This is an Engg Biotechnology project based on medicinal plant i.e singapore cherry or jamaican cherry tree (scientific name Muntingia calabure ), we did in 2013 in GMIT college Davangere, karanataka, India. i have complete project detail what we did..,

1. EVALUATION OF ANTIOXIDANT AND
ANTIMICROBIAL ACTIVITY OF LEAVES AND
FRUITS EXTRACT OF MUNTINGIA CALABURA
Submitted by: Guide:
Harish Kumar Ms. Triveni T G
Manjunath G Msc.MTech
Manjunatha P A Dept. of BT
Yashavantagouda S N G M I T College
Davangere, Karanataka

2. INTRODUCTION
1. India has one of the oldest, richest and most diverse cultural traditions
called folk traditions associated with the use of medicinal herbs and it is
still a living tradition in India.
2. Various herbal medicines derived from plant extracts are being used in
the treatment of a wide variety of clinical diseases
3. Plant secondary metabolites have provided an important source of drugs
since ancient times and now around half of the practical drugs used are
derived from natural sources
4. There is a need for research and developmental work in herbal medicine
because apart from the social and economic benefits, it has become a
persistent aspect of present day health care in developing countries

3. MUNTINGIA CALABURA
1. It is also called as Singapore cherry, Jamaica Cherry, Strawberry tree
& Jam tree
2. Fast growing fruit tree, Height: 7 to 12 m tall & Evergreen tree
3. Leaves are evergreen, ovate, long pointed at the apex, oblique at the
base and covered with tiny sticky hairs
4. It contain Small flowers with 5 green sepals and 5 white petals and
many prominent yellow stamens and slightly malodorous
5. Fruits are round 1-1.25 cm wide, with red or sometimes yellow,
smooth, thin, tender skin Light-brown, soft, juicy pulp, with very
sweet, musky, fig-like, fruits are edible
6. Fruits contain hundreds of tiny seeds which are minute, yellowish too
fine to be noticed in eating
7. Tree yields fruits all over the year

5.  Kingdom Plantae - Plants
 Subkingdom Tracheobionta - Vascular plants
 Superdivision Spermatophyta - Seed plants
 Division Magnoliophyta - Flowering plants
 Class Monocotyledonae- Dicotyledons
 Subclass Rosidae
 Order Malvales
 Family Muntinglaceae
 Genus Muntingia
 Species Calabura
CLASSIFICATION

6. AIMS AND OBJECTIVES
The present investigation is undertaken by utilizing the plant Muntingia
calabura with following objectives.
1. Extraction of the leaves and fruits with methanol.
2. Preliminary phytochemical analysis.
3. Find the Antioxidant property.
4. Find theAntibacterial activity & Antifungal activity.
5. Find the Anthelmintic activity.

7. 1. MATERIALS AND METHODS
Collection of material and Extraction procedure
– Collect the material (fruit and leaf)
– Wash
– Material Grinded using methanol as solvent
– Allow incubation for 1 week
– Filtration
– Dry the filtrate in room temperature
– Collect the sample and store

8. 1. Carbohydrates Molisch's test
2. Reducing Sugar Felhing's Test
3. Proteins Biuret test
4. Amino acids Ninhydrin Test
5. Test for Cardiac Glycosides .
a. Legal's test
b. Keller Killiani test
6. Test for anthraquinone Glycosides
a.Bomtrager s test
b.Modified Bomtranger's test
7. Test for Sapon in Glycosides
a. Foam test
8. Tests for flavonoids
a. Shinode's test
b. Extract + lead acetate
2. PHYTOCHEMICALANALYSIS

9. 9. Tests for alkaloids
a. Mayer's test
b. Wagner's Test
10. Tests for Tannis and Phenols
a. Fec13 solutions
b. Lead Acetate
c. Gelatin
d. Acetic acid
e. Potassium dichromate
f. dil Iodine soln
11. Test for potassium flame test
12. Test for sulphate
13. Test for carbonate
14. Test for nitrate
[8-11]

