RESULTS AND DISCUSSION OF VITEX NEGUNDO …shodhganga.inflibnet.ac.in/bitstream/10603/23543/13/13...Ethanol extract is obtained by solvent extraction method while petroleum ether,

CHAPTER-6 RESULTS AND DISCUSSION

Phytochemical and pharmacological investigation of anti-tubercular activity of the certain indigenous plants

199

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



200

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 + + -- -- + + + +



201

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 -- -- -- -- -- -- -- --



202

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



203

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 + + -- -- -- -- -- +



204

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 + + + + -- + + +



205

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.



206

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



207

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



208

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.



209

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.



210

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 ± 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*




multiple comparison tests. *p<0.05 and **p<0.01 was taken as the criterion of

significance when compared to control where n=3.



211

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



212

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.


extracts

Concentration

of test drug/

extracts (µg/ml)



(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**





213

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.



214

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.


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.



215

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.


by nitrate reductase assay method:

Sr.

no.


extracts

Concentration

of test drug/

extracts

(µg/ml)



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 + + +



216

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



217

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



218

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.



219


by BacT/Alert 3D system.

Sr.

no.


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**


test. *p<0.05 and **p<0.01 was taken as the criterion of significance when compared to

proportional control where n = 3.



220

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.



221

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.


by BacT/Alert 3 D system.

Sr.

no.


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**


test. *p<0.05 and **p<0.01 was taken as the criterion of significance when compared to

proportional control where n=3.



222

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.



223

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



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.



225

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


BacT/Alertmethod



226

150 µg/ml R S R R R S R R


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.



227

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)



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 + + +



228

11 Compound PE-34 50 -- -- --

100 -- -- --

12 Compound PEA-22 50 + + +

100 + + +


100 + + +


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



229

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.



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.



231

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.



232

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.



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.



234

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



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



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.


hexane and ethyl acetate (4:1) showed 4 spots and Rf values are 0.39, 0.47, 0.61

and 0.67.


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.


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.



237

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



238

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.



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 ---



240

Fig. no. 62: HPTLC study of pet. ether fraction of ethanolic extract.



241

Fig. no. 63: HPTLC study of chloroform fraction of ethanolic extract.

Fig. no. 64: HPTLC image of pet. ether and chloroform fractions



242

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.



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:



244

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



245

6.2.19: GC-MS STUDY:

URSOLIC ACID



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



247

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





6 Std. linoleic acid 1 0.76

7 Isolated linoleic acid --- 0.17796





248

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 %.



249

References:

1. Khandelwal KR, Practical Pharmacognosy. Nirali Prakashan, Pune. 2nd ed. 2002,

149-159.

2. Laszlo Adalbert, Raviglione Mario, Rieder Hans, Espinal Marcos. Aziz Mohamed

Abdel, Wrightabigail et al. Guidelines for Surveillance of Drug Resistance in

Tuberculosis. (WHO/CDS/CSR/RMD/2003.3). World Health Organization. 2nd

ed. 2003; 1-32.

3. Kulkarni SK, Handbook of Experimental Pharmacology. Vallabh prakashan,

New Delhi, 1999; 192.

4. Mahalakshmi R, Rajesh P, Balasubramanian V, Rajesh Kanan V.

Hepatoprotective activity on Vitex negundo Linn. (Verbanaceae) by using Wistar

albino rats in Ibuprofen induced model. International Journal of Pharmacology.

2010; 1-6.

5. Dr. Mukherjee PK, Quality control of herbal drugs –An approach to evaluation

of Botanicals. Business Horizons, Pharmaceutical publisher, 2008. 530-531.

6. Celio TH, Fernando RP, Clarice QF, Triterpenes and antitubercular activity of

Brysonima crassa. Quim. Nova, 2008; 31(7): 1719-1721.

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RESULTS AND DISCUSSION OF VITEX NEGUNDO …shodhganga.inflibnet.ac.in/bitstream/10603/23543/13/13...Ethanol extract is obtained by solvent extraction method while petroleum ether,