Antioxidant Activity and Irritation Test of Acanthus ilicifolius Linn. Extract

Transcription

1 Antioxidant Activity and Irritation Test of Acanthus ilicifolius Linn. Extract Theeraya Krisdaphong 1 Ampa Jimtaisong 1,* Surapol Natakankitkul 2 Narunan Wuttisin 1 1 School of Cosmetic Science, Mae Fah Luang University 2 Faculty of Pharmacy, Chiang Mai University * Corresponding Author ampa@mfu.ac.th Phone Fax 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 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 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 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.

2 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

3 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 and , the content of total phenolic compound present of gallic acid equivalent in the A. ilicifolius extract was % phenolic content of dry crude. (Waterman & Mole, 1994) 3. Determination of Flavonoids The absorbance of A. ilicifolius extract are and , 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 and , when referred to the standard curve (Figure 4.7), the content of total triterpenoid present of saponin equivalent in the A. ilicifolius extract was % 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 µg/ml whereas the IC50 values of standards were 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 :

4 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 % of polyphenol, of % 8.07flavonoid. The obtained crude extract showed satisfying free radical scavenging activity with IC50 equivalent of 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. REFERENCES Allen, C. F. H. (1943). 2,4-DINITROPHENYLHYDRAZINE. Organic Syntheses,2 (2), 228. Brand-Williams, W., Cuvelier, M. E. & Berset, C. (1995). Use of free radical method to evaluate antioxidant activity. Lebensmittel Wissenschaftund Technologie, 28(1), Chang, C. C., Yang, M. H., Wen, H. M. & Chern, J. C. (2002). Estimation of total flavonoid content in propolis by two complementary colorimetric methods. Journal of Food and Drug Analysis, 10(3), 2002, Griffin, S. G., Wyllie, S. G., Markham, J. L. & Leach, D. N. (1999). The role of structure and molecular properties of terpenoids in determining their antimicrobial activity. Flavour and Fragrance Journal, 14(5), Kanchanapoom, T., Kamel, M. S., Kasai, R., Yamasaki, K., Picheansoonthon, C. & Hiraga Y. (2001). Lignan glucoside from Acanthus ilicifolius. Phytochemistry, 56(4), p Khajure, P. V. & Rathod, J. L. (2010). Antimicrobial activity of extracts of Acanthus ilicifolius extracted from the mangroves of Karwar coast Karnataka, Pharmacology. Recent Research in Science and Technology, 6(2), Khajure, P. V. & Rathod, J. L. (2011). Potential anticancer activity of Acanthus ilicifolius extracted from the mangroves forest of Karwar, west coast of India. World Journal of Science and Technology, 1(1), 1-6. Kumar KT, M. S., Gorain, B., Roy, D. K., Zothanpuia, Samanta, S. K., Pal, M., Biswas, P., Roy, A., Adhikari, D., Karmakar, S. & Sen, T. (2008). Antiinflammatory activity of acanthus ilicifolius. J. Ethonopharmacol, 120(1), 7-12.

5 Ky, C.L., Louarn, J., Dussert, S., Guyot, B., Hamon, S. & Noirot, M. (2001). Caffeine, trigonelline, chlorogenic acids and sucrose diversity in wild Coffea arabica L. and C. canephora P. accessions. Food Chemistry Elsevier, 75(2), Lamson, D. W. & Matthew, S. B. (2000). Antioxidants and cancer III: quercetin. Alternative Medicine Review, 5(3), Miliauskasa, G., Venskutonisa, P. R. & Beek van, T.A. (2004). Screening of radical scavenging activity of some medicinal and aromatic plant extracts. Journal of Agricultural and Food Chemistry, 85(2), Mitchell, A. E., Hong, Y. J., Koh, E., Barrett, D. M., Bryant, D. E, Denison, R. F. & Kaffka, S. (2007). Ten-year comparison of the influence of organic and conventional crop management practices on the content of flavonoids in tomatoes. Journal of Agricultural and Food Chemistry, 55(15), Nunes, X. P., Silva, F. S., Almeida, J. R. G. D. S., Lima, J. T., Ribeiro, L. A. A., Quintans Júnior, L. J. & Barbosa Filho, J. M. (2011). Biological oxidations and antioxidant activity of natural products. Retrieved September 1, 2011, from in-nutrition-and-health/biological-oxidationsand-antioxidant-activity-of-natural-products Pietta, P. G. (2000). Flavonoids as antioxidants. J. Nat. Prod., 63(7), Raheleh, A., Hasanloo, T. & Khosroshahli, M. (2011). Evaluation of trigonelline production in Trigonella foenum-graecum hairy root cultures of two Iranian masses. POJ, 4(7), Safayhi, H. & Sailer, E. R. (1997). Anti-inflammatory actions of pentacyclic triterpenes. Planta Med, 63(6), Saroya, A. S. (2011). Ethnopharmacology of Acanthus ilicifolius Linn. Retrieved September 1, 2011, from Saroya, A. S. (Ed.). (2011). Herbalism, Phytochemistry and ethnopharmacology: Chapter 16 ethnopharmacology of Acanthus ilicifolius Linn. Punjab, India: Science Publishers. Singh A., Duggal S. & Suttee A. (2009). Acanthus ilicifolius Linn.-Lesser known medicinal plants with significant pharmacological activities. Ethnobotanical Leaflets., 13(1), p The European Cosmetic Toiletry and Perfumery Association (COLIPA). (1997). Toiletry and perfumery association. Product test guidelines for the assessment of human skin compatibility. COLIPA Guidelines, 1(1), 4-5. Van Acker, S. A. B. E., Van den Berg, D. J., Tromp, M. N. J. L., Griffioen, D. H., Van Bennekom, W. P. & Van der Vijgh, W. J. F. & Bast, A. (1996). Structural aspects of antioxidant activity of flavonoids. Free Radical Biology and Medicine, 20(3), Waterman, P. G. & Mole, S. (1994). Analysis of phenolic plant metabolites.oxford, UK: Blackwell Scientific. Wostmann, R. & Liebezeit, G. (2008). Chemical composition of the mangrove holly Acanthus ilicifolius (Acanthaceae). Senckenbergiana maritime, 38(1), 31-37