10. 3. ANTIOXIDANT ACTIVITY
• Various antioxidant activity methods have been used in food and natural
products to monitor and compare.
• In the present research program , in vitro antioxidant activity was determined
using following methods.
 Total phenolic assay
 1, 1-Diphenyl 2-Picryl Hydrazyl (DPPH) radical scavenging activity
 Hydroxyl radical scavenging assay
 Reducing power assay
 Preparation of Stock Solutions of extracts and ascorbic acid

11. A.Total phenolic assay
The phenolic content in the extract was estimated by Folin-Ciocalteu method
20μl extract+ 1.58ml water + 100μl of FC reagent
mix
Incubate for 8min at room temprature
Add 300μl of sodium carbonate solution
mix
solution was maintained at 200C for 2hours
Read the absorbance of each solution at 725nm
Plot a graph absorbance versus concentration.

12. B. 1, 1-Diphenyl 2-picryl Hydrazyl (DPPH) radical
scavenging assay
• Ascorbic acid was used as the standard control
1ml extract + 4ml of methanolic solution of DPPH
incubation at room temperature for 30minutes
record the absorbance at 517nm
• inhibition ratio was calculated using the formula,
• Plot a graph inhibition ratio verses concentration

13. C. Hydroxyl radical scavenging assay
 The scavenging activity for hydroxyl radical was measured by fenton
reaction.
1mM FeCl3 + 1mM 1,10-phenanthroline + 0.2M-phosphate
buffer (pH 7.8) + 0.17M H2O2 + extract at various conc.
Incubation at room temp for 5min
The absorbance was measured at 560nm.
 The hydroxy radical scavenging assay activity was calculate by the equation
i.e. % Inhibition = [ (A0-A) / A0 ] × 100
where, A0 = absorbance of the control (without samples)
A = absorbance of test samples.
mix

14. D. Reducing power assay
 The reducing power of the extract was determined by the method of
Oyaizu with slight modification.
The extract at various conc + 0.2M-phosphate buffer (pH 6.6)
+ 1% potassium hexacyanoferrate (k3 Fe(CN)6)
in water bath for 20min.
Then the reaction stopped by adding 10% trichloroacetic acid (TCA) soln.
The supernatant was mixed with distl water and 1% ferric chloride soln.
The absorbance of the reducing power assay was measured at 700nm.
Left for 10min
Centrifuge at 800rpm for 10min
Mix and Incubate at 50◦c

15.  Antibacterial activities of plant extracts are tested by agar well diffusion
method.
 The microorganisms used are Escherichia coIi, Bacillus subtillus,
Pseudomonas aerogenosa, Bacillus cereus. [15]
4. ANTIBACTERIALACTIVITY
Procedure :
Molten nutrient agar media is poured into sterile Petri plates.
kept for solidification
bacterial culture swab inoculated on to the surface medium using sterile
cotton swab.
Using a sterile cork-borer of 6 mm diameter, four holes were made
in to the set agar in Petri dishes containing the bacterial culture.

16. The wells are filled with varied concentrations of extract
Standard antibiotic Ciprofloxacin (5 mg/ml) was used as reference
or positive control.
Incubated for 24hrs at 370C of after loading of the extracts.
The plates are observed for clear zone formation around the well.
 Antimicrobial activities are expressed in millimetre

17.  Antifungal activities of plant extracts are tested by potato dextrose agar well
diffusion method. [15]
 The microorganisms used are T viride, cladospora, candida, and A.niger.
 Preparation of different concentration of fruit and leaf extract of Muntingia
calabura for antifungal screening in DMSO solution.
ANTIFUNGALACTIVITY
Procedure :
Media is poured into sterile Petri plates
fungal culture swab inoculated on to the surface medium using
sterile cotton swab.
Using a sterile cork-borer of 6 mm diameter, four holes were made
in to the set agar in Petri dishes containing the fungal culture.

18. The wells is filled with varied concentrations of extract
Standard antibiotic Fluconazole (5 mg/ml) was used as reference
or positive control.
Incubated for 72hrs at 370C of after loading of the extracts.
The plates are observed for clear zone formation around the well.
 Antimicrobial activities are expressed in millimetre.

