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COMPARISON OF FREE RADICAL SCAVENGING ACTIVITY AND CURCUMINOIDS CONTENT OF TURMERIC EXTRACTS USING DIFFERENT METHODS OF EXTRACTION

Werayut Pothitirat1, Roongtawan Supabphol2 and Wandee Gritsanapan1* Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand. 2 Department of Physiology, Faculty of Medicine, Srinakarintarawirot University, Bangkok, Thailand. *Corresponding Author, E-mail: pywgs@mahidol.ac.th
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Abstract
This investigation compared several extraction methods, i.e. maceration, percolation, soxhlet extraction and ultrasonic extraction, to find out the method promoted high content of curcuminoids, and high free radical scavenging activity, of the extracts from rhizomes of Curcuma longa L. Extracting solvents were 95% ethanol and methanol while quantitative analysis of each curcuminoid content was performed by high performance liquid chromatography with UV detection. Free radical scavenging activity of the extracts was investigated using DPPH-scavenging assay. TLC-fingerprints of each extract were also investigated. Soxhlet extraction method with methanol gave the highest yield (29.52 %, w/w) of crude extract, while ultrasonic extraction using 95% ethanol as a solvent gave the highest contents of curcumin (19.63 %, w/w), demethoxycurcumin (4.67 %, w/w) and bisdemethoxycurcumin (12.24 %, w/w) and also exhibited the strongest free radical scavenging activity (EC50 of 16.43 g/ml). Considering various factors involved in the extraction process, i.e. time consumed, extraction cost and yield of crude curcuminoids, the ultrasonic extraction with 95% ethanol appears to be the appropriate method for extraction of C. longa rhizome for high curcuminoids content and high free radical scavenging activity. Keywords : Curcuma longa, turmeric, extraction method, curcuminoid, free radical scavenging activity

Introduction
Turmeric (Curcuma longa L.) has been used for hundred years for a yellow coloring agent in food such as mustard, curries, butter, and yogurt, in medicinal preparations and cosmetic products. Recently, turmeric has been valued worldwide as a functional food because of its health promotion property (1). The yellow pigment of C. longa rhizome is known as curcuminoids which comprise three main components: curcumin, demethoxycurcumin and bisdemethoxycurcumin (Figure 1). Natural curcuminoids exhibit numerous biological activities including anti-cancer (2-3), anti-inflammatory (4-5), and antihepatotoxic activities (6). Some of these biological activities may derive from antioxidant property. Turmeric and curcuminoids have been reported to exhibit high radical scavenging activity (711). Biological activities and quality of turmeric-derived products are based on the content of curcuminoids. Appropriate extraction method of C. longa leads to high content of curcuminoids and better quality of the extract (12). Our previous study shows that percolations is the suitable method for extraction C. longa rhizomes with 95% ethanol for high total curcuminoids content, when determined by a UV spectrophotometer. In this study we compare two extracting solvents, methanol and 95% ethanol, by maceration, percolation, soxhlet extraction and ultrasonic extraction of C. longa rhizome for high contents of each curcuminoid determined by high performance liquid chromatography and high free radical scavenging activity determined by DPPH scavenging activity assay.

Compound Curcumin Demethoxycurcumin Bisdemethoxycurcumin Figure 1 Chemical structure of curcuminoids

