M. tuberculosis
The results revealed that HEA-2, CM-20, CM-24 and PE-34 inhibit growth of M.
tuberculosis which interpreted as –ve NRA. Therefore HEA-2, CM-20 and CM-24 posses
antitubercular activity at 100 µg/ml while PE-34 at 50 and 100 µg/ml.
6.2.11: ANTIMICROBIAL ACTIVITY:
Table no. 69: Activity of different extracts of Vitex negundo Linn. on micro-
organisms
Sr.
no.
Name of
Micro-
organisms
Media
Used
Zone of inhibition in mm
PET.
ET
CHN
BEN ETN MET AQU AMP
1 Staphylococ
cus aureus
Yoghel
johnson
agar
14 15 20 -- 8 -- 22
2 Pseudomon
as
aerugionsa
Cetrimid
e agar
15 10 20 11 13.3 12.3 21
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3 Candida
albicans
Saboura
ud
dextrose
agar
15 10 20 12 10 -- 21
--- indicates no zone of inhibition
Fig. no. 49: Effect of Vitex negundo Linn. extracts on Staphylococcus aureus.
Zone of inhibition observed for S. aureus by pet. ether, chloroform, benzene
and methanol extracts are 14, 15, 20 and 8 respectively. Hence benzene extract showed
maximum inhibition of growth of S. aureus followed by chloroform, pet. ether and
methanol extracts. Therefore benzene extract possesses significant antimicrobial activity.
Fig. no. 50: Effect of Vitex negundo Linn. extracts on pseudomonas aeruginosa.
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230
Zone of inhibition observed for P. aeruginosa by pet. ether, chloroform,
benzene, ethanol, methanol and aqueous extracts are 15, 10, 20, 11, 13.3 and 12.3
respectively which showed that the growth of P. aeruginosa moderately inhibited by
methanol, aqueous, ethanol and chloroform extracts and significantly inhibited by
benzene extract and followed by pet. ether extract. Therefore benzene extract was
possesses most significant while other possesses mild significant antimicrobial activity.
Ampicillin posses most significant antimicrobial activity compared to all extracts.
Fig. no. 51: Effect of Vitex negundo Linn. extracts on Candida albicans.
Zone of inhibition observed for C. albicans by pet. ether, chloroform, benzene,
ethanol and methanol 15, 10, 20, 12 and 10 respectively. Benzene extract showed
significant compared to standard ampicilin followed by pet. ether while other extracts
showed mild anti-microbial activity. From these results, it is concluded that benzene
extract possesses most significant anti-microbial activity.
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6.2.12 HEPATOPROTECTIVE ACTIVITY:
Table no. 70: Effect of ethanol extract of Vitex negundo Linn. leaves on Paracetamol
induced hepatotoxicity.
Groups SGOT
(units/ml)
SGPT
(units/ml)
ACP
(KA units)
ALP
(KA units)
Normal (D/W)
10 ml/kg
83.67
±1.856
67.00
±2.053
2.6130
±0.0853
10.2566
±0.0701
Control Paracetamol
(3 gm/kg)
166.17
±1.347
111.83
±2.167
4.6752
±0.0988
16.4480
±0.6181
Standard Silymarin
(100 mg/kg)
120.17
±2.056*
73.84
±1.740*
2.9703
±0.0711*
11.0038
±0.2348*
Ethanol extract
(300 mg/kg)
129.34
±0.9545*
74.50
±1.945*
3.0564
±0.0942*
11.8121
±0.0617*
Significance evaluated by one-way analysis of variance (ANOVA) followed by Dunnett’s
t-test control verses all. *P<0.05 is considered as criterion for significance. Values are
mean ± SEM, (n=6) SGOT-Serum Glutamate Oxaloacetate Transaminase, SGPT- Serum
Glutamate Pyruvate Transaminase, ACP-Acid Phosphatase, ALP- Alkaline Phosphatase
Fig. no. 52: Effect of Vitex negundo Linn. extracts on acid phosphatase level.
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Fig. no. 53: Effect of Vitex negundo Linn. extracts on alkaline phosphatase level.
