Antioxidant Activity and Irritation Test of Acanthus ilicifolius Linn. Extract
Theeraya Krisdaphong1 Ampa Jimtaisong1,* Surapol Natakankitkul2 Narunan Wuttisin1
School of Cosmetic Science, Mae Fah Luang University
2
Faculty of Pharmacy, Chiang Mai University
*
Corresponding Author Email : ampa@mfu.ac.th Phone +66 53916843 Fax +66 5391 6831
1
ABSTRACT
Acanthus ilicifolius Linn. was a tropical plant commonly found in Thailand yet it has been known
as a beneficial resource for antioxidant activity. This study aimed to evaluate antioxidant activity and
irritation of ethanolic A. ilicifolius leave extract. The major antioxidant compounds in the extract were
screened by thin layer chromatography (TLC) and measured by UV-spectrophotometer, which revealed
14.07% polyphenol, % 8.07flavonoid and % 20.96triterpenoid. The obtained crude extract indicated
satisfying free radical scavenging activity with IC50of 68.67 μg/ml in comparison to gallic acid (2.60
μg/ml), BHT (26.00 μg/ml) and quarcetin (6.12 μg/ml). A chromatogram obtained from High
Performance Liquid Chromatography (HPLC) illustrated the content of trigonelline at Rt of 6.321
minutes. The A. ilicifolius extract also presented no irritating effect upon twenty subjects after a 24 hours
single patch test measured at 30 minutes and 48 hours, the extract is therefore considered as safenatural
ingredient. Further study on the anti-inflammatory effect of the extract shall be considered.
Keywords: Acanthus ilicifolius/Polyphenol/Flavonoid/Triterpenoid/Antioxidant/Irritation test
INTRODUCTION
The development of traditional knowledge to suit new lifestyle would result huge benefit upon
livings. The global consumers were aware on the benefits of the natural products. The global market value
of Herbal/Traditional Products reached US$ 30 billion in the year 2010. Thus, the development of Thai
natural product industry to reach international standard would widen the current market share and increase
national income in the form of value-added products.
A. ilicifolius has been well known in a Thai traditional medicine as a treatment to eczema, which
is a form of dermatitis or inflammation of the epidermis layer of the skin. Eczema includes dryness and
recurring skin rashes that is observed as redness, swelling, itching and dryness. Thus, utilizing A.
ilicifolius as a cosmetic ingredient would be very beneficial in cosmetic industry refer to its antioxidant or
anti-inflammatory properties in the traditional data and its concept of natural origin in marketing term. In
order to safely utilize the A. ilicifolius in cosmetic application, this independent research focus on the
extraction of the A. ilicifolius by using ethanol as a solvent. The ethanolic fraction of the extract will be
tested on the antioxidant activities and further on to the anti-inflammatory activities.
MATERIALS
1. Chemical Reagents and Standards
1.1 Aluminium Chloride
1.2 Ethanol
1.3 Folin – Ciocalteu’s phenol reagent
1.4 Gallic acid
1.5 Methanol
1.6 2,2-diphenyl-1-picrylhydrazyl (DPPH)
1.7 Potassium acetate
1.8 Quercetin
1.9 Sodium Carbonate
EXPERIMENTAL
This study aimed to evaluate antioxidant activity and irritation of ethanolic A. ilicifolius leave
extract. The major antioxidant compounds in the extract were screened by thin layer chromatography
(TLC) and measure by UV-spectrophotometer, and then determination of total phenolic content, total
flavonoids, triterpenoid, free radical scavenging activity. A chromatogram obtained from High
Performance Liquid Chromatography (HPLC) and irritation test by covered patch test.
RESULTS
1. The Thin Layer Chromatography (TLC) Screening Test
1.1 The Screening test of Total Phenolic Content
The TLC plate from Toluene: Ethyl acetate system was observed. The unclear
blue spot, which indicate the present of phenolic compound, was observed under UV
visible light at 365 nm.
1.2 The Screening test of Total Flavonoids
The TLC plate from Ethyl acetate: Methanol: Water system was observed.
The yellowish orange spot, which indicate the present of flavonoid, was observed under
UV visible light at 365 nm.
1.3 The Screening test of Saponin
The TLC plate from Chlorofrom: Methanol: DI water system was observed.
A light blue spot, which indicate the present of saponin, was visibly observed.
2. Determination of Total Phenolic Content
The absorbance of A. ilicifolius extract are 0.3268 and 0.3268, the content of total
phenolic compound present of gallic acid equivalent in the A. ilicifolius extract was 14. 07%
phenolic content of dry crude. (Waterman & Mole, 1994)
3. Determination of Flavonoids
The absorbance of A. ilicifolius extract are 0.43120 and 0.4300, the content of total
flavonoids present of Quercetin equivalent in the A. ilicifolius extract was % 8.07 flavonoids of
dry crude.
4. Determination of Tritepenoids
The absorbance of A. ilicifolius extract were 0.3770 and 0.3503, when referred to the
standard curve (Figure 4.7), the content of total triterpenoid present of saponin equivalent in the
A. ilicifolius extract was 20. 96% triterpenoid of dry crude.
5. Antioxidant Activity of Acanthus ilicifolius Leaves Extract
Antioxidant activity of crude A. ilicifolius extract was considered as relatively satisfying
with the IC50 value of 68.67 μg/ml whereas the IC50 values of standards were 26.00 μg/ml for
BHT, 2.60 μg/ml for gallic acid and 6.12 μg/ml for quercetin.
6. High Performance Liquid Chromatography (HPLC)
The chromatogram of A. ilicifolius extracti llustrated the content of trigonelline content peak
presented at Rt of 6 min.
7. Irritation Test by Covered Patch Test
The readings at 30 minutes and 48 hours after removal of the patch produced
the following M.I.I. values :
CONCLUSION
A. ilicifolius leaves were proven in containing various beneficial compounds for antioxidant and
anti-inflammatory effects. The analysis of thin layer chromatography (TLC) and UVspectrophotometer
illustrated the content of major antioxidant compounds in the extract including % 14.07 of polyphenol, of
% 8.07flavonoid. The obtained crude extract showed satisfying free radical scavenging activity with IC50
equivalent of 68.67 μg/ml in comparison to gallic acid (2.60 μg/ml), quercetin (6.12 μg/ml) and BHT
(26.00 μg/ml) standards. Anti-inflammatory effect of the extract shall be further studied due to the high
content of % 20.96triterpenoids. Moreover, HPLC chromatogram of the extract also suggests the present
of trigonelline, which contains several therapeutic properties. The A. ilicifolius extract also shows no
irritating effect by 24 hours single patch test upon subjects after 30 minutes and 48 hours therefore is
considered as safe as a cosmetic ingredient. Nevertheless, repeated patch test shall be considered to
comply with international requirement. Due to the high content of triterpenoid and trigonelline of the
plant, further study on anti-inflammatory such as in-vitro analysis of COX-1/COX-2 should be considered
in order to obtain a new natural and sustainable source of cosmetic ingredient.
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