Is is a project proposal by which the antimicrobial and antioxidant property of Phyllanthus acidus will be explored that will open a new door for the innovation of new medicine.

1. Evaluation of
Antimicrobial and Antioxidant
Properties of Fruit Extract of
Phyllanthus acidus
PRESENTED BY
DR. SYEDA ZERIN IMAM
UNIT OF MICROBIOLOGY
FACULTY OF MEDICINE, AIMST UNIVERSITY
MALAYSIA
July 2016

2. Supervisor
Dr. Tahmina Monowar
Senior Lecturer
Unit of Microbiology
AIMST UNIVERSITY
Co-supervisors
1) Dr. Urmila Banik, FoM
2) Dr. Sridevi Chigurupati, FoP
Dr. Syeda Zerin Imam

3. BACKGROUND OF THE STUDY
Fruits are rich in various macro and micro nutrients which help to
maintain a sound health and prevent diseases. They have been used as
medication for various illnesses since the ancient time. Literature surveys
showed that Phyllanthus acidus known as Malay Gooseberry is a good
remedy for different types of ailments like emetic and purgative
(Lemmens et al., 1999), hypertension and respiratory (Sausa et al., 2007),
hepatoprotective (Lee et al., 2006), psoriasis (Burkill et al., 2002),anti-
diabetics. (Banik et al., 20 I 0), anti nociceptive (Catapan et al., 2000).
Poisoning, coughs, asthma and bronchitis, poulticing(bandaging) and
soles, cathartic(Laxative)(Caius et al., 2003), rehabilitation (Vongvanich et
al., 2000), addiction (Mahidol et al., 2002), liver tonic, laxative, urticaria,
eruptions & bronchial catarrh (Prasad D, 1986), sciatica, lumbago or
rheumatism, sudorific & gonorrhea, skin disorders (Morton et al., 1987).Dr. Syeda Zerin Imam

4. HYPOTHESIS
Phyllanthus acidus may have antimicrobial and antioxidant activity.
RESEARCH QUESTION
1. Does Phyllanthus acidus have antimicrobial property?
2. Does Phyllanthus acidus have antioxidant activity?
Dr. Syeda Zerin Imam

5. OBJECTIVE OF THE STUDY
General Objective:
To evaluate anti microbial and antioxidant property of Phyllanthus acidus
fruit extract.
Specific Objectives:
1. To evaluate antimicrobial property of Phyllanthus acidus fruit extract
using different solvents.
2. To evaluate antioxidant activity of Phyllanthus acidus fruit extract using
different solvents.
Dr. Syeda Zerin Imam

6. RESEARCH METHODOLOGY
• The antimicrobial activities of fruit extract of Phyllanthus acidus
will be tested in vitro against human pathogenic microorganisms
like:- ATCC strains of Staphylococcus aureus, Bacillus cereus, E coli,
Pseudomonas aeruginosa, Candida albicans by disc diffusion
method.
• Antioxidant potentiality of Phyllanthus acidus will be evaluated by
determining total flavonoid contents, ABTS assay and DPPH radical
scavenging activity.
Dr. Syeda Zerin Imam

7. Disc Diffusion Method
Flavonoid Content
DPPH Scavenging Activity ABTS Assay
Dr. Syeda Zerin Imam

8. PROBLEM STATEMENT
Recognizing the ongoing vulnerability to new disease threats from regions
with limited detection and response capacity it has become mandatory to
invent new drugs to fight the risk factors. There is an increased demand of
antimicrobial agents. Recent researches suggest that external
immunostimulants can induce immune responses against micro organisms
to prevent infections in immune-compromised conditions, such as aging,
cancers, sepsis, diabetes, etc (Goto et al., 2010). Along with that there is a
growing demand for natural antioxidants because of toxicological and
carcinogenic on animals (Amarowicz, 2000).
The plant, Phyllanthus acidus has been used traditionally for the treatment
of cathartic, emetic, coughs, hypertension, asthma and skin diseases. Due
to the high medicinal activity of Phyllanthus acidus. So it is suspected that it
contains active antimicrobial and antioxidant activity within itself. So it
should be investigated to explore the opportunities inside.
Dr. Syeda Zerin Imam

