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MATERIALS AND METHODS

COLLECTION OF PLANT MATERIALS


The leaves were collected from various parts of Chennai, Tamil Nadu.
DRYING:
The collected plant leaves are allowed for Shade drying for 4 days completely. The dried
leaves were ground into powder, which was used for further extraction.
EXTRACTION
About 60 gm of dry sample was weighed and macerated with 200 ml of hexane
separately and kept overnight in shaker. The extract was collected after filtration using
What man no:1 filter paper and was stored. Another 200 ml of chloroform was added to
the residual mixture and incubated in shaker for 24 hours and the extract was collected
again using a What man no:1 filter paper. This procedure was repeated once again with
methanol and the extract was evaporated, which was used for further phytochemical
analysis, and antimicrobial activity.
PHYTOCHEMICAL TESTS
1. TEST FOR CARBOHYDRATES:
To 2ml of plant extract, 1ml of Molischs reagent and few drops of conc. Sulphuric acid
were added. Presence of purple or reddish color indicates presence of carbohydrates.
2. TEST FOR TANNINS:

To 1ml

of plant extract, 2ml of 5% ferric chloride is added. Formation of dark blue

or greenish black indicates the presence of tannins.


3. TEST FOR SAPONINS:
To 2ml of plant extract, add 2ml of distilled water and shaken in graduated cylinder for
15min lengthwise. Formation of 1cm layer of foam indicates the presence of saponins.
4. TEST FOR FLAVONOIDS:
To 2ml of plant extract, 1ml of 2N NaOH was added. Presence of yellow color indicates
presence of flavonoids.
5. TEST FOR ALKALOIDS:
To 2ml of plant extract , 2ml of conc.Hcl and then few drops of Mayers reagent were
added this indicates the presence of alkaloids when green or white precipitate obtained.
6. TEST FOR QUINONES:
To 2ml of plant extract, added, 1ml of conc.sulphuric acid. Formation of red color
indicates the presence of quinones.
7. TEST FOR GLYCOSIDES:
To 2ml of extract, add 3ml of CHCl3 and 10% ammonia solution. Formation of pink
color indicates the presence of glycosides.
8. TEST FOR CARDIAC GLYCOSIDES:

To 0.5ml of extract, 2ml of glacial acetic acid and few drops of ferric chloride added.
This was under layered with 1ml of conc.sulphuric acid. Formation of brown ring at the
interface indicates presence of cardiac glycosides.
9. TEST FOR TERPENOIDS:
To 0.5ml of extract, 2ml of chloroform added and conc. Sulphuric acid was added.
Formation of red brown color at the interface indicates the presence of terpenoids.
10. TEST FOR TRITERPENOIDS:
To 1ml of extract, 1ml of Libemann Buchard reagent (glacial acetic acid and
conc.sulphuric acid) were added. Formation of blue or green color indicated the presence
of triterpenopids.
11. TEST FOR PHENOLS:
To 1ml of extract, 2ml of distilled water followed by few drops of 10% ferric chloride
was added. Formation of blue or green color indicates the presence of phenols.
12. TEST FOR COUMARINS:
To 1ml of extract, 1ml of 10% NaOH was added. Formation of yellow color indicates the
presence of coumarins.
13. NINHYDRIN TEST:
To 2ml of plant extract few drops of 0.2% Ninhydrin was added and heated for 5 minutes.
Formation of blue color indicates presence of proteins.

14. TEST FOR STEROIDS AND PHYTOSTEROIDS:


To 1ml of extract equal volume of chloroform is added and subjected with few drops of
conc.sulphuric acid , appearance of brown ring indicates the presence of steroids and
appearance of bluish brown ring indicates the presence of phytosteroids.
15. TEST FOR PHLOBATANNINS:
To 1ml of extract few drops of 2% Hydrochloric acid was added appearance of red color
precipitate indicates the presence of phlobatannins.
16. TEST FOR ANTHRAQUINONES:
To 1ml of extract few drops of 10% ammonia solution was added appearance of pink
color precipitate indicates the presence of anthraquinones.
ANTIMICROBIAL ACTIVITY
40 mg of extract was dissolved in 1 ml of Dimethyl sulphuric acid (DMSO), and it is
used for agar diffusion method.
The plant extracts were analyzed against four microbial strains by agar diffusion method.
The Molten Muller Hinton agar was prepared for 150 ml with distilled water. 25 ml of
MHA medium were poured into the sterile petri dish. After solidification, the sterile
cotton swab was dipped into the broth culture of following microorganisms
1.
2.
3.
4.