Fig. no. 54: Effect of Vitex negundo Linn. extracts on SGPT level.
Fig. no. 55: Effect of Vitex negundo Linn. extracts on SGOT level.
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233
Acetaminophen (N-acetyl-p-aminophenol, Paracetamol), a widely used analgesic
and antipyretic drug is known to cause hepatotoxicity in experimental animals and
humans at high doses. N-acetyl-p-benzoquinone-imine (NAPQI) is a highly reactive
arylating minor metabolite of paracetamol which is detoxified by conjugation with
glutathione. When a very large dose of paracetamol is taken, glucuronidation capacity is
saturated, more of the minor metabolite is formed-hepatic glutathione is depleted and this
metabolite binds covalently to proteins in liver cells causing necrosis. Toxicity thus
shows a threshold effect manifesting only when glutathione is depleted to a critical point.
Nucleophiles and increasing glutathione levels protects against the hepatic injury.
Administration of 3 gm/kg body weight of paracetamol to experimental animals
for 1day produced statistically significant rise in the enzymes levels, namely SGOT
(166.17 KA units/ml), SGPT (111.83 KA units/ml), ACP (4.6752 KA units), ALP
(16.4480 KA units) indicating the chemical induced hepatocellular toxicity. Pretreatment
of the animals with the 300 mg/kg p. o. of the ethanolic extract of leaves of Vitex negundo
Linn. showed the significant protection in the biochemical parameters like SGOT
(129.34), SGPT (74.50), ACP (3.0564) and ALP (11.8121) against paracetamol induced
elevations. Further there was increase in weight of the liver treated with the paracetamol
(10.69 gm) is seen as compared to the normal (7.02 gm). The treatment with the ethanolic
extract of Vitex negundo Linn. retains the liver weight (7.12 gm) near to the normal. The
inhibitory effect of the Vitex negundo Linn. on hepatotoxicity was compared to that of
positive control group.
6.2.13 HISTOPATHOLOGY OF THE PARACETAMOL MODEL:
The hepatoprotective effect of ethanolic extract of Vitex nigundo Linn. was further
confirmed by histopathological examination of the livers of normal, control, paracetamol
treated silymarin and paracetamol plus ethanolic extract treated groups are showed in
following fig.
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A] Control paracetamol (3gm/kg) B] Ethanol extract (300mg/kg)
C] Normal group (Distilled water) D] Standard silymarin (100mg/kg)
Fig. no. 56: Representative photomicrographs of histopathological changes showing
effect of test materials on the rats intoxicated with Paracetamol.
Liver section of control rat showing a normal hepatic architecture well brought
about from the central vein. The liver samples of paracetamol treated rats showed gross
necrosis of the centrilobular hepatocytes characterized by gross necrosis, degeneration,
karyolysis and eosinophilic infiltration which are significantly prevented by the treatment
with the ethanolic extract of Vitex negundo Linn. that showed the hepatoprotective
activity. The histopathological pattern of the livers of the rats treated with paracetamol
plus extract showed minimal necrosis in centrilobular and regeneration of hepatocytes.
Administration of leaves of ethanolic extract of Vitex negundo Linn. that showed
significant hepatoprotective activity; while qualitative phytochemical investigations on
CHAPTER-6 RESULTS AND DISCUSSION
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235
the ethanolic extract of Vitex negundo Linn. also showed test positive for flavonoids by
chemical tests. Further, it has been reported that the flavonoid constituents of the plant
possess antioxidant properties and was found to be useful in the treatment of liver
damage(4).
The administration of hepatoprotective drugs may induce the hepatocytes to resist
the toxic effect of paracetamol. The results indicated that the ethanolic extract of Vitex
negundo Linn. has significant hepatoprotective activity.
From the available data and the experimental results suggest the Vitex negundo
Linn. as a hepatoprotectant. However the mechanism of action and the active component
which is responsible for the actual hepatoprotectivity is not well known. The present
study was suggested that the ethanolic extract of Vitex negundo Linn. could have a
preventive activity towards paracetamol induced hepatotoxicity in albino rats. However
further exploration is needed in order to elucidate the components responsible for
hepatoprotection.
