HIMA HARIDASAN
Dept. Of Microbiology,
St. Mary’s college,
Thrissur- 680020,
University of Calicut
E-mail: [email protected]
Family : Rutaceae
Native to India.
Leaves - Indian diet to improve
appetite and digestion.
Deciduous to semi-evergreen
aromatic tree
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
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
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.
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)
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)
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)
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
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)
9. Detection of phytosterolsLibermann-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
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.
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
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
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.
Staphylococcus species inhibited by methanolicextracts of Murrayakoiengii 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.
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”.
THANK
YOU
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