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Phytochemicals and antioxidant capacity of Xao tam phan (Paramignya trimera) root as affected by various solvents and extraction methods
Industrial Crops and Products 67 (2015) 192–200
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Phytochemicals and antioxidant capacity of Xao tam phan (Paramignya trimera) root as affected by various solvents and extraction methods Van Tang Nguyen a,b,∗ , Michael C. Bowyer a , Quan Van Vuong a , Ian A.Van Altena a , Christopher J. Scarlett a,∗∗ a b
School of Environmental and Life Sciences, Faculty of Science and Information Technology, University of Newcastle, Ourimbah, NSW 2258, Australia Department of Food Technology, Faculty of Food Technology, Nha Trang University, No. 2 Nguyen Dinh Chieu, Nha Trang, Khanh Hoa 8458, Viet Nam
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Article history: Received 22 November 2014 Received in revised form 22 January 2015 Accepted 26 January 2015 Keywords: Xao tam phan Paramignya trimera Phytochemicals Antioxidant Solvent Extraction method
a b s t r a c t Xao tam phan (Paramignya trimera (Oliv.) Guillaum) is a Vietnamese traditionally medicinal plant used in the treatment of numerous cancers. The preparation of Xao tam phan extracts including solvent type and extraction method have significant effects on extraction efficiency, phytochemical profile and biological activity. This study aimed to investigate the effects of five various solvents (water, acetonitrile, methanol, ethyl acetate and hexane) and three different extraction methods (conventional, ultrasoundassisted and microwave-assisted) on phytochemical yield and antioxidant capacity of P. trimera root from Vietnam. The results indicate that methanol extracted the maximal yield of phytochemicals from P. trimera and exhibited the greatest antioxidant capacity, with eleven compounds were identified and quantified. Microwave-assisted extraction produced the maximal phytochemical yields (except for total flavonoids) and antioxidant capacity, when compared to conventional and ultrasound-assisted extractions. These data reveal that the use of methanol and microwave-assisted extraction are recommended for extraction of biologically active phytochemicals from P. trimera root for application in the nutraceutical and/or pharmaceutical industries. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Xao tam phan (Paramignya trimera (Oliv.) Guillaum) has a synonym of Atalantia trimera Oliv., which belongs to the Paramignya genus of Citrus family (Rutaceae) and is well known as a medicinal plant in Thailand and Vietnam (Nguyen et al., 2013). Recent studies on the P. trimera have revealed hepatoprotective and cytotoxic activity from a crude methanolic extract (Nguyen et al., 2013). Importantly, methanolic extract, n-hexane fraction and an individual compound isolated from P. trimera, named ostruthin (6-(3,7-dimethyl-2,6-octadienyl) 7-hydroxy-2H-1-benzopyran-2one) demonstrated cytotoxic activity against five cancer cell lines in vitro, especially, the partitioned n-hexane fraction and ostruthin
∗ Corresponding author. School of Environmental and Life Sciences, Faculty of Science and Information Technology, University of Newcastle, Brush Road, Ourimbah, NSW 2258, Australia. Tel.: +61 434238842; fax: +61 243484145. ∗∗ Corresponding author. Tel.: +61 243484680; fax: +61 243484145. E-mail addresses: [email protected] (V.T. Nguyen), [email protected] (C.J. Scarlett). http://dx.doi.org/10.1016/j.indcrop.2015.01.051 0926-6690/© 2015 Elsevier B.V. All rights reserved.
from the methanolic extract displayed activity against hepatocellular carcinoma (Hep-G2) and human epithelial cervical carcinoma (Hela) cell lines with IC50 values of 39.61 and 5.36 g/ml, respectively. Pham et al. (2013) also isolated and quantitatively analysed ostruthin to be a major compound in the P. trimera roots and stems, while Li et al. (2012) showed that ostruthin displayed potent activity against pancreatic cancer cell lines. These studies indicate that P. trimera contains key biologically active phytochemicals, however it is imperative to optimize extraction conditions to maximize the yield of phytochemicals extracted for potential medicinal exploitation. Solvent type and extraction method directly affects the extraction efficiency and relates to production costs such as extraction time, solvent volume, energy costs as well as the effect on humans and the environment (Dai and Mumper, 2010; Azmir et al., 2013; Kalia et al., 2008). In recent years, advanced extraction techniques have been developed and applied to optimize the extraction of phenolics from plant materials. For example, ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE) and techniques using compressed fluids such as subcritical water extraction (SWE), supercritical fluid extraction (SFE), pressurized fluid
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extraction (PFE) or accelerated solvent extraction (ASE) (Tiwari et al., 2011; Doughari, 2012) have proven to significantly reduce extraction time as well as gaining improved extraction efficiency. For this reason, careful consideration for the selection of solvent type and extraction method is a crucial step in the preparation of extracts from plant materials. Very few studies have reported the physicochemical properties and biological activity of P. trimera extracts and none have focused on selection of solvent and extraction method. With this in mind, our study investigated the effects of various solvent types and extraction methods on the phytochemical yield and antioxidant capacity of P. trimera root extracts. Our data reveal the optimal conditions for solvent type and extraction method for the preparation of crude P. trimera root extracts for potential application in the nutraceutical and/or pharmaceutical industries. 2. Materials and methods 2.1. Plant materials P. trimera root was collected from Ninh Hoa district, Khanh Hoa province, Vietnam in January 2014 and identified by the National Institute of Medicinal Materials, Ministry of Health, Vietnam. After collection, the fresh samples were rinsed in deionized water to remove sand and soil, drained and left to dry under the sun (34.5 ± 1 ◦ C) to constant weight, and then packed in vacuum sealed polyamide (PA) bags and stored at −18 ◦ C until used. The residual moisture of the dried samples was determined according to the AOAC official methods of analysis (AOAC, 1998) using a hot-air oven (Anax Pty Ltd., NSW, Australia) at 120 ◦ C for 5 h. 2.2. Analytical chemicals All chemicals used were of analytical grade. 2,2 -azino-bis3-ethylbenzothiazoline-6-sulphonic acid (ABTS), 1,1-diphenyl2-picryl-hydrazil (DPPH), Folin-Ciocalteu, trolox, neocuproine, 2,4,6-tripyridyl-s-triazine (TPTZ), iron (III) chloride and standard compounds including gallic acid, p-coumaric acid, caffeic acid, 5,7-dimethoxycoumarin, quercetin, kaempferol, rutin, syringic acid, chlorogenic acid, escin, (+)-catechin, (−)-epicatechin, (±)naringenin, myricetin, luteolin, apigenin, (−)-epigallocatechin gallate, -sitosterol--D-glucoside, gemcitabine, -sitosterol, and ascorbic acid were purchased from Sigma–Aldrich Pty Ltd. (NSW, Australia). Vanillin, potassium persulfate, methanol and ethanol were obtained from Merck (Darmstadt, Germany). Copper (II) chloride was purchased from Standard Laboratories (Victoria, Australia). Sodium acetate trihydrate was obtained from Government Stores Department (Australia). Ammonium acetate was purchased from BDH Chemicals (Victoria, Australia). Aluminium chloride (anhydrous) was obtained from Acros (New Jersey, USA). Sodium hydroxide was purchased from Ajax chemicals (NSW, Australia). Sodium carbonate anhydrous was purchased from Chem-supply (Gillman, South Australia). Sulfuric acid and hydrochloric acid were purchased from Ajax Finechemicals (NSW, Australia), and acetic acid was obtained from BDH Laboratory Supplies (Poole, England). 2.3. Preparation of extracts To select the best solvent for further study, five various solvents were evaluated including water (WT), acetonitrile (AT), methanol (MT), ethyl acetate (EA) and hexane (HX). Then, three different extraction methods were applied with selected solvent, i.e. conventional extraction (CE), ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE). The extracts of P. trimera root were prepared according to the method described by Nguyen
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et al. (2011) with some modifications. The extraction procedures were as follows: For effect of solvents: 0.2 g of dried sample was extracted with 20 mL of various solvents applying UAE using an ultrasonic cleaner Soniclean 1000HD (Soniclean Pty Ltd., South Australia) at a power of 150 W and 45 ◦ C for 60 min. For effect of extraction methods: 0.2 g of dried sample was extracted with 20 mL of selected solvent, the extraction process included two stages: the first stage was set at room temperature (20 ± 1 ◦ C) for 20 min, and the second stage was extracted using the various methods for 60 min. CE was carried out using a waterbath (Buchi, Flawil, Switzerland) at 45 ◦ C, UAE was performed using an ultrasonic cleaner Soniclean 1000HD at a power of 150 W and 45 ◦ C, and MAE was carried out using a microwave oven (Sharp Carousel, Sharp Corporation, Thailand) at a power of 360 W, with the initial irradiation time of 15 s, then 5 s for each further 5 min (total irradiation time was applied to be 70 s). After extraction, the extracts were rapidly cooled to room temperature by ice water and then filtered through qualitative no. 1 filter papers (Bacto Laboratories Pty Ltd., NSW, Australia) to obtain extracts for further analysis. To determine the extraction yield, 2 mL of extract was dried in a hot-air oven (Anax Pty Ltd., NSW, Australia) at 100 ◦ C for 4 h to a constant weight (extraction yield = g of dried extract/100 g of dried sample).