Anti-tubercular activities of all the extracts were evaluated; it was found that 150
µg/ml ethanolic extract of leaves of Vitex negundo Linn. found more potent than other
extracts hence; ethanolic extract was used for separation and isolation of active
constituents. TLC of this extract was performed by using many mobile phases but does
not indicated much constituents in TLC of the ethanolic extract.
Literature reported that triterpenes were mostly soluble in petroleum ether and
chloroform and triterpenes have anti-tubercular activity(5) and 200 µg/ml of pet. ether
extract and chloroform extract also showed anti-tubercular activity. Hence separation of
phytoconstituents from ethanolic extract in pet. ether was carried out by separating funnel
and collected the pet. ether fraction. Then same residue was dissolved in chloroform and
obtained chloroform fraction by similar method. Further the remaining residue was
dissolved in methanol and methanol fraction collected by similar method by using
separating funnel.
6.2.14 IDENTIFICATION AND SEPERATION OF ACTIVE CONSTITUENTS:
Anti-tubercular activities of pet. ether, chloroform and methanol fractions were
performed by nitrate reductase assay method and BacT/Alert method. The results
observed that 150 µg/ml of only pet. ether and chloroform fractions showed anti-
tubercular activity. Hence these two fractions were used for separation and isolation of
CHAPTER-6 RESULTS AND DISCUSSION
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236
phytoconstituents by column chromatography method while methanol fraction did not
show any anti-tubercular activity.
6.2.15 TLC STUDY:
1. TLC of chloroform fraction of ethanolic extract was performed in mobile phase
chloroform and methanol (15:1) and developed by anisaldehyde-sulphuric acid
reagent showed 5 spots and Rf values are 0.25, 0.39, 0.54, 0.62 and 0.85 while
pet. ether fraction showed 6 spots and Rf values are 0.30, 0.37, 0.44, 0.51, 0.61
and 0.68.
2. TLC of chloroform fraction of ethanolic extract was performed in mobile phase
hexane and ethyl acetate (4:1) showed 4 spots and Rf values are 0.39, 0.47, 0.61
and 0.67.
3. TLC of chloroform fraction of ethanolic extract was performed in mobile phase
benzene and ethyl acetate (4:1) showed 5 spots and Rf values are 0.10, 0.25, 0.42,
0.55 and 0.76 while pet. ether fraction showed 4 spots and Rf values are 0.18,
0.33, 0.53 and 0.64.
4. TLC of chloroform fraction of ethanolic extract was performed in mobile phase
pet. ether and ethyl acetate (4:1) showed 4 spots and Rf values are 0.25, 0.37, 0.55
and 0.67 while pet. ether fraction showed 6 spots and Rf values are 0.18, 0.29,
0.53, 0.68, 0.72 and 0.83.
From the above results of TLC, Rf values of chloroform and pet. ether fraction
matches to betulinic acid, ursolic acid and β-sitosterol which is reported in literatuer(6).
Therefore column chromatography of both fractions and ethanolic extract were carried
out by using different proportions of solvents. A total of 14 compounds collected from
different column chromatography and anti-tubercular activity was performed for all 14
compounds by nitrate reductase assay which showed in table no. 68 and results revealed
that HEA-2, CM-20 and CM-24 all posses antitubercular activity at 100 µg/ml while PE-
34 at 50 and 100 µg/ml.
Further HPTLC of these compounds was performed which showed PE-34 was a
single isolated compound. However others showed as mixtures that containing more than
one compound. Therefore only PE-34 was characterized by IR, H-NMR and GC-MS.
HPTLC of std. β -sitosterol with chloroform and pet. ether fractions of ethanolic extract
were performed which showed as follows.
CHAPTER-6 RESULTS AND DISCUSSION
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6.2.16 HPTLC STUDY:
Fig. no. 57: HPTLC study of standard β-sitosterol at 574 nm.
Fig. no. 58: HPTLC study of chloroform fraction of ethanolic extract at 574 nm
CHAPTER-6 RESULTS AND DISCUSSION
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Fig. no. 59: HPTLC study of petroleum ether fraction of ethanolic extract at 574
nm.