19. 5. ANTHELMINTHIC ACTIVITY
• Five serial suspensions of extracts are prepared in soluble solvent ranging
from 2% to 10 % (20mg/ml to 100mg/ml).
• The standard reference drug Albendazole is prepared at 2%.
• Thus a total of twelve groups comprising five tests for each extract (leaf and
fruit extract of Muntingia calabura) and one negative control (Solvent) and
one positive control (Albendazole) are subjected to evaluation of
anthelmintic property.
• Each group consists of four approximately equal sized earthworms, which
are released in to a large Petri plate containing 50ml of each suspension.
• Observations are made for the time taken to paralyze, and death of
individual worms. [16]

20. RESULTS AND DISCUSSION
Qualitative analysis of phytochemicals of muntingia calabura of leaves and fruits
SI.
No.
Tests Leaves
(extract)
Fruits
(extract)
1 Carbohydrates Molisch's test
+ +
2
Reducing Sugar Felhing's Test
+ +
3 Proteins Biuret test - -
4 Aminoacids Ninhydrin Test -
+
5 Test for Cardiac Glycosides .
a. Legal's test
+ +
b. Keller Killiani test
- +
6 Test for anthraquinone Glycosides
a.Bomtrager s test - +
b.Modified Bomtranger's test
+
+
7 Test for Sapon in Glycosides
a. Foam test
+ -

21. 8 Tests for flavonoids
a. Shinode's test
- -
b. Extract + lead acetate +
+
9
Tests for alkaloids
a. Mayer's test + +
b. Wagner's Test
+ +
10 Tests for Tannis and Phenols
a. Fec13 solutions + +
b. Lead Acetate - -
c. Gelatin + +
d. Acetic acid - -
11 Test for potassium flame test + +
Test for sulphate . - -
Test for carbonate
- -
Test for nitrate
- -

22. Con. of Galic
acid (mg/lit)
Vol. of Galic
acid (µl)
Distl. Water
(ml)
FC reagent
(µl)
Shakefor3mins
Na2co3 (µl)
2hrsforincubation
O.D. at 725nm
0 20 1.58 100 300 -
50 20 1.58 100 300 0.44
100 20 1.58 100 300 0.66
150 20 1.58 100 300 0.81
200 20 1.58 100 300 1.01
250 20 1.58 100 300 1.24
300 20 1.58 100 300 1.48
500 20 1.58 100 300 1.86
Total phenolic assay of Muntingia calabura of leaves and fruits
Fruit – 0.7310
Leaf – 0.8997
ANTIOXIDANT PROPERTY

23. Conc.(mg/lit) Ascorbic acid (std.) Leaf Fruit
00 - - -
50 19.86 63.54 -1.44
100 50.33 82.49 5.48
150 70.73 84.69 11.67
200 84.56 85.60 36.34
250 94.12 86.94 40.29
DPPH scavanging assay of Muntingia calabura of leaves and fruits

24. Conc.(mg/lit) Ascorbic acid (std.) Leaf Fruit
00 - - -
50 0.228 0.283 0.42
100 0.19 0.233 0.419
150 0.184 0.205 0.35
200 0.146 0.195 0.261
250 0.123 0.179 0.167
Hydroxy radical scavanging assay of Muntingia calabura of leaves and fruits
Hydroxy radical scavanging Assay
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0μg 50μg 100μg 150μg 200μg 250μg
Concentraion in microgram
Std
leaf
fruit

25. Concentration (mg/lit) Ascorbic acid
(std.)
Leaf Fruit
00 - - -
50 1.521 0.41 0.30
100 2.321 1.42 0.39
150 2.869 2.61 0.54
200 2.992 2.99 0.82
250 3.521 3.20 1.27
Reducing power assay of Muntingia calabura of leaves and fruits
Reducing Power assay
0
0.5
1
1.5
2
2.5
3
3.5
4
0μg 50μg 100μg 150μg 200μg 250μg
Concentration in μg
OpticalDensistyat700nm
Std
leaf
fruit

