Comparison of different

extraction methods to
study the antioxidant activity
of Centella asiatica

Name: Tharshini R.Ramays

ID: 1107162002
Supervisor: Ms Shamala Marimuthu
  Is a prostrate, faintly aromatic, stoloniferous, glabrous, striated

 Attains height up to 15cm (6 inches)

 Flourishes extensively in shady, marshy, damp and wet places. Eg.paddy

fields, river banks

 Consists of biochemical component i.e. Secondary metabolite

 Triterpenes of Centella asiatica consists of Asiatic acid, madecassic acid,

asiaticosside, madecassoside, brahmoside, brahmic acid, brahminoside,
isothankunisode, cenellicacid, centic acid

 High total phenolic content contributed by flavonoids such as quercetin,

kaempherol, catechin, rutin, apigenin, naringin

 Rich in Vitamin A, Vitamin B, Vitamin B₂, niacin, carotene, Vitamin C

Centella asiatica @
Indian Pennywort
Literature review
Centella asiatica - taxonomy classification
• Treating skin problems
• To heal wounds
• For revitalizing the brain and nerve cell
• Consist of bioactive compounds
• Good effect on antibacterial and fungicidal activity against
bacteria and fungi
• Promote collagen synthesis
• Promote fibroblast proliferation
• Antioxidant, anti-inflammatory
• Memory enhancing
Antioxidant
activity
 Prevent oxidation of other molecules
 Oxidation of molecule result in free radical
 Free radical is unstable atom seek for electron
and become pair
 This damage the other cell and leads to other
disease


Antioxidant in synthetic and natural
Synthetic antioxidant is not safe
 The carcinogenic properties leads to liver damage
 Natural source of antioxidant is more developed
 Plants are good source of antioxidant
 Best way of treat disease through traditional medicine
 Centella asiatica treats fever, jaundice, dysentery,
diarrhea, mental illness
 Problem Statement
Problem statement
o Recently, the demand for natural antioxidant has increase, due to consumers health concerns about safety of
synthetic antioxidants. This prompted the evaluation of plants as a source of potential therapeutic agents with
antioxidant activity based on their medicinal uses.

Significance
o This study is designed to compare and identify the extraction method which gives the greatest yield and the
extract obtained is further used to test the antioxidant activity of Centella asiatica leave by carrying out DPPH
free radical scavenging activity and reducing power assay.
 Research OBJECTIVES
• To extract Centella asiatica leaves using soxhlet, maceration and ultrasound-assisted
extraction method

• To compare the yield of different extraction method which are soxhlet, maceration and
ultrasound – assisted extraction method

• To identify the phytochemicals properties of Centella asiatica by conducting thin layer

chromatography (TLC)

• To analyze the antioxidant properties of Centella asiatica leaves using DPPH free radical
scavenging activity assay and reducing power assay
Methodology
Extraction Phytochemical Antioxidant
Sample analysis assay
collection
•Soxhlet
•Maceration Thin layer
chromatography •DPPH free radical
•Ultrasound-
assisted scavenging activity
•Reducing power assay

Figure 1.0 shows the flow chart of methods that will be carry out in this study
1.0) Sample collection (Dukic, 2017)
Plant material will be bought from local market.

 Centella asiatica will be washed with running tap water and

rinsed with distilled water.

Then, cleaned plant material will stored in the 50℃ controlled

oven to dry for 3 days.

The dried plant material will be grinded using mortar and

pestle.

 The grinded powder will be stored for further use.

2.0) Extraction
2.1) Soxhlet Extraction (Dukic, 2017)
10g of grinded sample will be placed in the Soxhlet
apparatus

Extraction will be carry out for 8 hrs using 96% ethanol

Extract will be filtered through filter

paper and evaporated under vacuum and dried at 60℃

The dried extract will be stored in dark glass

bottle at 4℃
2.2) Maceration (Dukic, 2017)
2. The extraction process will
be carried out under laboratory
condition at 22℃ in a
sheltered dry place for seven
days with occasional shaking

3. Extract will be filtered

through extract paper

5. The dried extract will be

stored in a dark glass bottle at
4℃

1. Plant sample(10g) will be

extracted using 96% ethanol
(100ml) 4. The filterate will be concentrated into
dry mass by rotary evaporater under
vacuum dried at 60℃
2.3) Ultrasound-assisted extraction (UAE)
(Dukic, 2017)
Sample (10g) will be placed in
volumetric flask

100ml of (96% ethanol)

solvent will be added

Mixture will be sonicated

for 30min –frequency 35kHz
-ultrasound power 90% (216W)
 3.0) Phytochemical analysis
3.1) Thin Layer chromatography (Gujetti, 2013)
• Silica gel 6OF254, 7x6 cm will be used where the marking will be done with soft
pencil
• Glass Capillary will be used to spot the sample for TLC
Sample volume : 1micro-litre
Capillary distance: 1cm
Solvent system : Hexane : Acetic acid (9:1)
• After the run, plates dried and sprayed with freshly prepared iodine reagent to
detect the band on TLC plate.
• The movement of active compound is expressed by retention factor (Rf)
 4.0) Antioxidant Assay
4.0) Antioxidant assay
4.1) DPPH free radical scavenging activity
4.1) DPPH free radical
assay scavenging
(Sugunabai, 2015)
activity assay (Sugunabai, 2015) The mixture will be shaken vigorously and
allow to stand in dark for 30min.
2ml of tested sample and positive
controls will be mixed with 2ml of
DPPH solution

The absorbance at 517nm will be

measured with spectrophotometer
and all the test is repeated in
triplicate
DPPH free radical
scavenging activity will be
calculated using
4.2) Reducing power assay (Sugunabai, 2015)
FRAP Reagent included
1. 400µl sample solution will be added to 3ml freshly o 300mM acetate buffer
o 10mM TPTZ solution in
prepared FRAP reagent 40mM HCl
o 20mM FeCl₃∙6H₂O solution
2. This mixture will be incubated for 30min at 37℃ at Ratio : (10 :1 :1)
water bath in the dark
3. The absorbance of sample will be measured at 593nm
using spectrophotometer
4. Trolox will be used as standard solution.
5. FRAP result expressed in micromole trolox equivalent
per g of dry weight. (µmol eq. trolox/g)
Expected outcome
• Extraction method: Soxhlet extraction method is expected to give the higher yield
compared to two other method which is maceration and ultrasound-assisted extraction.

• Thin layer chromatography: Rf Values (Sanjay, 2013)

- Flavonoids: ±0.75, Alkaloids: ±0.50, Terpenoids: ± 0.70

• Antioxidant activity assay: (Rahman, 2013)

Extracts 96% Ethanol extract

Reducing power (AE) 40.37±0.73

DPPH scavenging Activity (IC50) 35.56±1.24

 TABLE OF RESEARCH
ACTIVITIES
Time Interval
Project Activities
W1 W2 W3 W4 W5 W6 W7 W8 W9 W10 W11 W12

Literature Review

Preparation

Extraction method

Antioxidant analysis

Thesis writing
 References
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