R1 OMe H H

R2 OMe OMe H

Materials and methods


Plant material The rhizomes of C. longa (aged 12 months) were collected from the Khao-Wong District, Surat Thani province in April 2004. The sample was identified by comparison with the specimen at the Forest Herbarium, Department of National Park, Wildlife and Plant Conservation, Ministry of Natural Resources and Environment, Bangkok, Thailand. The voucher specimen (No. WCL 042004) was deposited at the Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand. Fresh rhizome was cut into small pieces and dried in the sun for 2 days. The dried sample was further dried in a hot air oven at 50 C for 24 hours, then ground into powder and passed through a sieve no. 20 with nominal mesh aperture size 2 mm in diameter. Chemicals Curcumin, demethoxycurcumin and bisdemethoxycurcumin were isolated in our laboratory from C. longa extract. The identification of these compounds were confirmed using 1H and 13C NMR comparing with the reference (13). Ascorbic acid, trolox (vitamin E) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical were purchased from Sigma-Aldrich Chemical company (St. Louis, Mo., USA). All solvents used were AR/HPLC grade. The 95 % ethanol was obtained from the Excise Department, Bangkok, Thailand. It was distilled twice and stored in dark flasks until use. Extraction procedures Maceration (12) The dried powder (30 g) was separately extracted with 95% ethanol and methanol (each 300 ml) on a shaker with 210 rpm at room temperature for 2 days. The extract was filtered through Whatman no. 1 filter paper. Other portions of the solvent were added to the marc and the extraction was repeated until the extractant was colorless. The extracts were combined and filtered. The filtrates were concentrated under reduced pressured at 50 C using a rotary vacuum evaporator. The crude extract was then evaporated on a boiling water bath until constant weight was obtained. The experiment was performed in triplicate. Percolation (12) The extract was separately prepared by percolating 30 g of the dried powder with 95% ethanol (5,450 ml) and methanol (5,450 ml) at room temperature (flow rate 1.2 ml/ min) until the extractant was colorless. The extract was filtered and the filtrate was concentrated and evaporated under the same condition as described in the maceration method. The experiment was performed in triplicate. Soxhlet extraction (12) The extract was separately prepared by extracting 30 g of the turmeric powder with 95% ethanol (300 ml) and methanol (300 ml) using a soxhlet apparatus until the extractant was colorless. The extract was filtered and the filtrate was concentrated and evaporated under the same condition as described before. The experiment was performed in triplicate. Ultrasonic extraction Dried turmeric powder (30 g) and 900 ml (300 x 3) of 95% ethanol were placed in a 500-ml Erlenmeyer flask. The sonication was performed for 6 h (2 h x 3) using an ultrasonic bath model DSC104 (Thailand; total nominal power: 3 50 W; and internal dimensions: 30 15 25 cm) operating at 40 kHz frequency. At the end of each extraction, the extract was then separated from the residual plant material by vacuum filtration. The extract was filtered through Whatman no. 1 filter paper. Other portions of solvent were added to the marc and the extraction was repeated until the extractant was colorless. The extract was filtered and the filtrate was concentrated and evaporated under the same condition as in maceration method.

High performance liquid chromatographic procedure A HPLC method was performed on a Shimadzu SCL-10A HPLC system, equipped with a model LC-10AD pump, UV-vis detector SPD-10A, Rheodyne injector fitted with a 20 l loop and auto injector SIL-10A. A Hypersil BDS C18 column (250x 4.6 mm, 5 m size) was used. The elution was carried out with gradient solvent systems with a flow rate of 0.8 ml/min at ambient temperature. The mobile phase was consisted of 0.25 % acetic acid (A) and acetonitrile. The mobile phase was prepared daily, filtered through a 0.45 m and sonicated before use. The wavelength of the UV-vis detector was set at 425 nm. Quantitative levels of curcuminoids were determined using the above solvents with programmed at 50 % acetonitrile in A for 0-8 min. The gradient was set at 50 to 40 % acetonitrile for 8-10 min, constant at 40 % acetonitrile for 10-15 min and from 40 to 50 % acetonitrile for 15-16 min and then constant at 50 % acetonitrile for 16-24 min. The compounds were quantified using CLASS VP software. Each curcuminoid content was calculated using its calibration curve with regard to the dilution factor. The contents of curcumin, demethoxycurcumin and bisdemethoxycurcumin were expressed as gram per 100 grams of the extract. Each determination was carried out in triplicate and the results were shown as the mean of each curcuminoid content (%w/w SD). Thin layer chromatographic fingerprints Ten milligram of each extract was separately dissolved in 10 ml of methanol to give a 1.0 mg/ml concentration. A 4.0-l volume of sample solution was applied as a band of length 8.0 mm on a precoated silica gel aluminium plate 60F254 (2010 cm) using a Camag Linomat 5 syringe. The plate was developed with chloroform : benzene : methanol (80 : 15 : 5). The developing distance was 8.0 cm. After removing the plate from the chamber, the plate was dried using an air dryer and examined under ultraviolet (254 and 366 nm). hRf values were determined comparing with reference standards. Video densitometry of the chromatoplate was carried out using CAMAG Reprostar 3 with cabinet cover and mounted digital camera. Determination of antioxidant activity by the DPPH scavenging assay Stock solution of the sample (5 mg/ml) was diluted to make a dilution series of 1.96 to 500 g/ml. The DPPH scavenging reaction was performed when DPPH solution (1.52x10-4 M) was added to the sample solution in the same volume (750 l). An aliquot of the mixture was measured for absorbance at 517 nm after 30 min of the reaction by a UV-visible spectrophotometer (Perkin-Elmer, USA). The corresponding blank readings were also taken and percent inhibition was then calculated as follows: Ablank Aextract X 100 Ablank EC50 value, the concentration of sample required for 50 % scavenging of the DPPH free radical, was determined from the curve of percent scavenging plotted against the concentration. Each determination was carried out in triplicate, and the average of EC50 value was then calculated. DPPH scavenging activity of the extracts were expressed as the mean of EC50 SD. Vitamin C and Vitamin E (trolox) were used as standard antioxidant compounds. % Inhibition =