26. ANTHELMINTIC ACTIVITY
The paralysis time for Muntingia calabura extract of leaves and fruits
Paralysis time
Conc. Std. (in min) Leaf (in min) Fruit (in min)
0%
2% 10.52 12.4 37.1
4% 11.22 30
6% 10.52 21.5
8% 9.42 15.4
10% 7.59 13.18
Anthelimentic-Paralysis
0
5
10
15
20
25
30
35
40
0% 2% 4% 6% 8% 10%
concentration
Paralysis(inMin)
std (2%)
Leaf
Fruit

27. ANTHELMINTIC ACTIVITY
Death time
Conc. Std. (in min) Leaf (in min) Fruit (in min)
0%
2% 14.52 14.3 55.56
4% 13.43 53.53
6% 13.07 46.9
8% 11.58 44.55
10% 10.23 42.02
Anthelimentic-Death
0
10
20
30
40
50
60
0% 2% 4% 6% 8% 10%
Concentraion
Death(inMin)
std (2%)
Leaf
Fruit
The death time for Muntingia calabura extract of leaves and fruits

28. ANTIBACTERIALACTIVITY
Escherichia coli Bacillus subtillus Pseudomonas aerogenosa Bacillus cereus
 Fig 1. Shows inhibitory zone of different bacteria for the Muntingia calabura extract

29. Table 3. The different inhibitory zone at
different concentrations of fruit and leaf
extracts with comparing to E.coli
Leaf Fruit
Con 2.33 2.33
Std 14.67 14.67
2% 5.5 3.583
4% 7.33 5
6% 8.58 6.56
8% 9.5 7.33
Leaf Fruit
Con 1 1
Std 14.67 14.67
2% 7.33 4.375
4% 9.08 8.25
6% 9.58 8.625
8% 10.16 9.5
Antibacterial activity against B.
substilis
0
2
4
6
8
10
12
14
16
Leaf Fruit
Axis Title
AxisTitle
Con
Std
2%
4%
6%
8%
Antibacterial activity against E.coli
0
2
4
6
8
10
12
14
16
Leaf Fruit
Type of sample
zoneofinhibitionin
mm
Con
Std
2%
4%
6%
8%
Figure 3. Antibacterial activity against E.coli
Table 2. The different inhibitory zone at
different concentrations of fruit and leaf
extracts with comparing to B.substillis.
2. Bacillus substillis 3. Escherichia coli
Figure 2. Antibacterial activity against B.substillis

30. Leaf Fruit
Con 2.33 2.33
Std 14.25 14.25
2% 7 4.1
4% 8.33 5.25
6% 10.26 7.83
8% 11.08 9
Leaf Fruit
Con 2.67 2.67
Std 16.5 16.5
2% 6.5 3.91
4% 8.16 6.08
6% 9.5 7.25
8% 10.25 8.33
Antibacterial activity againstP .aueroginosa
0
2
4
6
8
10
12
14
16
Leaf Fruit
Type of Sample
zoneofinhibitioninmm
Con
Std
2%
4%
6%
8%
Antibacterial activity against S.aureus
0
2
4
6
8
10
12
14
16
18
Leaf Fruit
Type of sample
zoneofinhibitioninmm
Con
Std
2%
4%
6%
8%
Table 4. Different inhibitory zone at different
concentrations of fruit and leaf extracts with
comparing to P.auerogenosa
Table 5. Different inhibitory zone at
different concentrations of fruit and leaf
extracts with comparing to B.cereus
Figure 5. Antibacterial activity against B.cereusFigure 4. Antibacterial activity against P.aueroginosa
4. Pseudomonas aueroginosa 5. Bacillus cereus