Escherichia coli
Pseudomonas aeroginosa
Staphylococcus aureus
Aeromonas hydrophilae

The entire agar surface of each plate was inoculated with the 1ml swab, first in horizontal
direction and then in vertical direction, which ensure the even distribution of organisms
over the agar surface. Then 4 wells are cut using the well cutter. After that 50 l of the
control (ciprofloxacin), leaf extracts such as hexane, ethyl acetate, chloroform and
methanol were poured into the each wells and then the plates were incubated at 37C for
24 hours. Then the zone of inhibition was measured in millimeter.

RESULTS
Extraction
The plant leaves were shade dried and extracted using three solvents Hexane,
Chloroform and Methanol. The yield was tabulated in Table 1, 2, 3, 4.
Table1. AMOUNT OF YIELD Wrightia tinctoria
Amount of leaf powder
60 g
60 g
60 g

Solvent
Hexane
Chloroform
Methanol

Yield (g)
1.002
1.325
3.985

Table2. AMOUNT OF YIELD Eupatorium triplinerve


Amount of leaf powder
60 g
60 g
60 g

Solvent
Hexane
Chloroform
Methanol

Yield (g)
1.876
2.654
3.674

Phytochemical analysis
The extracts were analysed for the presence of various phytochemicals like 1.
for carbohydrates,
cardiac glycosides,

tannins, saponins,

flavonoids,

Test

alkaloids, quinones, glycosides,

terpenoids, triterpenoids, phenols, coumarins, Steroids and

phytosteroids, phlobatannins, anthraquinones. The results are documented in tables 5, 6,


7, 8.
Table 6 Phytochemical analysis of Wrightia tinctoria

PHYTOCHEMICAL TEST
(Wrightia tinctoria)

Petroleum ether

Ethyl acetate

Ethanol

Carbohydrate test

Tannins test

Saponin test

Flavonoid test

Alkaloid test

Quinones

Glycosides test

Cardiac glycosides test

Terpenoids test

Triterpenoids

Phenols

Coumarins

+ (Phytosteroids)

+ (Phytosteroids)

+ (Steroids)

Phlobatannins

Anthraquinones

Steroids and Phytosteroids

+ Present; Absent

Table 7 Phytochemical analysis of Eupatorium triplinerve


PHYTOCHEMICAL TEST

(Eupatorium triplinerve)

Petroleum ether

Ethyl acetate

Ethanol

Carbohydrate test

Tannins test

Saponin test

Flavonoid test

Alkaloid test

Quinones

Glycosides test

Cardiac glycosides test

Terpenoids test

Triterpenoids

Phenols

Coumarins

+ (Phytosteroids)

+ (Phytosteroids)

+ (Steroids)

Phlobatannins

Anthraquinones

Steroids and Phytosteroids

+ Present; Absent

Antibacterial activity
The antibacterial activities of the plant extracts were understood using the well diffusion
assay. The results are documented in tables 9, 10, 11, 12.
Table 10 Antibacterial activity of Wrightia tinctoria

E.coli
K.pneumo
niae
S. aureus
P.aerogino
sa

Wrightia tinctoria (mm)


Hexan Chlorofo Methan
e
rm
ol
14
18
22

PC
32

11
11

16
20

18
17

30
28

12

18

18

26

Table 11 Antibacterial activity of Eupatorium triplinerve

E.coli
K.pneumo
niae
S. aureus
P.aerogino
sa

Eupatorium triplinerve
(mm)
Hexan Chlorofo Methan
e
rm
ol
12
12
18

PC
32

10
10

14
16

12
18

30
28

11

15

14

26