9. LITERATURE REVIEW
The Botanical Classification of the Plant
Kingdom Plantae
Division Mgnoliophyta
Class Mgnoliopsida
Order. Mglgiphiales
Family Phyllanthaceae
Tribe Phyllanthaceae
SubtribeFlueggeinae
Genus Phyllanthus
Species Phyllanthusacidus
Phyllanthus acidus
Dr. Syeda Zerin Imam

10. Phyllanthus acidus is a small, glabrous tree up to 10 m tall with
phyllanthoid branching, bark rough, grey, with prominent lenticels;
cataphylls not persistent, blackish-brown, their stipules triangular-ovate;
deciduous branch lets ascending, 20-52 cm long, with 25-40 leaves. Fruit
drupaceous(seed in side the fruit), oblate(flattened in the polar position),
1-1.5 cm x (1.2-)1.5-2(-2.5) cm when fresh, shallowly 6- or 8-lobed,
greenish yellow to creamy-white; flesh firm, sour with a hard, bony,
grooved stone containing 6-8 smooth seeds. Phyllanthus, the generic
name is derived from the Greek ‘phullon’-leaf and ‘anthos’-flowers from
the fact that members of this genus have flowers in dense clusters in leaf
axils. The tree prefers hot, humid tropical lowlands.
The mature sour fruits may be eaten fresh but usually they are sprinkled
with salt to neutralize the acidity. Used in cooking to flavor dishes, the
fruits are excellent raw materials for processing into pickle and sweetened
dried fruits; fruit juice is used in cold drinks and fruit to make vinegar.Dr. Syeda Zerin Imam

11. In Malaysia, ripe and unripe fruit are served as a relish, syrup or
sweet preserve. The fruits, combined with other fruits are used in
chutney or jam, because of their setting properties. Young leaves
are cooked as a vegetable in Indonesia, Thailand and India. The
tree is used as fuel wood. The wood is fairly hard, strong, tough
and durable if seasoned. It is used for utensils and other small
objects. The bark is used in India as a tanning agent.
Extract from the plant has shown nematicidal activity against the
pine wood nematode, Bursaphelenchusxylophilus. The juice of the
root bark is weakly poisonous.
Dr. Syeda Zerin Imam

12. Food value of P. acidus is
Moisture 91.90 g
Protein 0.155 g
Fat 0.52 g
Fiber 0.80 g
Ash 0.51 g
Calcium 5.40 mg
Phosphorus 17.90 mg
Iron 3.25 mg
Carotene 0.019 mg
Thiamine 0.025 mg
Riboflavin 0.013 mg
Niacin 0.292 mg
Ascorbic Acid 4.60 mg
Dr. Syeda Zerin Imam
Food Value of 100gm of edible portion
Food Value per 100 g of Edible PortionFood Value per 100 g of Edible PortionFood Value per 100 g of Edible PortionFood Value per 100 g of Edible PortionFood Value per 100 g of Edible PortionFood Value per 100 g of Edible Portion

13. But it has intense medicinal activity. The latex is credited with emetic
and purgative activity. In Indonesia the bark is heated with coconut oil
and spread on eruptions on feet and hands. An infusion of the root is
taken to alleviate asthma in Java. In Borneo, roots are used in the
treatment of psoriosis of the feet. A leaf decoction is applied to
urticaria, a decoction of the bark is used to treat bronchial catarrh in
Philippines. The fruit is used as a laxative in Myanmar. In India, the
fruits are taken as a liver tonic to enrich the blood.
From the biochemical research of the Phyllanthus acidus we could find
out a level of biomechanical parameters in the fruits of Phyllanthus
acidus. (M. Suriyavathana and P.Subha. 2011). Those are as follows:
Dr. Syeda Zerin Imam

