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

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

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 tree3. Leaves are evergreen, ovate, long pointed at the apex, oblique at the

base and covered with tiny sticky hairs4. It contain Small flowers with 5 green sepals and 5 white petals and

many prominent yellow stamens and slightly malodorous5. 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

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

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. 

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

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. PHYTOCHEMICAL ANALYSIS

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]

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

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.

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

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

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

  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. ANTIBACTERIAL ACTIVITY

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. 

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

 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.

ANTIFUNGAL ACTIVITY

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. 

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.

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]            

                           

RESULTS AND DISCUSSIONQualitative analysis of phytochemicals of muntingia calabura of leaves and fruits

SI.

No.

Tests Leaves

(extract)

Fruits

(extract)

1 Carbohydrates Molisch's test+ +

2Reducing 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

+ -

8 Tests for flavonoids    

 a. Shinode's test

- -

 b. Extract + lead acetate +

+

9Tests 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

- -

Con. of Galic acid (mg/lit)

Vol. of Galic acid (µl)

Distl. Water (ml)

FC reagent (µl)

Shake for 3 mins

Na2co3 (µl) 2 hrs for

incubation

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

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

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

00.050.1

0.150.2

0.250.3

0.350.4

0.45

0μg 50μg 100μg 150μg 200μg 250μg

Concentraion in microgram

Std

leaf

fruit

 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

00.5

11.52

2.53

3.54

0μg 50μg 100μg 150μg 200μg 250μg

Concentration in μg

Opti

cal D

ensi

sty

at 7

00nm

Std

leaf 

fruit

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

0510

15202530

3540

0% 2% 4% 6% 8% 10%

concentration

Para

lysi

s (in

Min

)

std (2%)

Leaf 

Fruit

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

Dea

th (i

n M

in)

std (2%)

Leaf 

Fruit

The death time for Muntingia calabura extract of leaves and fruits

ANTIBACTERIAL ACTIVITY

Escherichia coli Bacillus subtillus Pseudomonas aerogenosa Bacillus cereus

Fig 1. Shows inhibitory zone of different bacteria for the Muntingia calabura extract

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

02

468

1012

1416

Leaf  Fruit

Axis Title

Axis

Title

Con

Std 

2%

4%

6%

8%

Antibacterial activity against E.coli

0246810121416

Leaf  Fruit

Type of sample

zone

of i

nhib

ition

in

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

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

68

10

12

14

16

Leaf  Fruit

Type of Sample

zone

of i

nhib

ition

in m

m

Con

Std 

2%

4%

6%

8%

Antibacterial activity against S.aureus

024681012141618

Leaf  Fruit

Type of sample

zone

of i

nhib

ition

in m

m

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

Fruit

Inhibition Zone in mm

B.subtilis E.coli P.auerogenosa

B.cereus

Control2.33 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.auerogenosa

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

zone

of i

nhib

ition

in

mm

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

zone of inhibition

in

m m

Con

Std 

2%

4%

6%

8%

Table 7. Antibacterial Activity of Muntingia calabura of leaves

ANTIFUNGAL ACTIVITY

T. Viridae Cladospora Candida A. niger

Figure 1. Shows inhibitory zone of different fungi for the Muntingia calabura extract

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

zone

of i

nhib

ition

in

mm

Con

Std 

5%

10%

15%

20%

2. T Viridae

Antifungal activity against Trichoderma viridae

0

5

10

15

20

Leaf  Fruit

Type of sample

zone

of i

nhib

ition

in

mm

Con

Std 

5%

10%

15%

20%

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

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

zone

of i

nhib

ition

in

mm

Con

Std 

5%

10%

15%

20%

Antifungal activity against Cladosopora sps.

0

5

10

15

20

Leaf  Fruit

Type of sample

zone

of i

nhib

ition

in

mm

Con

Std 

5%

10%

15%

20%

4. Cladospora 5. Aspergellus nigerTable 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

FruitInhibition Zone in mm

T. viridae C. albicans cladospora A.nigerControl

1.48 1.75 1.65 2.3Std(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.nigerControl

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

zone

of i

nhib

ition

in

mm

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

zone

of i

nhib

ition

in

mm

Con

Std 

5%

10%

15%

20%

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.

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 81-208-1784-24.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.

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