Comparative study of semi-purification methods of Caesalpinia sappan L. extract : Thin layer chromatography and free radical scavenging activity

Dusadee Charnvanich

Abstract


Keywords: Caesalpinia sappan L.; Brazilin; Protosappanin A; DPPH; TLC fingerprint


Objectives: The purpose of this study was to compare thin layer chromatography (TLC) fingerprints and free radical scavenging activities of semi-purified Caesalpinia sappan L. extracts.  
Methods: Sappanwood crude extract was prepared by maceration of heartwood with 95% ethanol. The crude extract was semi-purified by partition method and ion-exchange chromatography. For partition method, the crude extract was partitioned with deionized water, dichloromethane and ethyl acetate, respectively. For ion-exchange chromatography, Diaion® HP-20 was used to semi-purify the extract. Brazilin and protosappanin A reference standards were isolated from ethyl acetate fraction by vacuum liquid chromatography on silica gel and Shephadex LH20. The identification of brazilin and protosappanin A were performed by HR-ESIMS and NMR. All extracts and standards was evaluated for TLC fingerprint. The free radical scavenging activity of all extracts was determined using DPPH assay.
Results: Ethyl acetate fraction had the highest percentage of yield (71.05%) followed by Diaion® HP-20 fraction (69.5%), water fraction (12.69%) and dichloromethane fraction (5.02%), respectively. The isolation and identification of protosappanin A and brazilin were approved by NMR and MS data with a previous report. All semi-purified extracts showed that brazilin was a major band, whereas protosappanin A was a minor band in TLC fingerprint. The water fraction had significantly the highest DPPH scavenging activity (IC50 = 1.19 ± 0.02 µg/ml) followed by Diaion® HP-20 (IC50 = 2.03 ± 0.04 µg/ml), ethyl acetate fraction (IC50 = 2.08 ± 0.02 µg/ml), crude extract (IC50 = 2.72 ± 0.03 µg/ml) and dichloromethane fraction (IC50 = 3.95 ± 0.05 µg/ml), respectively.
Conclusion: The ethyl acetate fraction and Diaion® HP-20 fraction had high percentage of yields and similar DPPH scavenging activity. Both fractions showed improved antioxidant activity when compared to crude extract. The TLC finger print showed that brazilin was a major compound of sappanwood. In further study, brazilin content of various sappanwood extracts will be evaluated by using high pressure liquid chromatography.



Full Text:

76-79

References


Washiyama, M., et al. (2009). Anti-inflammatory constituents of sappan lignum. Biological and Pharmaceutical

Bulletin 32(5): 941-944.

Yu-Ping, C., L. L., Yu-Hong, Z, Jing, W., Yong, J., P.-F., Tu. (2008). Chemical constituents from Sappan Lignum.

Journal of Chinese Pharmaceutical Science. 17: 82-86.

Temsiririrkkul, R., Punsrirat, J., Ruangwises, N., Wongkrajang, Y., Nakornchai, S. Determination of

haematoxylin and brazilin in Caesalpinia sappan extract from various locations in Thailand by high performance

liquid chromatography. Planta Medica 73(9):901.

Santos-Buelga, C., et al. (2012). Extraction and isolation of phenolic compounds.Methods Mol Biol 864: 427-

Huh, Y., Hong, T., Hong, W. (2004). Effective extraction of oligomeric proanthocyanidin (OPC) from wild grape

seeds. Biotechnology and Bioprocess Engineering. 9:471-5.

Seidel, V. (2012). Initial and bulk extraction of natural products isolation. In: Sarker, S.D., Nahar, L., (eds).

Natural Products Isolation. pp. 27-41. Humana Press.

Nirmal, N.P., Panichayupakaranant, P. (2014). Anti-propionibacterium acnes assay-guided purification of

brazilin and preparation of brazilin rich extract from Caesalpinia sappan heartwood. Pharmaceutical Biology.

:1204-7.

Miliauskas, G., Venskutonis, P.R., Van Beek, T.A. (2004). Screening of radical scavenging activity of some

medicinal and aromatic plant extracts. Food Chemistry. 85:231-7.

Batubara, I., et al. (2009). Brazilin from Caesalpinia sappan wood as an antiacne agent. Journal of Wood

Science 56(1): 77-81.

Nirmal, N. P., et al. (2015). Brazilin from Caesalpinia sappan heartwood and its pharmacological activities: A

review. Asian Pacific Journal of Tropical Medicine 8(6): 421-430.

Wetwitayaklung, P., Phaechamud, T., Keokitichai, S., (2013). The antioxidant activity of Caesalpinia sappan

L. heartwood in various ages.

Senthilkumar, N., et al. (2012). Biochemical estimation and antimicrobial activities of the extracts of

Caesalpinia Sappan Linn. Bangladesh Journal of Scientific and Industrial Research 46(4): 8.

Chung, K.-T., et al. (1998).Tannins and human health: A Review. Critical Reviews in Food Science and

Nutrition 38(6): 421-464.

Amarowicz, R. and A. Troszyn´ska (2003). Antioxidant activity of extract of pea and its fractions of low

molecular phenolics and tannins. Polish Journal of Food and Nutrition Sciences 12(Suppl.1): 10-15.

Karamać, M. (2009). In-vitro study on the efficacy of tannin fractions of edible nuts as antioxidants. European

Journal of Lipid Science and Technology 111(11): 1063-1071.


Refbacks

  • There are currently no refbacks.