14. Spice Carbohydrate
(mg/g)
Starch
(mg/g)
Protein
(mg/g)
Cholesterol
(mg/g)
Ascorbic
acid (mg/g)
Calcium
(mg/g)
Phosphoro
us
(mg/g)
Iron
(mg/g)
Phyllanthus
acidus
20.75±0.012 0.498±0.0
06
4.013±0.0
12
0.208±0.00
02
2.505±0.00
41
3.807±0.00
16
0.504±0.00
33
0.635±0.00
41
Values are mean ± S.D. , Statistical significance: p<0.01, p<0.005
In this research Phyllanthus acidus was recommended as a good source of
natural antioxidants.
Traditionally Phyllanthus acidus has been used for the remedy of various
diseases incorporated with infections and degeneration of cellular activity. So
it is an important source of antimicrobial and antioxidant medications to treat
the respective.
Dr. Syeda Zerin Imam

15. FLOW CHART OF THE
PROJECT
ISOLATION OF THE SELECTED
BACTERIAS
MAINTANANCE OF THE BACTERIA
USING NUTRIENT AGAR
EXTRACT PREPARATION OF THE
FRUIT OF PHYLLANTHUS ACIDUS
PREPARATION OF THE
EXPERIMENT DISC
DISC DIFFUSION TEST
DETERMINATION OF MIC
DETERMINATION OF
TOTAL FLAVONOIDS
CONTENTS
DPPH RADICAL
SCAVANGING
ACTIVITY
ABTS
STATISTICAL
ANALYSIS
ANTIMICROBIAL
STUDY
ANTIOXIDANT
STUDY

16. METHODS
Sample collection:
Fruits of Phyllanthus acidus will be collected locally.
ANTI MICROBIAL TEST
Bacterial isolates and their maintenance:
ATCC strains of Staphylococcus aureus, Bacillus cereus, E coli, Pseudomonas
aeruginosa, Candida albicans will be collected from Laboratory. All the
bacterial strains will be cultured in nutrient agar and incubated at 37oC for
24 h and Candida albicans will be cultured in Sabourauds Dextrose Agar and
will be incubated in 30oC. Mueller Hinton Agar (MHA) will be used to
determine the MIC and the antibacterial activity of the extract.
.
Dr. Syeda Zerin Imam

17. Extracts to be used
Aqueous, ethanol, ethyl acetate , Di ethyl ether and methanol extracts
will be used for the purpose.
Extract
Preparation
Disc
Preparation
Disc Diffusion
test
Determination
of MIC
Statistical
Analysis
Dr. Syeda Zerin Imam

18. ANTI OXIDANT TEST
Determination of total flavonoids content
Kumaran and Karunakaran method will be used to determine the
total flavonoids content (Kumuran et al, 2007).
DPPH radical scavenging activity
DPPH radical scavenging activity will be investigated according to the
method of Sreejayan and Rao, (Sreejayan N. et al., 1996).
ABTS Assay
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) or ABTS is
chemical compound used to observe the reaction kinetics of
specific enzymes. For ABTS assay, the procedure followed the
method of Arnao et al. (2001) with some modifications.
Dr. Syeda Zerin Imam

19. EXPECTED RESULT/ BENEFITS
Identification of antimicrobial sensitivity of the fruit of Phyllanthus acidus
and
observing the free radical activity to expolre the antioxidant activity of the fruit
Dr. Syeda Zerin Imam
SPECIFIC OR POTENTIAL APPLICATIONS
1. Identification of medically advantageous fruit among the bulk of available fruit in the
market.
2. People who like sour fruits Phyllanthus acidus can be an advantageous fruit for them.
3. Due to the medicinal activity of Phyllanthus acidus, it can be produce d for
pharmacological purpose and supplied to the market commercially.

20. Dr. Syeda Zerin Imam

21. Stages 2016 2017
1 2 3 4 5 6 7 8 9 10 11 12
May Jun July Aug Sep Oct Nov Dec Jan Feb Mar Apr
Project Design
Literature update
Sample Collection
Extract preparation
Antimicrobial Study
Antioxidant Activity
Data Analysis
Thesis Writing and Submission
Thesis Defense
GANTT CHART
Dr. Syeda Zerin Imam

22. Dr. Syeda Zerin Imam