Results and discussion


The yield of crude extract (gram of extract/100 grams of sample) and free radical scavenging activity of the extracts prepared by four different extraction methods were shown in Table 1. All extraction methods using methanol as a solvent gave more yield of crude extracts than using 95% ethanol, but the methanol extracts gave the lower content of total curcuminoids and less scavenging activity. Thus, 95 % ethanol is the appropriate solvent for extraction of curcuminoids from C. longa rhizome. Although, the ultrasonic extraction with 95% ethanol gave the lowest yield of crude extract (24 % dry wt.), it promoted the highest contents of curcumin (19.63 %, w/w), demethoxycurcumin (4.67 %, w/w), bisdemethoxycurcumin (12.24 %, w/w) and total curcuminoids (36.54 %, w/w). The extract obtained from this extraction method also exhibited the strongest free radical scavenging activity (EC50 of 16.43 g/ml). HPLC chromatograms in Figure 2 showed that a major component of the extracts from all extraction methods was curcumin. Compared with percolation and other methods, ultrasonic extraction was simple, convenient, carried low cost in terms of solvent used, and less time consumed. This method promotes better penetration of solvent into plant particles and using low extraction temperature that effects the stability of active components (14). However, other modern extraction methods have been developed for plant extraction, i.e. microwave assisted extraction (15) and pressurized liquid extraction (16). There modern methods should be considered for extracting higher quality and quantity of C. longa rhizome for pharmaceutical production.

Table 1 Yield of crude extracts, free radical scavenging activity, and contents of curcumin, demethoxycurcumin, and bisdemethoxycurcumin determined by HPLC of C. longa rhizome using different extracting methods and solvents Extraction Methanol ME PE SE 95% ethanol ME PE SE USE 25.34 0.71 26.37 1.50 26.67 1.46 24.00 2.00 3000 5450 300 900 696 / RT 119 / RT 90 / BP 6 / RT 20.72 0.78 17.22 0.16 21.75 0.09 16.43 0.39 13.28 0.37 16.56 0.16 13.22 0.33 19.63 0.89 2.28 0.23 3.02 0.12 2.85 0.05 4.67 0.37 8.98 0.42 7.46 0.19 7.14 0.33 12.24 0.42 24.54 1.02 27.04 0.47 23.21 0.71 36.54 1.68 26.76 0.33 29.21 0.32 29.52 0.95 3000 5450 300 696 / RT 119 / RT 90 / BP 36.19 0.75 23.51 0.74 21.72 0.71 10.73 0.39 8.71 0.09 11.76 0.59 3.03 0.10 1.89 0.13 2.40 0.14 8.05 0.12 6.58 0.13 7.96 0.24 21.81 0.61 17.18 0.35 22.12 0.97 % Yield (w/w) of crude extract Solvent used (ml) Extraction time (h) / T (C) EC50 (g/ml) C Curcuminoid content by HPLC (% w/w in ext) DMC BDMC Total

ME = maceration, PE = percolation, SE = soxhlet extraction, USE = ultrasonic extraction, T = temperature, RT = room temperature (30 C), BP = boiling point, C = curcumin, DMC = demethoxycurcumin, BDMC = bisdemethoxycurcumin

Figure 2 HPLC chromatograms of C. longa rhizome extracts obtained using different extraction methods.
A = maceration with methanol; B = maceration with 95 %ethanol; C = percolation with methanol; D = percolation with 95 %ethanol; E = soxhlet extraction with methanol; F = soxhlet extraction with 95 %ethanol; G = ultrasonic extraction with 95 %ethanol; 1 = curcumin, 2 = demethoxycurcumin, 3 = bisdemethoxycurcumin

TLC fingerprints of the extracts from all extraction methods, using chloroform : benzene : methanol (80 : 15 : 5) as a mobile phase (Figure 3), showing 3 main components, curcumin, demethoxycurcumin, and bisdemethoxycurcumin.