2.4. Determination of phytochemical compounds 2.4.1. Total phenolic content (TPC) TPC of P. trimera extracts was determined using the Folin–Ciocalteu method as previously described by Nguyen et al. (2011) and Vuong et al. (2013) with some modifications. Briefly, 0.5 mL of the extracts were diluted 2 times and mixed with 2.5 mL of 10% (v/v) Folin–Ciocalteu reagent in distilled water. The mixture was left to settle for 6 min, then 2 mL of 7.5% (w/v) Na2 CO3 solution was added and incubated in the dark at room temperature for 1 h. The absorbance of mixture was measured at 765 nm using a UV–vis spectrophotometer (Cary 50 Bio Varian, Australia). Methanol and gallic acid were used as a control and standard. TPC was expressed in mg gallic acid equivalents (GAE)/g dried sample.
2.4.2. Total flavonoid content (TFC) TFC of P. trimera extracts was performed according to the method described by Vuong et al. (2013) with some modifications. 0.5 mL of extract was mixed with 2 mL of distilled water and 0.15 mL of 5% (w/v) Na2 CO3 solution and incubated in the dark at room temperature for 6 min. 0.15 mL of 10% (w/v) AlCl3 solution was then added and incubated for a further 6 min. Finally, 2 mL of 4% (w/v) NaOH solution and 0.7 mL of distilled water were added and incubated for 15 min. The absorbance of mixture was measured at 510 nm using a UV–vis spectrophotometer. Methanol and rutin were used as a control and standard, respectively. TFC was expressed as mg of rutin equivalents (RE)/g dried sample.
2.4.3. Proanthocyanidin content Proanthocyanidin content of P. trimera extracts was determined using the method described by Vuong et al. (2013) with some modifications. Briefly, 0.5 mL of extract was mixed with 3 mL of 4% (w/v) vanillin solution, then 1.5 mL of concentrated HCl was added and the mixture was incubated in the dark at room temperature for 15 min. The absorbance of mixture was measured at 500 nm using a UV–vis spectrophotometer. Methanol and catechin were used as a control and standard, respectively. Proanthocyanidin content was expressed as mg of catechin equivalents (CE)/g dried sample.
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2.4.4. Saponin content Saponin content of P. trimera extracts was determined according to the method described by Vuong et al. (2013) with the modified procedure to reduce analytical time. Briefly, 0.5 mL of the extract was mixed with 0.5 mL of 8% (w/v) vanillin solution, then 5 mL of 72% (v/v) H2 SO4 solution was added. The mixture was then incubated at 70 ◦ C for 10 min and rapidly cooled by ice water to room temperature. The absorbance of mixture was measured at 560 nm using a UV–vis spectrophotometer. Methanol and escin were used as a control and standard, respectively. Saponin content was expressed as mg of escin equivalents (EE)/g dried sample.
2.4.5. Identification and quantification of phytochemical compounds Phytochemical compounds in the P. trimera extracts were identified and quantified based on the methods described by Maity et al. (2013) and Plaza et al. (2014) with some modified procedures using HPLC system (Shimadzu, Japan). Briefly, the external standard solutions were prepared by dissolving standard compounds in methanol at a concentration of 200 g/mL, then 200 L of each standard solution was taken to make a mixed standard solution. Trolox was used as an internal standard. The extracts and standard solutions were then filtered through 0.45 m nylon membranes (phenex syringe filters) and 20 L of extracts or standard solutions were individually injected by an auto injector (SIL-10AV VP) onto a phenosphere-NEXT 5 u C18 column (250 mm × 4.6 mm × 5 m, Phenomenex, Torrance, CA, USA), which was maintained at 35 ◦ C by a column oven (CTO-10A VP). The mobile phase consisted of 0.2% orthophosphoric acid in distilled water (A) and 100% acetonitrile (B). Flow rate was set at 1 mL/min with the gradient as follows: 0–5 min, 0% B; 5–20 min, 20% B; 20–30 min, 30% B; 30–110 min, 30% B; 110–120 min, 0% B. Phytochemical compounds were detected at 210 nm using a UV–vis detector SPD-10AV VP. Identification of individual phytochemical compounds was achieved by comparing retention times (tR ) of compounds in the P. trimera extracts with those of commercial standard compounds, including gallic acid, p-coumaric acid, caffeic acid, 5,7-dimethoxycoumarin, quercetin, kaempferol, rutin, syringic acid, chlorogenic acid, escin, (+)-catechin, (−)-epicatechin, (±)-naringenin, myricetin, luteolin, apigenin, (−)-epigallocatechin gallate, -sitosterol--D-glucoside, gemcitabine, -sitosterol, and ascorbic acid, in terms of individual standards, mixed standard solution and mix of extract with each standard (1:1 v/v). Quantification of individual phytochemical compounds was performed using the standard equations based on peak area of the commercial standard compounds and the individual phytochemical compounds in the P. trimera extracts.
2.5.2. DPPH radical scavenging capacity (DRSC) DRSC of P. trimera extracts was measured based on the method reported by Vuong et al. (2013) and Pisoschi and Negulescu (2011) with some modifications. Briefly, a stock solution of 0.024% (w/v) DPPH (1,1-diphenyl-2-picryl-hydrazil) in methanol was prepared and stored at −18 ◦ C. Before use, a working solution was prepared by diluting 1.0 mL of stock solution with 45 mL of methanol to obtain an absorbance of 1.1 ± 0.02 at 515 nm. For the DPPH reaction, 0.15 mL of the extract was mixed with 2.850 mL of the working solution and incubated for 3 h in the dark at room temperature. The absorbance of mixture was measured at 515 nm using a UV–vis spectrophotometer. The results were expressed as mg trolox equivalents (TE)/g dried sample. 2.5.3. Cupric ion reducing antioxidant capacity (CUPRAC) CUPRAC of P. trimera extracts were analyzed using the method described by Vuong et al. (2013) and Pisoschi and Negulescu (2011). Briefly, 1.0 mL of 10 mM CuCl2 solution was mixed with 1.0 mL of 7.5 mM neocuproine and 1.0 mL of 7.7% (w/v) NH4 Ac solutions. 1.1 mL of the extract was then added and incubated in the dark at room temperature for 1.5 h. The absorbance of mixture was measured at 450 nm using a UV–vis spectrophotometer. The results were expressed as mg trolox equivalents (TE)/g dried sample. 2.5.4. Ferric reducing antioxidant power (FRAP) FRAP of P. trimera extracts was determined based on the method reported by Kamonwannasit et al. (2013) and Pisoschi and Negulescu (2011) with some modifications. Reagent A: 300 mM acetate buffer solution, pH 3.6; Reagent B: 10 mM TPTZ (2, 4, 6tripyridyl-s-triazine) solution in 40 mM HCl; and Reagent C: 20 mM ¨ FeCl3 Y6H 2 O solution. Before use, the fresh FRAP solution was prepared by mixing reagents A, B and C at a ratio of 10:1:1. For the FRAP reaction, 0.15 mL of the extract was mixed with 2.850 mL of the fresh FRAP solution and incubated in the dark at room temperature for 30 min. The absorbance of mixture was read at 593 nm using a UV–vis spectrophotometer. The results were expressed as mg trolox equivalents (TE)/g dried sample. 2.6. Statistical analysis All experiments were performed at least in triplicate. The data were analyzed using SPSS software (Version 16.0, Chicago, IL, U. S. A.) and expressed as mean ± standard deviation (n = 3). Statistical comparisons were made using one-way analysis of variance (ANOVA) and Tukey HSD tests. Differences were considered to be significant when P-values were below 0.05 (P < 0.05). 3. Results and discussion
2.5. Determination of antioxidant capacity 2.5.1. ABTS antioxidant capacity (AC) AC of P. trimera extracts was determined using the ABTS assay described by Kamonwannasit et al. (2013) and Pisoschi and Negulescu (2011) with some modifications. Briefly, a stock solution was prepared by mixing 7.4 mM ABTS•+ (2,2’-azinobis-3-ethylbenzothiazoline-6-sulphonic acid) and 2.6 mM K2 S2 O8 solutions (1:1 ratio) and then incubated in the dark at room temperature for 12 h, then stored at −18 ◦ C. Before use, a working solution was prepared by mixing 1 mL of stock solution with 60 mL of methanol to obtain an absorbance of 1.1 ± 0.02 at 734 nm. For the ABTS reaction, 0.15 mL of the extract was mixed with 2.85 mL of the working solution and incubated for 2 h. The absorbance of mixture was measured at 734 nm using a UV–vis spectrophotometer. The results were expressed as mg trolox equivalents (TE)/g dried sample.