31. Fruit
Inhibition Zone in mm
B.subtilis E.coli P.auerogenos
a
B.cereus
Control
2.33 1 2.33 2.67
Std(Ciprofloxin)
14.67 14.67 14.25 16.5
2%
3.583 4.375 4.1 3.91
4%
5 8.25 5.25 6.08
6%
6.56 8.625 7.83 7.25
8%
7.33 9.5 9 8.33
Leaf
Inhibition Zone in mm
B.subtilis E.coli P.auerogenos
a
B.cereus
Control 2.33 1 2.33 2.67
Std(Ciprofloxin) 14.67 14.67
14.25
16.5
2% 5.5 7.33 7 6.5
4% 7.33 9.08 8.33 8.16
6% 8.58 9.58 10.26 9.5
8% 9.5 10.16 11.08 10.25
Antibacterial activity of leaf extract
on different pathogens
0
5
10
15
20
B substilis Ecoli P aueroginosa S aureus
Names of Pathogenic genic bacteria
zoneofinhibition
inmm
Con
Std
2%
4%
6%
8%
Table 6. Antibacterial Activity of
Muntingia calabura of fruits
Antibacterial activity of Fruit extract
on different pathogens
0
5
10
15
20
B substilis Ecoli P aueroginosa B cereus
Names of Pathogenic bacteria
zo
ne
of
in
hi
bi
ti
o
n
in
m
m
Con
Std
2%
4%
6%
8%
Table 7. Antibacterial Activity of
Muntingia calabura of leaves

32. ANTIFUNGALACTIVITY
T. Viridae Cladospora Candida A. niger
Figure 1. Shows inhibitory zone of different fungi for the Muntingia calabura extract

33. Leaf Fruit
Con 1.48 1.48
Std 15.64 15.64
2% 7.75 5.47
4% 9.34 6.47
6% 12.67 9.58
8% 14.6 11.05
Leaf Fruit
Con 1.75 1.75
Std 13.33 13.33
2% 7.25 4.21
4% 9.5 5.25
6% 10.5 8
8% 11.5 8.25
Antifungal activity against
Candida albicans
0
5
10
15
20
Leaf Fruit
Type of sample
zoneofinhibition
inmm
Con
Std
5%
10%
15%
20%
2. T Viridae
Figure 2. Antifungal activity against T.viride.
Table 2. The different inhibitory zone at
different concentrations of fruit and leaf
extracts with comparing to T viride.
Figure 3. Antifungal activity against C. albicans
Table 3. The different inhibitory zone at
different concentrations of fruit and leaf
extracts with comparing to C.albicans
3. Candida

34. Leaf Fruit
Con 1.65 1.65
Std 16.33 16.33
2% 7.75 3
4% 9.75 4.5
6% 11 8.25
8% 15.25 10.75
Leaf Fruit
Con 2.3 2.3
Std 18.25 18.25
2% 8.25 4.5
4% 9.75 7.25
6% 11.25 8.75
8% 12.55 9.75
Antifungal activity against
Aspergellus niger
0
5
10
15
20
25
Leaf Fruit
Type of sample
zoneofinhibition
inmm
Con
Std
5%
10%
15%
20%
Antifungal activity against
Cladosopora sps.
0
5
10
15
20
Leaf Fruit
Type of sample
zoneofinhibition
inmm
Con
Std
5%
10%
15%
20%
4. Cladospora 5. Aspergellus niger
Table 4. The different inhibitory zone at
different concentrations of fruit and leaf
extracts with compared to Cladospora
Table 5. The different inhibitory zone at
different concentrations of fruit and leaf
extracts with comparing to A.niger
Figure 5. Antifungal activity against A.niger.Figure 4.Antifungal activity against cladospora

35. Fruit
Inhibition Zone in mm
T. viridae C. albicans cladospora A.niger
Control
1.48 1.75 1.65 2.3
Std(fluconozol)
15.64 13.33 16.33 18.25
5% 5.47 4.21 3 4.5
10% 6.47 5.25 4.5 7.25
15% 9.58 8 8.25 8.75
20% 11.05 8.25 10.75 9.75
Table 6. Antifungal activity of Mutingia calabura
of fruits.
Leaf
Inhibition Zone in mm
T. viridae C. albicans cladospora A.niger
Control
1.48 1.75 1.65 2.3
Std(fluconozol)
15.64 13.33 16.33 18.25
5% 7.75 7.25 7.75 8.25
10% 9.34 9.5 9.75 9.75
15% 12.67 10.5 11 11.25
20% 14.6 11.5 15.25 12.55
Table 7. Antifungal activity of Muntingia calabura
of leaf.
Antifungal activity of leaf extract on
different pathogens
0
5
10
15
20
25
T. viridae C. albicans cladospora A.niger
Names of Pathogenic Fungi
zoneofinhibition
inmm
Con
Std
5%
10%
15%
20%
Antifungal activity of fruit extract on
different pathogens
0
5
10
15
20
25
T. viridae C. albicans cladospora A.niger
Names of Pathogenic Fungi
zoneofinhibition
inmm
Con
Std
5%
10%
15%
20%