Figure 3 Thin layer chromatographic fingerprints of C. longa rhizome extracts obtained from different extraction methods
Absorbent : silica gel 60 GF254; Solvent system: chloroform/benzene/methanol (80 : 15 : 5); Detector: UV 366 T1 = maceration with methanol; T2 = maceration with 95 %ethanol; T3 = percolation with methanol; T4 = percolation with 95 %ethanol; T5 = soxhlet extraction with methanol; T6 = soxhlet extraction with 95 %ethanol; T7 = ultrasonic extraction with 95 %ethanol; C = curcumin, DMC = demethoxycurcumin, BDMC = bisdemethoxycurcumin

Conclusions
Considering various factors involved in the process, i.e. time consumed, extraction costs and yield of curcuminoids, ultrasonic extraction with 95% ethanol appears to be the recommended method, more appropriate than percolation which we recommended before (12), for extraction of the rhizomes of C. longa to promote high curcuminoids content and high free radical scavenging activity.

Acknowledgements
The authors acknowledge the Drug Program of National Research Council of Thailand (NRCT), and Mahidol University, Bangkok, Thailand for financial support for this project.

References
1. Ammon HPT and Wahl MA, Pharmacology of Curcuma longa, Planta medica 57 (1991):1-7. 2. Wan JS., Chien CS., Ming JD., et al., Cytotoxicity of curcuminoids and some novel compounds from Curcuma zedoaria, Journal of Natural Product 61 (1998): 1531-1534. 3. Ruby AJ., Kuttan G., Dinesh Babu K., et al., Anti-tumour and antioxidant activity of natural curcuminoids Cancer Letters 94, (1995): 79-83. 4. Masuda T., Jitoe A., Isobe J., et al., Anti-oxidative and anti-inflammatory curcumin-related phenolics from rhizomes of Curcuma domestica, Phytochemistry 32 (1993): 1557-1560. 5. Ramsewak RS., DeWitt DL., and Nair MG, Cytotoxicity, antioxidant and anti-inflammatory activities of curcumin I-III from Curcuma longa, Phytomedicine 7 (4) (2000): 303-308. 6. Kiso Y., Suzuki Y., Watanabe N., et al., Antihepatotoxic principles of Curcuma longa rhizomes, Planta medica 49 (1983):185-187. 7. Mongkolsilp S, Pongbupakit I, and Sitthithaworn W, Antioxidant activity of medicinal plants used in primary health care, The Thai Journal of Pharmacuetical Sciences 27(Suppl) (2003): 72. 8. Kaur C and Kapoor CH, Anti-oxidant activity and total phenolic content of some Asian vegetables, International Journal of Food Science and Technology 37 (2002): 153-161. 9. Scartezziini P and Speroni E, Review on some plants of Indian traditional medicine with antioxidant activity, Journal of Ethnopharmacology. 2000; 71:23-43. 10. Sreejayan N and Rao MK, Free radical scavenging activity of curcuminoid, Arzneimittelforschung 46 (1996): 169-171. 11. Eun-Kyoung S, Hyun C, et al., Diarylheptanoids with free radical scavenging and hepatoprotective activity in vitro from Curcuma longa, Planta Medica 67 (2001): 876-877.

12. Pothitirat W, Gritsanapan W, Extraction method for high curcuminoid content from Curcuma longa, Mahidol University Journal of Pharmacuetical Science 31(3-4) (2004): 44-47. 13. Guddadarangavvanahally KJ., Lingamullu JMR., and Kunnumpurath KS, Improved HPLC method for the determination of curcumin, demethoxycurcumin, and bisdemethoxycurcumin, Journal of Agricultural and Food Chemistry 50 (2002): 3668-3672. 14. Soares Melecchi MI, Peres VF, Dariva C, Zini CA, Abad FC, Martinez MM, Caramao EB. , Optimization of the sonication extraction method of Hibiscus tiliaceus L. flowers, Ultrason Sonchem 13 (2006): 242-250. 15. Wang Q, Ma S, Fu B, Lee FSC, Wang X, Development of multi-stage countercurrent extraction technology for the extraction of glycyrrhizic acid (GA) from licorice (Glycyrrhiza uralensis Fisch) Biochemical Engineering Journal 21 (2004): 285-292. 16. Ong ES, Len SM, Pressurized hot water extraction of berberine, baicalein and glycyrrhizin in medicinal plants, Analytica Chimica Acta 482 (2003): 81-89.