3.1. Effects of solvents and extraction methods on the extraction and phytochemical yields 3.1.1. Extraction yield Extraction yield refers to the percentage of crude extract obtained from a dried plant sample through a solvent extraction procedure for further isolation and utilization. The residual moisture of dried sample after drying was determined to be 8.74%. Table 1 shows that of the five solvents used, water produced the highest extraction yield (11.48 g dried extract/100 g dried sample). This yield was not however found to be significantly different from the methanol extract (11.04 g dried extract/100 g dried sample). Both the aqueous and methanol extracts were found to be significantly greater (P < 0.05) than those of acetonitrile, ethyl acetate and hexane extracts (2.31, 2.32 and 2.08 g dried extract/100 g dried sample, respectively). These findings are supported by Rahman et al. (2013), who demonstrated the phytochemical content and
V.T. Nguyen et al. / Industrial Crops and Products 67 (2015) 192–200 Table 1 Extraction yield of P. trimera root obtained by five solvents and three extraction methods. Solvent and extraction method
Water Acetonitrile Methanol Ethyl acetate Hexane Conventional Ultrasound-assisted Microwave-assisted
Extraction yield (g dried extract/100 g dried sample) 11.48 2.31 11.04 2.32 2.08 12.02 10.93 14.39
± ± ± ± ± ± ± ±
0.95a, * 0.08b 0.23a 0.48b 1.17b 1.09AB 1.97B 0.32A
* Means and standard deviations from three replicates. Different letters as indicated by lowercase (for solvent) and uppercase (for extraction method) in each group were significantly different (P < 0.05).
antioxidant potential of Colubrina asiatica were affected by extraction solvent polarities, such as 100% ethanol, 50% ethanol, and water. Extraction yields showed a clear correlation with solvent character, with the polar, protic solvents (methanol and water) extracting significantly greater yield than their aprotic counterparts. The importance of protic solvent character is illustrated by the difference in obtained yield for the methanol and acetonitrile extracts. These two solvents possess similar dielectric constant values (methanol: 32.63, acetonitrile: 37.5; Maryott and Smith, 1951), considered an accepted measure of polarity, yet the protic character of methanol produces a fivefold increase in relative extraction yield. We propose that the efficiency of methanol as a solvent relates to its intermediate polarity, which allows it to solvate low molecular weight organics compounds possessing protonatable functional groups (e.g. COOH, OH). Based on the results obtained, methanol was selected as the solvent for the enhanced extraction procedures. MAE produced a higher extraction yield (14.39 g dried extract/100 g dried sample) relative to CE and UAE (12.02 and 10.93 g dried extract/100 g dried sample, respectively, Table 1). While statistical analysis found a significant difference between the efficiencies of MAE and UAE methods, neither was significantly different to the CE method. This finding is in similar to research conducted by Dhanani et al. (2013), who reported that MAE of Withnaia somnifera using ethanol and mix of water and ethanol (9:1 v/v) exceeded yields obtained by CE and UAE. Compared to other plant species, P. trimera produced lower yield than that of papaya leaf (19.15 g dried extract/100 g dried sample) as indicated by Vuong et al. (2013). Chisté et al. (2014) reported the yield of solid extract from Capsicum villosum fruit pulps obtained the highest level by mix of ethanol and ethyl acetate (1:1 v/v; 46.4 g dried extract/100 g dried sample), followed by ethyl acetate, water, ethanol, and mix of ethanol and water (1:1 v/v; 41.9, 14.7, 11.1 and 10.8 g dried extract/100 g dried sample, respectively). These data indicate that the extraction yield of P. trimera was greatly affected by solvents, as well as extraction methods. 3.1.2. Total phenolic content (TPC) Phenolic compounds in plants have strong links to their antioxidant and anticancer activities (Khoddami et al., 2013; Dai and Mumper, 2010). Fig. 1A indicates that among the five solvents used, methanol extracted the highest TPC from P. trimera (33.36 mg GAE/g dried sample) and it was significantly higher than those obtained from water and acetonitrile (25.06 and 22.33 mg GAE/g dried sample, respectively), while TPCs levels extracted by ethyl acetate and hexane were very low (3.32 and 4.58 mg GAE/g dried sample, respectively). It appeared that the TPC obtained from MAE (42.25 mg GAE/g dried sample) was higher than those from EC and
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Table 2 Phytochemical content of methanolic extract of P. trimera root obtained by microwave-assisted extraction. Retention time (min) Phytochemical compound Content(mg/g dried sample) 4.075 32.517 33.622 34.232 35.929 36.508 38.258 43.100 64.400 80.785 88.517 a
gallic acid chlorogenic acid caffeic acid (−)-epicatechin syringic acid p-coumaric acid rutin myricetin quercetin (+)-catechin kaempferol
2.79 7.72 0.34 1.22 0.66 2.14 2.27 1.03 0.57 1.16 16.72
± ± ± ± ± ± ± ± ± ± ±
0.17a 0.37 0.03 0.05 0.08 0.01 0.01 0.04 0.09 0.04 0.10
Means and standard deviations were of three replicates.
UAE (39.86 and 37.91 mg GAE/g dried sample, respectively), however any differences observed were not significant. Table 3 shows the correlation amongst three of the four assay methods (DRSC, CUPRAC and FRAP) for TPC was strong (R2 = 0.81, 0.91 and 0.98, respectively), with only AC showing poor reliability (R2 = 0.31). Vuong et al. (2013) reported the TPC of papaya leaf extracted by various solvents decreased in the order water, methanol, acetone and ethanol (23.06, 15.03, 10.71 and 9.43 mg GAE/g dried sample, respectively), while Poh-Hwa et al. (2011) indicated the methanolic extracts of Phyllanthus species (50.12–68.70 mg GAE/g dried sample) had higher TPC than water extracts (41.19–55.38 mg GAE/g dried sample). The TPC of Phyllanthus amarus (mg/100 g dried sample) gradually reduced by various solvents (methanol: 212.67, ethanol: 208.09 and petroleum ether: 203.54) as reported by Sen and Batra (2013), while Shahriar et al. (2013) found the TPC in W. somnifera root extracts decreased in the order chloroform, petroleum ether, methanol, ethanol and n-hexane (60.99, 56.58, 23.86, 5.46 and 1.43 mg GAE/g dried sample, respectively). The relatively low levels of TPC obtained for ethanol in this study are brought into question by recently published findings on the same plant species by Dhanani et al. (2013) who obtained significantly higher TPC by MAE, CE and UAE (40.96, 35.93 and 24.72 mg GAE/g dried sample, respectively). Garofulic´ı et al. (2013) similarly indicated the MAE of anthocyanins and phenolic acids from Prunus cerasus var. obtained higher efficiency than that by CE. These phenomena may be explained by microwaves causing a localized temperature rise in plant tissue, leading to cellular disruption and consequently the migration of phenolic compounds to the surrounding solvent. In comparison with other plants, the TPC of P. trimera was much higher than that of sage plants (Ibtissem et al., 2013) (approximately 2.3 mg GAE/g dried sample), Oolong tea (Nguyen et al., 2011) and papaya leaf (Vuong et al., 2013) (13.58 and 15.03 mg GAE/g dried sample, respectively), however it was much lower than those of P. amarus, P. urinaria and P. niruri (PohHwa et al., 2011) (50.24, 62.56 and 68.70 mg GAE/g dried sample, respectively). Our findings show a link between solvent character and TPC extraction efficiency from P. trimera, with methanol was the most effective solvent because of its protic character and intermediate polarity which enables effective solvation of carboxyl and Table 3 The correlations between phytochemicals and antioxidant capacity of P. trimera root extracts obtained by five solvents and three extraction methods. Correlations (R2 )a
TPC
TFC
Proanthocyanidins
Saponins
AC DRSC CUPRAC FRAP
0.31 0.83 0.91 0.98
0.14 0.77 0.83 0.61
0.27 0.77 0.63 0.40
0.07 0.76 0.64 0.56
a
Based on values obtained from five solvents and three extraction methods.