36. CONCLUSION
The clinical efficacy of many existing antibiotics is being treatened by
the emergence of multidrug-resistant pathogens. Plants due to their
medicinal values could be suitably exploited for preventing and curing
the diseases. The present study indicates the potential usefulness of
methanol extract Muntingia calabura in the treatment of pathogenic
diseases, helminthiasis and various other disesases. The plant extact
shows anthelminthic activity, antifungal activity and antibacterial
activity. The present work on invitro antibacterial, antifungal and
antihelmentic evaluation of muntingia extract forms a primary
platform for further pharmological studies and research.

37. Thomson, W.A.R., 1978. Medicines from the Earth. Maidenhead, United Kingdom.
McGraw-Hill Book Co.
1.Stockwell, C., 1988. Nature’s pharmacy. London, United Kingdom. Century
Hutchinson Ltd.
2.Newman, D.J., G.M. Cragg and K.M. Snader, 2000.The influence of natural products
upon drug discovery. Nat. Prod. Res., 17: 215-234.
3.Clifford, Terry (2003). Tibetan Buddhist Medicine and Psychiatry. 42. Motilal
Banarsidass Publications. ISBN 81-208-1784-2
4.Sharma, A. K. (2003). "Panchkarma Therapy in Ayurvedic Medicine". In Mishra,
Lakshmi Chandra. Scientific Basis for Ayurvedic Therapies. Boca Raton, FL: CRC
Press. p. 43. ISBN 0-8493-1366-X.
5.Srivastava, J., J. Lambert and N. Vietmeyer, 1996.Medicinal plants: An expanding
role in development.World Bank Technical Paper. No. 320.
6.Uniyal, S.K., K.N. Singh, P. Jamwal and B. Lal, 2006. T Traditional use of medicinal
plants among the tribal. communities of Chhota Bhangal, Western Himalayan J.
Ethnobiol. Ethnomed., 2: 1-14.
REFERENCE

38. 8. Harborne, J.B., 1984. Phytochemical methods: A guide to modern techniques
of plant analysis, 2 ed., Chapman & Hall, New York, p. 85.
9. Trease. G.E., and Evans, W.C., 1989. Pharmacognosy, 13th ed., ELBS
Publication, Delhi, PP. 171
10. P.A. Egwaikhide and 2C.E. Gimba, Analysis of the Phytochemical Content
and Anti-microbial Activity of Plectranthus glandulosis Whole Plant. Middle-
East Journal of Scientific Research 2 (3-4): 135-138, 2007
11. Mehta Kavit1, Patel B.N.1 and Jain B.K.2 Phytochemical analysis of leaf
extract of Phyllanthus fraternus, Research Journal of Recent Sciences, Vol.
2(ISC-2012),
12-15 (2013)
12. P. Mandal, T. K. Misra,1 and I. D. Singh1,Antioxidant Activity in the Extracts
of Two Edible Aroids Indian J Pharm Sci v.72(1); Jan-Feb 2010 PMC2883208
13. Dhalwal K, Deshpande YS, Purohit AP, Kadam SS. Evaluation of the
antioxidant activity of Sida cordifolia. J Pharm Biol. 2005;43:754–61
14. Hasan, CM. Begum, S.N. Illias, M. and Hussain, A. 1988. Antibacterial
activities on the leaves and stem bark of Cassia alata. Bangladesh J. Bot. 17(2):
135-139.
15. Bate-Smith EC., J Soc Bot, 58: 95-103,(1962)