1. HIMA HARIDASAN
Dept. Of Microbiology,
St. Mary’s college,
Thrissur- 680020,
University of Calicut
E-mail: himaharidasan93@gmail.com

2.  Family : Rutaceae
 Native to India.
 Leaves - Indian diet to improve
appetite and digestion.
 Deciduous to semi-evergreen
aromatic tree

3. TRADITIONAL USES
 Analgesic - pain-killing
 Febrifuge - reduce fever
 Stomachic - improves
appetite and digestion
 Carminative - relieves
flatulence
 Treatment of dysentery
& skin eruptions
PROPERTIES
 Anti-oxidative –
inhibits oxidation damages
 Cytotoxic – cell-toxic
 Antimicrobial
 Anti-ulcer
 Positive inotropic - modifies
the force or speed of muscle
contraction
 Cholesterol reducing
activities

4. 1)Preliminary phytochemical analysis
2)Antimicrobial analysis
MATERIALS AND METHODS
1)PLANT MATERIAL
2)PREPARATION OF EXTRACT
a) The powdered leaves (30g) - methanol extraction
b) Filteration - whatman filter paper
c) Filtrate – evaporation (65ºc)
d) Organic semisolid mass was dried

5. The bacterial cultures: –
o Gram positive -
Staphylococcus and Bacillus
o Gram negative - Escherichia
coli, Pseudomonas, Proteus
and Klebsiella (obtained from
Poly clinic, Thrissur).
o Were grown and maintained in
nutrient broth medium at 37ºc.
The fungal cultures :-
o Aspergillus flavus, Alternaria
and Pencillium ( isolated by
air sampling in Microbiology
laboratory, St.Mary’s college,
Thrissur).
o Were maintained on
sabourauds dextrose agar
(SDA) slants.

6. 1.Detection of Tannins
By Ferric chloride test –
extract + distilled water
(5mg) (5ml)
mixture
neutral 5%
feCl3 soln
added
blue green color
(presence of tanin)
2.Detection of Phenols
By Lead acetate test –
extract + distilled water
(5mg) (5ml)
10% Pb acetate
soln (3ml) added
bulky white precipitates
(presence of phenols)

7. 3. Detection of flavanoids
The chloroform extract of the
solution
Treated with
NH4OH soln
Yellow fluorescence
(presence of flavanoids)
4. Detection of Terpenoids
Chloroform (2ml) + conc.
H2SO4
added carefully to
0.5ml of extract
red brown color at the
interface
(presence of terpenoid)

8. 5. Detection of Quinone
Conc. H2SO4 (1ml)
added to
plant extract (1ml)
Red colour
(presence of Quinones)
6. Detection of steroids and
phytosteroids
Plant exract + chloroform
(0.5 ml) (0.5 ml)
added few drops of
conc. H2SO4
brown ring / bluish- brown
ring
(steroids) (phytosteroids)

9. 7. Detection of carbohydrate
Molisch’s test –
Molisch’s reagent + extract solution
(2 drops) (1 ml)
mixed well
Inclined the tube
Added conc. H2SO4(1ml) along the
sides of the tube
Reddish violet ring at the junction of
two liquids
(presence of carbohydrate)
8. Detection of Proteins
The extract + distilled water
(10ml)
filtered through
Whattmann No.1 filter paper
filtrate
Used to tests for proteins and
aminoacids

10. 8.a) Biuret test –
Filtrate + 2% CUSO4 soln
(aliquot) (a drop)
added
95% ethanol(1ml)
added
excess of potassium
hydroxide pellets
The pink color in ethanol layer
(presence of proteins)
8.b) Detection of aminoacids
Ninhydrin test –
Ninhydrin solution + Filtrate
(2 drops) (2 ml)
Purple colour
(presence of amino acids)

11. 9. Detection of phytosterols
Libermann-Burchard’s test –
Extract +dry chloroform
Dissolved in a dry test tube
Acetic anhydride (several drops) was added
Conc.H2SO4 (2 drops) was added
Mixed carefully
After the reaction finished
Cholesterol conc. was measured using spectrophotometry

12. Method : Well diffusion Assay
Procedure :-
 Swabbing
 Short incubation
 Wells cutting
 100μg, 200μg, 300μg and 400μg of 0.5g leaf extract
prepared in 1000ml of methanol – loaded.
 Centre well- 100μl methanol, control.
 Incubation
 Zone of inhibition diameter - recorded.

13. 1)PHYTOCHEMICAL SCREENING
SL.NO. PHYTOCHEMICALS OBSERVATION INFERENCE
1 Tanin blue green color Present
2 Phenol bulky white
precipitate
Present
3 Flavanoid yellow fluorescence Present
4 Terpenoids red brown colour at
the interface
Present
5 Quinone red colour Present

14. SL.NO PHYTOCHEMICALS OBSERVATION INFERENCE
6 Steroid &
phytosteroid
brown and bluish
brown ring
Present
7 Carbohydrate reddish violet ring Present
8 Protein pink colour Present
9 Amino acid purple colour Present
10 Phytosterols No deep green colour Absent
Phytochemical compounds in the methanolic extract of Murraya koiengii leaves

15. Sl.
No.
TEST
MICROORGANISM
ZONE OF
INHIBITION
1 Staphylococcus Present
2 Bacillus Present
3 Proteus Present
4 Escherichia coli Absent
5 Pseudomonas Present
6 Klebsiella Absent
ZONE OF INHIBITION (mm) FOR DIFFERENT QUANTITY OF
EXTRACT
100μg 200 μg 300 μg 400 μg
20mm 25 mm 33 mm 40 mm
_ 14mm 16mm 21mm
_ 14mm 25mm 34mm
_ _ _ _
_ 20mm 25mm 30mm
_ _ _ _
Antibacterial efficiency of methanolic extracts of Murraya koiengii leaves.

16. Staphylococcus species
inhibited by methanolic
extracts of Murraya
koiengii leaves.
 The three Gram positive
bacteria showed
sensitivity against the
methanolic extract
 Staphylococcus sp.,
emerged as the most
susceptible.
 Among the fungal strains,
none of the tested ones
showed considerable
inhibition by the
methanolic extract.

17.  So, Murraya koenigii is a common remedy among the
various ayurvedic practitioners for treatment of
diversity of ailments.
 So, it is used in foods.
Hippocrates, the father of medicine proclaimed
“Let food be thy medicine and medicine be thy food”.

18. THANK
YOU