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Fig. 1. (A) Total phenolic content (TPC); (B) total flavonoid content (TFC); (C) proanthocyanidin content; and (D) saponin content of P. trimera root obtained by five solvents and three extraction methods. Different letters within the columns denotes a significant difference between groups for TPC, proanthocyanidins and saponins (P < 0.05) and TFC (P < 0.0005). WT: water; AT: acetonitrile, MT: methanol; EA: ethyl acetate and HX: hexane. CE: conventional extraction, UAE: ultrasound-assisted extraction and MAE: microwave-assisted extraction.
phenol-containing species. Besides, the efficiency of extracting TPC from P. trimera was also affected by extraction methods. 3.1.3. Total flavonoid content (TFC) TFC of P. trimera extracted by various solvents as follows: methanol > acetonitrile > hexane > water > ethyl acetate (Fig. 1B). Methanolic extract possessed significantly higher TFC (14.11 mg RE/g dried sample; P < 0.0005) as compared to extracts from acetonitrile, hexane, water and ethyl acetate (12.00, 3.76, 2.72 and 2.27 mg RE/g dried sample, respectively). No significant differences were observed for TFC between the three extraction methods, with a range of 13.63–15.74 mg RE/g dried sample. Table 3 indicates that the TFC of P. trimera had strong correlations with its DRSC and CUPRAC (R2 = 0.77 and 0.83, respectively) as compared to AC and FRAP (R2 = 0.14 and 0.61, respectively). Shahriar et al. (2013) reported the TFC of W. somnifera root extracts reduced by various solvents in the order chloroform, petroleum ether, methanol, ethanol and n-hexane (122.09, 92.14, 88.76, 66.01 and 44.95 mg quercetin equivalent/g dried sample, respectively). Chisté et al. (2014) found the TFC of C. villosum fruit pulps (mg catechin equivalent/g dried sample) gradually reduced from a mix of ethanol and water (1:1 v/v; 3.8) to water, ethanol, a mix of ethanol and ethyl acetate (1:1 v/v; 2.5, 0.3 and 0.04, respectively), and lastly to ethyl acetate (0.03). Similarly, Vuong et al. (2013) also indicated the TFC of papaya leaf (mg catechin equivalent/g dried sample) extracted by ethanol was the highest (17.07), followed by acetone (16.41), methanol (11.96) and water (6.44). Garofulic´ı et al. (2013) found the total anthocyanins and total phenolic acids efficiency from P. cerasus var. obtained by MAE
were higher than those gained by CE. As seen with the TPC, these data indicate that the TFC extractability from P. trimera strongly depended on solvent type as well as extraction methods, with higher levels generally obtained in the polar, protic organic solvents. 3.1.4. Proanthocyanidin content Proanthocyanidin content isolated from P. trimera was found to be highest in ethyl acetate extracts (5.28 mg CE/g dried sample), followed by methanol, acetonitrile, water and hexane (5.00, 3.92, 2.99 and 1.77 mg CE/g dried sample, respectively, Fig. 1C). Methods of CE, MAE and UAE did not significantly differ in proanthocyanidin content (5.66, 5.51 and 5.54 mg CE/g dried sample, respectively). Table 3 shows the correlations between proanthocyanidin content and antioxidant capacity of P. trimera extracts in order by DRSC (0.77), followed by CUPRAC (0.63), FRAP (0.40) and AC (0.27). Vuong et al. (2013) found the maximum proanthocyanidin content of Carica papaya leaf was following extraction with ethanol (7.91 mg CE/g dried sample) as compared to those extracted by acetone, methanol and water (6.39, 4.86 and 1.91 mg CE/g dried sample, respectively). These results indicate that solvent type had significant effects on extraction efficiency of proanthocyanidins from P. trimera, however extraction methods did not greatly influence proanthocyanidin content from this sample. 3.1.5. Saponin content Saponins contain many valuable phytochemicals such as triterpenes, lupeol, triterpene glycosides, sterols, glycyrrhizic acid and triterpene scaffolds that display antiviral, antimicrobial,
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Fig. 2. (A) ABTS antioxidant capacity (AC); (B) DPPH radical scavenging capacity (DRSC); (C) cupric ion reducing antioxidant capacity (CUPRAC); and (D) ferric reducing antioxidant power (FRAP) of P. trimera root obtained by five solvents and three extraction methods. Different letters within the columns denotes a significant difference between groups for AC, DRSC and CUPRAC (P < 0.05) and FRAP (P < 0.005). WT: water; AT: acetonitrile, MT: methanol; EA: ethyl acetate and HX: hexane. CE: conventional extraction, UAE: ultrasound-assisted extraction and MAE: microwave-assisted extraction.
antioxidative, anti-hyperglycaemic, hepatoprotective, cardioprotective, antiherbivore/cytotoxic, immunomodulatory, antidiarrhoeal, anti-HIV, antitumour, antihepatitis B and C, antiinflammatory and anti-cancer properties (Amoros et al., 1987; Yoshikawa et al., 2003; Lacaille-Dubois, 2005; Mandal et al., 2006; Podolak et al., 2010; Tiwari et al., 2011; Osbourn et al., 2011; Doughari, 2012; Weng et al., 2014). The most efficient extraction of saponins from P. trimera was achieved using methanol (414.49 mg EE/g dried sample). This result was significantly better than the other solvents trialed, with ethyl acetate, water, hexane and acetonitrile producing yields of 221.85, 155.30, 141.31 and 114.33 mg EE/g dried sample, respectively (Fig. 1D). Extraction method produced significantly different outcomes across the three methods, with MAE (500.18 EE/g dried sample) proving most efficient, followed by CE and UAE (401.29 and 321.76 mg EE/g dried sample, respectively). Correlations between saponin content and the various antioxidant assay methods of P. trimera (Table 3) proved to be variable, very weak correlation for AC (R2 = 0.07) and modest correlations for FRAP, CUPRAC and DRSC (R2 at 0.56, 0.64 and 0.76, respectively), suggesting that saponins make a limited contribution to antioxidant capacity. Vuong et al. (2013) reported saponin content from C. papaya leaf with various solvents decreased in the order of ethanol, methanol, acetone and water (82.88, 49.14, 31.75 and 26.36 mg EE/g dried sample, respectively), while Patel et al. (2011) reported high levels of saponins and tannins (24.05 and 17.50%, respectively) to be isolated from P. amarus. Comparatively, these data indicate that the P. trimera root is extremely rich in saponins, and that both solvent type and extraction method significantly affected saponin yield.
3.1.6. Phytochemical compounds in the P. trimera extracts Generally, saponins have high molecular weight (Doughari, 2012). Fig. 3 illustrates an HPLC chromatogram of the standard compounds (Fig. 3A) and the methanolic extract (Fig. 3B) of P. trimera. Based on the retention times (tR : min) of the individual compounds in the P. trimera extract, and the commercial standard compounds used, eleven compounds were identified and quantified in the P. trimera methanolic extract (Table 2) including (1) gallic acid, (2) chlorogenic acid, (3) caffeic acid, (4) (−)-epicatechin, (5) syringic acid, (6) p-coumaric acid, (7) rutin, (8) myricetin, (9) quercetin, (10) (+)-catechin and (11) kaempferol. In which, kaempferol content was the highest (16.72 mg/g dried sample), followed by chlorogenic acid, gallic acid, rutin and p-coumaric acid (7.72, 2.79, 2.27 and 2.14 mg/g dried sample, respectively), and the least content was caffeic acid (0.34 mg/g dried sample). However, many other major compounds in the P. trimera extracts have yet to be identified and quantified in this study, highlighting the need for further research. Pham et al. (2013) reported that ostruthin is a major constituent in the P. trimera roots and stems. These results show that P. trimera root is a very rich source of phytochemicals, and therefore warrants further investigation for their potential application in the nutraceutical and pharmaceutical industries. 3.2. Effects of solvents and extraction methods on antioxidant capacity 3.2.1. ABTS antioxidant capacity (AC) Fig. 2A shows the AC of P. trimera extracts obtained by five various solvents and three different extraction methods. Of the five solvents tested, methanol and water produced the highest AC
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Fig. 3. HPLC chromatograms of commercial standard compounds (A) and methanolic extract of P. trimera root (B), with UV–vis detector was set at 210 nm.