Fig. no. 60: HPTLC image of std. β-sitosterol, chloroform fraction and pet. ether
fraction at 574 nm.
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239
Fig. no. 61: HPTLC image of std. β-sitosterol , chloroform fraction and pet. ether
fraction at 574 nm.
Table no.71: Identification and estimation of β -sitosterol of Vitex negundo Linn. By
HPTLC:
Sr.
no.
Sample name Rf value Area β -sitosterol
content (%w/w)
1 Pet. ether fraction 0.28 2492.8 6.463
2 Chloroform fraction 0.32 2260.9 5.862
3 Std. β-sitosterol 0.29 7713.1 ---
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Fig. no. 62: HPTLC study of pet. ether fraction of ethanolic extract.
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241
Fig. no. 63: HPTLC study of chloroform fraction of ethanolic extract.
Fig. no. 64: HPTLC image of pet. ether and chloroform fractions
CHAPTER-6 RESULTS AND DISCUSSION
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Fig. no. 65: HPTLC STUDY OF ISOLATED PE-34 (URSOLIC ACID)
6.2.17 IR STUDY:
Fig. no. 66: IR spectrum of isolated ursolic acid.
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243
Table no. 72: IR study of ursolic acid
Functional group Wave number(cm-1)
Free OH stretching 3450
Aliphatic CH stretching 2923
CH-stretching 2852
C=O stretching 1709
C=C stretching 1657
C-O group 1384.64
6.2.18 NMR STUDY:
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Fig. no. 67: NMR study of isolated ursolic acid
Table no.73: NMR study of isolated ursolic acid.
Carbon number δ ppm values Description of functional groups
12 5.575 1 H
3 4.15 OH
1 and 3 3.569 H, H
1 and 18 2.095 H, H
27 1.030 CH3 group
23,24,25,26,29 and 30 0.898-0.826 Multiplet CH3 group
2,3,5-7,21,22,28 and 29 1.03-1.60 Multiplet CH2 group
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6.2.19: GC-MS STUDY:
URSOLIC ACID
CHAPTER-6 RESULTS AND DISCUSSION
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246
Peak Justification
323 C+
CH3
CH3
CH3
CH3
O
263
CH3
CH3
CH3
O
OH
CH2
+
203
CH3
CH3
CH3
CH+
179
C+
CH 3
CH 2
CH 3
CH 3
O
161
CH3
CH2
+
CH3
CH3
141 CH+
CH3
CH3
CH3
OH
126
CH+
CH+
CH3
CH3
OH
CHAPTER-6 RESULTS AND DISCUSSION
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84
CH2
+
CH3
CH2
+
67 CH
+
CH3
CH2
55 CH3
CH2
+
Fig. no. 68: GC-MS study of isolated ursolic acid
6.2.20: DEVELOPMENT OF NEW ANALYTICAL METHOD BY DOUBLE
BEAM UV-SPECTROPHOTOMETER:
Different following dilutions were prepared of std. linoleic acid and absorbance
was recorded by using double beam UV-spectrophotometer. Absobance was recorded
also of isolated linoleic acid.
Table no. 74: Absorbance data of different concentrations of std. linoleic acid and
isolated compound linoleic acid.
Sr. no. Name of sample Concentration in
mg/ml
Absorbance
1 Std. linoleic acid 0.1 0.07
2 Std. linoleic acid 0.2 0.12
3 Std. linoleic acid 0.3 0.18
4 Std. linoleic acid 0.4 0.23
5 Std. linoleic acid 0.5 0.38
6 Std. linoleic acid 1 0.76
7 Isolated linoleic acid --- 0.17796
8 Isolated linoleic acid --- 0.22134
9 Isolated linoleic acid --- 0.11358
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Fig. no.69: Absorbance of different concentrations of std. linoleic acid.
The result of linoleic acid content was obtained from regression equation of
calibration curve y= 0.768x-0.027.Average concentration of isolated linoleic acid is found
87.09 %.
CHAPTER-6 RESULTS AND DISCUSSION
Phytochemical and pharmacological investigation of anti-tubercular activity of the certain indigenous plants
249
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