(174.31 mg and 171.42 mg TE/g dried sample, respectively), followed by acetonitrile, ethyl acetate and hexane extracts (129.12, 123.95 and 45.51 mg TE/g dried sample, respectively). The three extraction methods were found to be significantly different, with AC from CE (196.72 mg TE/g dried sample) being greater than both UAE and MAE (162.62 and 91.08 mg TE/g dried sample, respectively). Correlations between AC and the respective compound groups (TPC, TFC, proanthocyanidins and saponins) was found to be uniformly weak, with R2 values of 0.31, 0.14, 0.27 and 0.07, respectively (Table 3). Dhanani et al. (2013) indicated that the AC of an ethanolic extract from W. somnifera was the highest as compared to extracts from a mix of water and ethanol (9:1 v/v) and water, while the AC of the extract from MAE with ethanol was the best, followed by CE and UAE. Kamonwannasit et al. (2013) indicated the aqueous extract of Aquilaria crassna leaves exhibited substantial AC as compared to BHT (IC50 = 218.93 and 83.09 g/ml, respectively). These results illustrate that both solvent type and extraction method significantly affected the AC of P. trimera. 3.2.2. DPPH radical scavenging capacity (DRSC) The highest level of DRSC was obtained from the methanol extract (66.99 mg TE/g dried sample), which was significantly
higher than acetonitrile, ethyl acetate, water and hexane extracts (35.59, 29.81, 26.82 and 0.43 mg TE/g dried sample, respectively; Fig. 2B). Negligible DRSC activity in the hexane extract was indicative of the absence of lipophilic radical scavengers in P. trimera. No significant differences in DRSC were observed among extracts from the three extraction methods, with 73.17 mg, 71.66 and 66.72 mg TE/g dried sample recorded for CE, MAE and UAE, respectively. Table 3 indicates strong correlations between the TPC, TFC, proanthocyanidins and saponins and DRSC of P. trimera (R2 = 0.83, 0.77, 0.77 and 0.76, respectively), suggesting a similar in vitro radical scavenging capacity for each compound class. This result is similar to the findings by Vijayakumar et al. (2013), who showed the DRSC of various extracts of Illicium griffithii seeds reduced in order by methanol, ethyl acetate and hexane, and Shahriar et al. (2013) indicated chloroform, petroleum ether and methanolic extracts from W. somnifera root possessed maximum DRSC, whereas ethanol and n-hexane extracts produced the least DRSC. The DRSC of P. amarus Schum and Thonn extracts decreased in order by methanol, ethanol and petroleum ether as found by Sen and Batra (2013). Maity et al. (2013) indicated the DRSC of P. amarus root extracts reduced in the order ethyl acetate, water, crude and petroleum ether with IC50 values of 18.43, 29.52, 35.48 and 96.05 g/mL, respectively. Dhanani et al. (2013) reported that
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the ethanolic extract of W. somnifera by MAE possessed the maximal DRSC, followed by CE and least by UAE. The finding illustrates the DRSC of P. trimera strongly depended on solvents but it was weakly affected by extraction methods. 3.2.3. Cupric ion reducing antioxidant capacity (CUPRAC) Fig. 2C indicates the CUPRAC of P. trimera extracts were significantly different between five extraction solvents (18.72, 14.10, 10.22, 7.47 and 1.93 mg TE/g dried sample for methanol, acetonitrile, water, ethyl acetate and hexane, respectively). Between three different extraction methods, the MAE possessed the highest CUPRAC (21.61 mg TE/g dried sample), but was not significantly different to CE or UAE (19.45 and 19.21 mg TE/g dried sample, respectively). Correlation between CUPRAC and TPC of P. trimera was strong (R2 = 0.91; Table 3), but was less so for TFC, saponins and proanthocyanidins (R2 = 0.83, 0.64, and 0.63, respectively). Vuong et al. (2013) reported higher CUPRAC in polar extracts of Carica papaya leaf varying in the order by water, methanol, acetone and ethanol (166.66, 158.91, 133.18 and 122.47 g TE/g dried sample, respectively). By contrast, Shahriar et al. (2013) found higher CUPRAC in less polar extracts of W. somnifera root, with the chloroform extract being comparable to ascorbic acid, BHA and BHT, followed by petroleum ether, methanol, ethanol and n-hexane. The results for P. trimera extracts indicate that solvent type greatly affected the CUPRAC of P. trimera but was only marginally improved by altering the extraction method. 3.2.4. Ferric reducing antioxidant power (FRAP) The FRAP antioxidant assay (Fig. 2D) displayed a similar profile to the CUPRAC assay, save for higher relative activity in polar extracts, suggesting a greater sensitivity towards polar solventsoluble compounds. Methanol again processed the highest CUPRAC (71.82 mg TE/g dried sample) while the hexane extract (7.12 mg TE/g dried sample), as with the other assay methods, again proved to be the least active extract. There were no significant differences in FRAP between hexane and ethyl acetate extracts (16.34 mg TE/g dried sample). MAE generated a marginally improvement in the FRAP of the P. trimera methanolic extract (88.98 mg TE/g dried sample) over both UAE and CE (80.61 and 79.80 mg TE/g dried sample, respectively). Correlations between the FRAP assay and the phytochemical profile of the P. trimera extracts showed significant variability, ranging from R2 = 0.98 for TPC to R2 = 0.40 in the case of proanthocyanidins. Vijayakumar et al. (2013) reported the methanolic extracts of I. griffithii seeds had the highest FRAP (0.297 mM Fe(II)/g) as compared to ethyl acetate and hexane (0.142 and 0.108 mM Fe(II)/g, respectively), but they were much lower than the known antioxidant standards of BHT and ascorbic acid (2.312 and 2.270 mM Fe(II)/g, respectively) at 300 g/ml. Kalia et al. (2008) indicated that the FRAP of Potentilla atrosanguinea was gradually reduced depending on the extraction methods: soxhlet, microwave, ultrasound and maceration (41.43, 26.88, 19.41 and 13.64 mg of TE/g dried sample, respectively). Of these, the solvent type greatly affected the FRAP of P. trimera and methanol was the best solvent for retaining the FRAP of P. trimera, while the MAE was a superior extraction method in retaining the FRAP of P. trimera. 4. Conclusion This study demonstrates that solvent type and extraction method had great effects on the phytochemical yield and antioxidant activity of P. trimera root extracts. Of these, methanol and microwave-assisted extraction are the best selections for the extraction of biologically active phytochemicals from P. trimera root. Investigation of extraction conditions is of great interest to those researchers, not just interested in the biological activity of P.
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trimera, but also those interested in the preparation of any natural product extracts and their potential application for the functional food and pharmaceutical industries. In further study, we optimize the microwave-assisted extraction parameters for saponins and antioxidant capacity from P. trimera root.
Conflict of Interest The authors declare no conflicts of interest.
Acknowledgments We sincerely acknowledge the following funding support: Ramaciotti Foundation (ES2012/0104); Cancer Australia and Cure Cancer Australia Foundation (1033781). The authors kindly thank the Vietnamese Government through the Vietnam International Education Development-Ministry of Education and Training (Project 911) and the University of Newcastle for awarding a VIEDTUIT scholarship to Van Tang Nguyen. The authors also would like to thank Mr Duy An Nguyen for kindly providing the P. trimera roots and Mr Thanh Son Le for the supporting references from the National Institute of Medicinal Materials, Vietnam.
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Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop
Phytochemicals and antioxidant capacity of Xao tam phan (Paramignya trimera) root as affected by various solvents and extraction methods Van Tang Nguyen a,b,∗ , Michael C. Bowyer a , Quan Van Vuong a , Ian A.Van Altena a , Christopher J. Scarlett a,∗∗ a b
School of Environmental and Life Sciences, Faculty of Science and Information Technology, University of Newcastle, Ourimbah, NSW 2258, Australia Department of Food Technology, Faculty of Food Technology, Nha Trang University, No. 2 Nguyen Dinh Chieu, Nha Trang, Khanh Hoa 8458, Viet Nam
a r t i c l e
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Article history: Received 22 November 2014 Received in revised form 22 January 2015 Accepted 26 January 2015 Keywords: Xao tam phan Paramignya trimera Phytochemicals Antioxidant Solvent Extraction method
a b s t r a c t Xao tam phan (Paramignya trimera (Oliv.) Guillaum) is a Vietnamese traditionally medicinal plant used in the treatment of numerous cancers. The preparation of Xao tam phan extracts including solvent type and extraction method have significant effects on extraction efficiency, phytochemical profile and biological activity. This study aimed to investigate the effects of five various solvents (water, acetonitrile, methanol, ethyl acetate and hexane) and three different extraction methods (conventional, ultrasoundassisted and microwave-assisted) on phytochemical yield and antioxidant capacity of P. trimera root from Vietnam. The results indicate that methanol extracted the maximal yield of phytochemicals from P. trimera and exhibited the greatest antioxidant capacity, with eleven compounds were identified and quantified. Microwave-assisted extraction produced the maximal phytochemical yields (except for total flavonoids) and antioxidant capacity, when compared to conventional and ultrasound-assisted extractions. These data reveal that the use of methanol and microwave-assisted extraction are recommended for extraction of biologically active phytochemicals from P. trimera root for application in the nutraceutical and/or pharmaceutical industries. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Xao tam phan (Paramignya trimera (Oliv.) Guillaum) has a synonym of Atalantia trimera Oliv., which belongs to the Paramignya genus of Citrus family (Rutaceae) and is well known as a medicinal plant in Thailand and Vietnam (Nguyen et al., 2013). Recent studies on the P. trimera have revealed hepatoprotective and cytotoxic activity from a crude methanolic extract (Nguyen et al., 2013). Importantly, methanolic extract, n-hexane fraction and an individual compound isolated from P. trimera, named ostruthin (6-(3,7-dimethyl-2,6-octadienyl) 7-hydroxy-2H-1-benzopyran-2one) demonstrated cytotoxic activity against five cancer cell lines in vitro, especially, the partitioned n-hexane fraction and ostruthin
∗ Corresponding author. School of Environmental and Life Sciences, Faculty of Science and Information Technology, University of Newcastle, Brush Road, Ourimbah, NSW 2258, Australia. Tel.: +61 434238842; fax: +61 243484145. ∗∗ Corresponding author. Tel.: +61 243484680; fax: +61 243484145. E-mail addresses: [email protected] (V.T. Nguyen), [email protected] (C.J. Scarlett). http://dx.doi.org/10.1016/j.indcrop.2015.01.051 0926-6690/© 2015 Elsevier B.V. All rights reserved.
from the methanolic extract displayed activity against hepatocellular carcinoma (Hep-G2) and human epithelial cervical carcinoma (Hela) cell lines with IC50 values of 39.61 and 5.36 g/ml, respectively. Pham et al. (2013) also isolated and quantitatively analysed ostruthin to be a major compound in the P. trimera roots and stems, while Li et al. (2012) showed that ostruthin displayed potent activity against pancreatic cancer cell lines. These studies indicate that P. trimera contains key biologically active phytochemicals, however it is imperative to optimize extraction conditions to maximize the yield of phytochemicals extracted for potential medicinal exploitation. Solvent type and extraction method directly affects the extraction efficiency and relates to production costs such as extraction time, solvent volume, energy costs as well as the effect on humans and the environment (Dai and Mumper, 2010; Azmir et al., 2013; Kalia et al., 2008). In recent years, advanced extraction techniques have been developed and applied to optimize the extraction of phenolics from plant materials. For example, ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE) and techniques using compressed fluids such as subcritical water extraction (SWE), supercritical fluid extraction (SFE), pressurized fluid
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extraction (PFE) or accelerated solvent extraction (ASE) (Tiwari et al., 2011; Doughari, 2012) have proven to significantly reduce extraction time as well as gaining improved extraction efficiency. For this reason, careful consideration for the selection of solvent type and extraction method is a crucial step in the preparation of extracts from plant materials. Very few studies have reported the physicochemical properties and biological activity of P. trimera extracts and none have focused on selection of solvent and extraction method. With this in mind, our study investigated the effects of various solvent types and extraction methods on the phytochemical yield and antioxidant capacity of P. trimera root extracts. Our data reveal the optimal conditions for solvent type and extraction method for the preparation of crude P. trimera root extracts for potential application in the nutraceutical and/or pharmaceutical industries. 2. Materials and methods 2.1. Plant materials P. trimera root was collected from Ninh Hoa district, Khanh Hoa province, Vietnam in January 2014 and identified by the National Institute of Medicinal Materials, Ministry of Health, Vietnam. After collection, the fresh samples were rinsed in deionized water to remove sand and soil, drained and left to dry under the sun (34.5 ± 1 ◦ C) to constant weight, and then packed in vacuum sealed polyamide (PA) bags and stored at −18 ◦ C until used. The residual moisture of the dried samples was determined according to the AOAC official methods of analysis (AOAC, 1998) using a hot-air oven (Anax Pty Ltd., NSW, Australia) at 120 ◦ C for 5 h. 2.2. Analytical chemicals All chemicals used were of analytical grade. 2,2 -azino-bis3-ethylbenzothiazoline-6-sulphonic acid (ABTS), 1,1-diphenyl2-picryl-hydrazil (DPPH), Folin-Ciocalteu, trolox, neocuproine, 2,4,6-tripyridyl-s-triazine (TPTZ), iron (III) chloride and standard compounds including gallic acid, p-coumaric acid, caffeic acid, 5,7-dimethoxycoumarin, quercetin, kaempferol, rutin, syringic acid, chlorogenic acid, escin, (+)-catechin, (−)-epicatechin, (±)naringenin, myricetin, luteolin, apigenin, (−)-epigallocatechin gallate, -sitosterol--D-glucoside, gemcitabine, -sitosterol, and ascorbic acid were purchased from Sigma–Aldrich Pty Ltd. (NSW, Australia). Vanillin, potassium persulfate, methanol and ethanol were obtained from Merck (Darmstadt, Germany). Copper (II) chloride was purchased from Standard Laboratories (Victoria, Australia). Sodium acetate trihydrate was obtained from Government Stores Department (Australia). Ammonium acetate was purchased from BDH Chemicals (Victoria, Australia). Aluminium chloride (anhydrous) was obtained from Acros (New Jersey, USA). Sodium hydroxide was purchased from Ajax chemicals (NSW, Australia). Sodium carbonate anhydrous was purchased from Chem-supply (Gillman, South Australia). Sulfuric acid and hydrochloric acid were purchased from Ajax Finechemicals (NSW, Australia), and acetic acid was obtained from BDH Laboratory Supplies (Poole, England). 2.3. Preparation of extracts To select the best solvent for further study, five various solvents were evaluated including water (WT), acetonitrile (AT), methanol (MT), ethyl acetate (EA) and hexane (HX). Then, three different extraction methods were applied with selected solvent, i.e. conventional extraction (CE), ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE). The extracts of P. trimera root were prepared according to the method described by Nguyen
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et al. (2011) with some modifications. The extraction procedures were as follows: For effect of solvents: 0.2 g of dried sample was extracted with 20 mL of various solvents applying UAE using an ultrasonic cleaner Soniclean 1000HD (Soniclean Pty Ltd., South Australia) at a power of 150 W and 45 ◦ C for 60 min. For effect of extraction methods: 0.2 g of dried sample was extracted with 20 mL of selected solvent, the extraction process included two stages: the first stage was set at room temperature (20 ± 1 ◦ C) for 20 min, and the second stage was extracted using the various methods for 60 min. CE was carried out using a waterbath (Buchi, Flawil, Switzerland) at 45 ◦ C, UAE was performed using an ultrasonic cleaner Soniclean 1000HD at a power of 150 W and 45 ◦ C, and MAE was carried out using a microwave oven (Sharp Carousel, Sharp Corporation, Thailand) at a power of 360 W, with the initial irradiation time of 15 s, then 5 s for each further 5 min (total irradiation time was applied to be 70 s). After extraction, the extracts were rapidly cooled to room temperature by ice water and then filtered through qualitative no. 1 filter papers (Bacto Laboratories Pty Ltd., NSW, Australia) to obtain extracts for further analysis. To determine the extraction yield, 2 mL of extract was dried in a hot-air oven (Anax Pty Ltd., NSW, Australia) at 100 ◦ C for 4 h to a constant weight (extraction yield = g of dried extract/100 g of dried sample).
2.4. Determination of phytochemical compounds 2.4.1. Total phenolic content (TPC) TPC of P. trimera extracts was determined using the Folin–Ciocalteu method as previously described by Nguyen et al. (2011) and Vuong et al. (2013) with some modifications. Briefly, 0.5 mL of the extracts were diluted 2 times and mixed with 2.5 mL of 10% (v/v) Folin–Ciocalteu reagent in distilled water. The mixture was left to settle for 6 min, then 2 mL of 7.5% (w/v) Na2 CO3 solution was added and incubated in the dark at room temperature for 1 h. The absorbance of mixture was measured at 765 nm using a UV–vis spectrophotometer (Cary 50 Bio Varian, Australia). Methanol and gallic acid were used as a control and standard. TPC was expressed in mg gallic acid equivalents (GAE)/g dried sample.
2.4.2. Total flavonoid content (TFC) TFC of P. trimera extracts was performed according to the method described by Vuong et al. (2013) with some modifications. 0.5 mL of extract was mixed with 2 mL of distilled water and 0.15 mL of 5% (w/v) Na2 CO3 solution and incubated in the dark at room temperature for 6 min. 0.15 mL of 10% (w/v) AlCl3 solution was then added and incubated for a further 6 min. Finally, 2 mL of 4% (w/v) NaOH solution and 0.7 mL of distilled water were added and incubated for 15 min. The absorbance of mixture was measured at 510 nm using a UV–vis spectrophotometer. Methanol and rutin were used as a control and standard, respectively. TFC was expressed as mg of rutin equivalents (RE)/g dried sample.
2.4.3. Proanthocyanidin content Proanthocyanidin content of P. trimera extracts was determined using the method described by Vuong et al. (2013) with some modifications. Briefly, 0.5 mL of extract was mixed with 3 mL of 4% (w/v) vanillin solution, then 1.5 mL of concentrated HCl was added and the mixture was incubated in the dark at room temperature for 15 min. The absorbance of mixture was measured at 500 nm using a UV–vis spectrophotometer. Methanol and catechin were used as a control and standard, respectively. Proanthocyanidin content was expressed as mg of catechin equivalents (CE)/g dried sample.
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2.4.4. Saponin content Saponin content of P. trimera extracts was determined according to the method described by Vuong et al. (2013) with the modified procedure to reduce analytical time. Briefly, 0.5 mL of the extract was mixed with 0.5 mL of 8% (w/v) vanillin solution, then 5 mL of 72% (v/v) H2 SO4 solution was added. The mixture was then incubated at 70 ◦ C for 10 min and rapidly cooled by ice water to room temperature. The absorbance of mixture was measured at 560 nm using a UV–vis spectrophotometer. Methanol and escin were used as a control and standard, respectively. Saponin content was expressed as mg of escin equivalents (EE)/g dried sample.
2.4.5. Identification and quantification of phytochemical compounds Phytochemical compounds in the P. trimera extracts were identified and quantified based on the methods described by Maity et al. (2013) and Plaza et al. (2014) with some modified procedures using HPLC system (Shimadzu, Japan). Briefly, the external standard solutions were prepared by dissolving standard compounds in methanol at a concentration of 200 g/mL, then 200 L of each standard solution was taken to make a mixed standard solution. Trolox was used as an internal standard. The extracts and standard solutions were then filtered through 0.45 m nylon membranes (phenex syringe filters) and 20 L of extracts or standard solutions were individually injected by an auto injector (SIL-10AV VP) onto a phenosphere-NEXT 5 u C18 column (250 mm × 4.6 mm × 5 m, Phenomenex, Torrance, CA, USA), which was maintained at 35 ◦ C by a column oven (CTO-10A VP). The mobile phase consisted of 0.2% orthophosphoric acid in distilled water (A) and 100% acetonitrile (B). Flow rate was set at 1 mL/min with the gradient as follows: 0–5 min, 0% B; 5–20 min, 20% B; 20–30 min, 30% B; 30–110 min, 30% B; 110–120 min, 0% B. Phytochemical compounds were detected at 210 nm using a UV–vis detector SPD-10AV VP. Identification of individual phytochemical compounds was achieved by comparing retention times (tR ) of compounds in the P. trimera extracts with those of commercial standard compounds, including gallic acid, p-coumaric acid, caffeic acid, 5,7-dimethoxycoumarin, quercetin, kaempferol, rutin, syringic acid, chlorogenic acid, escin, (+)-catechin, (−)-epicatechin, (±)-naringenin, myricetin, luteolin, apigenin, (−)-epigallocatechin gallate, -sitosterol--D-glucoside, gemcitabine, -sitosterol, and ascorbic acid, in terms of individual standards, mixed standard solution and mix of extract with each standard (1:1 v/v). Quantification of individual phytochemical compounds was performed using the standard equations based on peak area of the commercial standard compounds and the individual phytochemical compounds in the P. trimera extracts.
2.5.2. DPPH radical scavenging capacity (DRSC) DRSC of P. trimera extracts was measured based on the method reported by Vuong et al. (2013) and Pisoschi and Negulescu (2011) with some modifications. Briefly, a stock solution of 0.024% (w/v) DPPH (1,1-diphenyl-2-picryl-hydrazil) in methanol was prepared and stored at −18 ◦ C. Before use, a working solution was prepared by diluting 1.0 mL of stock solution with 45 mL of methanol to obtain an absorbance of 1.1 ± 0.02 at 515 nm. For the DPPH reaction, 0.15 mL of the extract was mixed with 2.850 mL of the working solution and incubated for 3 h in the dark at room temperature. The absorbance of mixture was measured at 515 nm using a UV–vis spectrophotometer. The results were expressed as mg trolox equivalents (TE)/g dried sample. 2.5.3. Cupric ion reducing antioxidant capacity (CUPRAC) CUPRAC of P. trimera extracts were analyzed using the method described by Vuong et al. (2013) and Pisoschi and Negulescu (2011). Briefly, 1.0 mL of 10 mM CuCl2 solution was mixed with 1.0 mL of 7.5 mM neocuproine and 1.0 mL of 7.7% (w/v) NH4 Ac solutions. 1.1 mL of the extract was then added and incubated in the dark at room temperature for 1.5 h. The absorbance of mixture was measured at 450 nm using a UV–vis spectrophotometer. The results were expressed as mg trolox equivalents (TE)/g dried sample. 2.5.4. Ferric reducing antioxidant power (FRAP) FRAP of P. trimera extracts was determined based on the method reported by Kamonwannasit et al. (2013) and Pisoschi and Negulescu (2011) with some modifications. Reagent A: 300 mM acetate buffer solution, pH 3.6; Reagent B: 10 mM TPTZ (2, 4, 6tripyridyl-s-triazine) solution in 40 mM HCl; and Reagent C: 20 mM ¨ FeCl3 Y6H 2 O solution. Before use, the fresh FRAP solution was prepared by mixing reagents A, B and C at a ratio of 10:1:1. For the FRAP reaction, 0.15 mL of the extract was mixed with 2.850 mL of the fresh FRAP solution and incubated in the dark at room temperature for 30 min. The absorbance of mixture was read at 593 nm using a UV–vis spectrophotometer. The results were expressed as mg trolox equivalents (TE)/g dried sample. 2.6. Statistical analysis All experiments were performed at least in triplicate. The data were analyzed using SPSS software (Version 16.0, Chicago, IL, U. S. A.) and expressed as mean ± standard deviation (n = 3). Statistical comparisons were made using one-way analysis of variance (ANOVA) and Tukey HSD tests. Differences were considered to be significant when P-values were below 0.05 (P < 0.05). 3. Results and discussion
2.5. Determination of antioxidant capacity 2.5.1. ABTS antioxidant capacity (AC) AC of P. trimera extracts was determined using the ABTS assay described by Kamonwannasit et al. (2013) and Pisoschi and Negulescu (2011) with some modifications. Briefly, a stock solution was prepared by mixing 7.4 mM ABTS•+ (2,2’-azinobis-3-ethylbenzothiazoline-6-sulphonic acid) and 2.6 mM K2 S2 O8 solutions (1:1 ratio) and then incubated in the dark at room temperature for 12 h, then stored at −18 ◦ C. Before use, a working solution was prepared by mixing 1 mL of stock solution with 60 mL of methanol to obtain an absorbance of 1.1 ± 0.02 at 734 nm. For the ABTS reaction, 0.15 mL of the extract was mixed with 2.85 mL of the working solution and incubated for 2 h. The absorbance of mixture was measured at 734 nm using a UV–vis spectrophotometer. The results were expressed as mg trolox equivalents (TE)/g dried sample.
3.1. Effects of solvents and extraction methods on the extraction and phytochemical yields 3.1.1. Extraction yield Extraction yield refers to the percentage of crude extract obtained from a dried plant sample through a solvent extraction procedure for further isolation and utilization. The residual moisture of dried sample after drying was determined to be 8.74%. Table 1 shows that of the five solvents used, water produced the highest extraction yield (11.48 g dried extract/100 g dried sample). This yield was not however found to be significantly different from the methanol extract (11.04 g dried extract/100 g dried sample). Both the aqueous and methanol extracts were found to be significantly greater (P < 0.05) than those of acetonitrile, ethyl acetate and hexane extracts (2.31, 2.32 and 2.08 g dried extract/100 g dried sample, respectively). These findings are supported by Rahman et al. (2013), who demonstrated the phytochemical content and
V.T. Nguyen et al. / Industrial Crops and Products 67 (2015) 192–200 Table 1 Extraction yield of P. trimera root obtained by five solvents and three extraction methods. Solvent and extraction method
Water Acetonitrile Methanol Ethyl acetate Hexane Conventional Ultrasound-assisted Microwave-assisted
Extraction yield (g dried extract/100 g dried sample) 11.48 2.31 11.04 2.32 2.08 12.02 10.93 14.39
± ± ± ± ± ± ± ±
0.95a, * 0.08b 0.23a 0.48b 1.17b 1.09AB 1.97B 0.32A
* Means and standard deviations from three replicates. Different letters as indicated by lowercase (for solvent) and uppercase (for extraction method) in each group were significantly different (P < 0.05).
antioxidant potential of Colubrina asiatica were affected by extraction solvent polarities, such as 100% ethanol, 50% ethanol, and water. Extraction yields showed a clear correlation with solvent character, with the polar, protic solvents (methanol and water) extracting significantly greater yield than their aprotic counterparts. The importance of protic solvent character is illustrated by the difference in obtained yield for the methanol and acetonitrile extracts. These two solvents possess similar dielectric constant values (methanol: 32.63, acetonitrile: 37.5; Maryott and Smith, 1951), considered an accepted measure of polarity, yet the protic character of methanol produces a fivefold increase in relative extraction yield. We propose that the efficiency of methanol as a solvent relates to its intermediate polarity, which allows it to solvate low molecular weight organics compounds possessing protonatable functional groups (e.g. COOH, OH). Based on the results obtained, methanol was selected as the solvent for the enhanced extraction procedures. MAE produced a higher extraction yield (14.39 g dried extract/100 g dried sample) relative to CE and UAE (12.02 and 10.93 g dried extract/100 g dried sample, respectively, Table 1). While statistical analysis found a significant difference between the efficiencies of MAE and UAE methods, neither was significantly different to the CE method. This finding is in similar to research conducted by Dhanani et al. (2013), who reported that MAE of Withnaia somnifera using ethanol and mix of water and ethanol (9:1 v/v) exceeded yields obtained by CE and UAE. Compared to other plant species, P. trimera produced lower yield than that of papaya leaf (19.15 g dried extract/100 g dried sample) as indicated by Vuong et al. (2013). Chisté et al. (2014) reported the yield of solid extract from Capsicum villosum fruit pulps obtained the highest level by mix of ethanol and ethyl acetate (1:1 v/v; 46.4 g dried extract/100 g dried sample), followed by ethyl acetate, water, ethanol, and mix of ethanol and water (1:1 v/v; 41.9, 14.7, 11.1 and 10.8 g dried extract/100 g dried sample, respectively). These data indicate that the extraction yield of P. trimera was greatly affected by solvents, as well as extraction methods. 3.1.2. Total phenolic content (TPC) Phenolic compounds in plants have strong links to their antioxidant and anticancer activities (Khoddami et al., 2013; Dai and Mumper, 2010). Fig. 1A indicates that among the five solvents used, methanol extracted the highest TPC from P. trimera (33.36 mg GAE/g dried sample) and it was significantly higher than those obtained from water and acetonitrile (25.06 and 22.33 mg GAE/g dried sample, respectively), while TPCs levels extracted by ethyl acetate and hexane were very low (3.32 and 4.58 mg GAE/g dried sample, respectively). It appeared that the TPC obtained from MAE (42.25 mg GAE/g dried sample) was higher than those from EC and
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Table 2 Phytochemical content of methanolic extract of P. trimera root obtained by microwave-assisted extraction. Retention time (min) Phytochemical compound Content(mg/g dried sample) 4.075 32.517 33.622 34.232 35.929 36.508 38.258 43.100 64.400 80.785 88.517 a
gallic acid chlorogenic acid caffeic acid (−)-epicatechin syringic acid p-coumaric acid rutin myricetin quercetin (+)-catechin kaempferol
2.79 7.72 0.34 1.22 0.66 2.14 2.27 1.03 0.57 1.16 16.72
± ± ± ± ± ± ± ± ± ± ±
0.17a 0.37 0.03 0.05 0.08 0.01 0.01 0.04 0.09 0.04 0.10
Means and standard deviations were of three replicates.
UAE (39.86 and 37.91 mg GAE/g dried sample, respectively), however any differences observed were not significant. Table 3 shows the correlation amongst three of the four assay methods (DRSC, CUPRAC and FRAP) for TPC was strong (R2 = 0.81, 0.91 and 0.98, respectively), with only AC showing poor reliability (R2 = 0.31). Vuong et al. (2013) reported the TPC of papaya leaf extracted by various solvents decreased in the order water, methanol, acetone and ethanol (23.06, 15.03, 10.71 and 9.43 mg GAE/g dried sample, respectively), while Poh-Hwa et al. (2011) indicated the methanolic extracts of Phyllanthus species (50.12–68.70 mg GAE/g dried sample) had higher TPC than water extracts (41.19–55.38 mg GAE/g dried sample). The TPC of Phyllanthus amarus (mg/100 g dried sample) gradually reduced by various solvents (methanol: 212.67, ethanol: 208.09 and petroleum ether: 203.54) as reported by Sen and Batra (2013), while Shahriar et al. (2013) found the TPC in W. somnifera root extracts decreased in the order chloroform, petroleum ether, methanol, ethanol and n-hexane (60.99, 56.58, 23.86, 5.46 and 1.43 mg GAE/g dried sample, respectively). The relatively low levels of TPC obtained for ethanol in this study are brought into question by recently published findings on the same plant species by Dhanani et al. (2013) who obtained significantly higher TPC by MAE, CE and UAE (40.96, 35.93 and 24.72 mg GAE/g dried sample, respectively). Garofulic´ı et al. (2013) similarly indicated the MAE of anthocyanins and phenolic acids from Prunus cerasus var. obtained higher efficiency than that by CE. These phenomena may be explained by microwaves causing a localized temperature rise in plant tissue, leading to cellular disruption and consequently the migration of phenolic compounds to the surrounding solvent. In comparison with other plants, the TPC of P. trimera was much higher than that of sage plants (Ibtissem et al., 2013) (approximately 2.3 mg GAE/g dried sample), Oolong tea (Nguyen et al., 2011) and papaya leaf (Vuong et al., 2013) (13.58 and 15.03 mg GAE/g dried sample, respectively), however it was much lower than those of P. amarus, P. urinaria and P. niruri (PohHwa et al., 2011) (50.24, 62.56 and 68.70 mg GAE/g dried sample, respectively). Our findings show a link between solvent character and TPC extraction efficiency from P. trimera, with methanol was the most effective solvent because of its protic character and intermediate polarity which enables effective solvation of carboxyl and Table 3 The correlations between phytochemicals and antioxidant capacity of P. trimera root extracts obtained by five solvents and three extraction methods. Correlations (R2 )a
TPC
TFC
Proanthocyanidins
Saponins
AC DRSC CUPRAC FRAP
0.31 0.83 0.91 0.98
0.14 0.77 0.83 0.61
0.27 0.77 0.63 0.40
0.07 0.76 0.64 0.56
a
Based on values obtained from five solvents and three extraction methods.
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Fig. 1. (A) Total phenolic content (TPC); (B) total flavonoid content (TFC); (C) proanthocyanidin content; and (D) saponin content of P. trimera root obtained by five solvents and three extraction methods. Different letters within the columns denotes a significant difference between groups for TPC, proanthocyanidins and saponins (P < 0.05) and TFC (P < 0.0005). WT: water; AT: acetonitrile, MT: methanol; EA: ethyl acetate and HX: hexane. CE: conventional extraction, UAE: ultrasound-assisted extraction and MAE: microwave-assisted extraction.
phenol-containing species. Besides, the efficiency of extracting TPC from P. trimera was also affected by extraction methods. 3.1.3. Total flavonoid content (TFC) TFC of P. trimera extracted by various solvents as follows: methanol > acetonitrile > hexane > water > ethyl acetate (Fig. 1B). Methanolic extract possessed significantly higher TFC (14.11 mg RE/g dried sample; P < 0.0005) as compared to extracts from acetonitrile, hexane, water and ethyl acetate (12.00, 3.76, 2.72 and 2.27 mg RE/g dried sample, respectively). No significant differences were observed for TFC between the three extraction methods, with a range of 13.63–15.74 mg RE/g dried sample. Table 3 indicates that the TFC of P. trimera had strong correlations with its DRSC and CUPRAC (R2 = 0.77 and 0.83, respectively) as compared to AC and FRAP (R2 = 0.14 and 0.61, respectively). Shahriar et al. (2013) reported the TFC of W. somnifera root extracts reduced by various solvents in the order chloroform, petroleum ether, methanol, ethanol and n-hexane (122.09, 92.14, 88.76, 66.01 and 44.95 mg quercetin equivalent/g dried sample, respectively). Chisté et al. (2014) found the TFC of C. villosum fruit pulps (mg catechin equivalent/g dried sample) gradually reduced from a mix of ethanol and water (1:1 v/v; 3.8) to water, ethanol, a mix of ethanol and ethyl acetate (1:1 v/v; 2.5, 0.3 and 0.04, respectively), and lastly to ethyl acetate (0.03). Similarly, Vuong et al. (2013) also indicated the TFC of papaya leaf (mg catechin equivalent/g dried sample) extracted by ethanol was the highest (17.07), followed by acetone (16.41), methanol (11.96) and water (6.44). Garofulic´ı et al. (2013) found the total anthocyanins and total phenolic acids efficiency from P. cerasus var. obtained by MAE
were higher than those gained by CE. As seen with the TPC, these data indicate that the TFC extractability from P. trimera strongly depended on solvent type as well as extraction methods, with higher levels generally obtained in the polar, protic organic solvents. 3.1.4. Proanthocyanidin content Proanthocyanidin content isolated from P. trimera was found to be highest in ethyl acetate extracts (5.28 mg CE/g dried sample), followed by methanol, acetonitrile, water and hexane (5.00, 3.92, 2.99 and 1.77 mg CE/g dried sample, respectively, Fig. 1C). Methods of CE, MAE and UAE did not significantly differ in proanthocyanidin content (5.66, 5.51 and 5.54 mg CE/g dried sample, respectively). Table 3 shows the correlations between proanthocyanidin content and antioxidant capacity of P. trimera extracts in order by DRSC (0.77), followed by CUPRAC (0.63), FRAP (0.40) and AC (0.27). Vuong et al. (2013) found the maximum proanthocyanidin content of Carica papaya leaf was following extraction with ethanol (7.91 mg CE/g dried sample) as compared to those extracted by acetone, methanol and water (6.39, 4.86 and 1.91 mg CE/g dried sample, respectively). These results indicate that solvent type had significant effects on extraction efficiency of proanthocyanidins from P. trimera, however extraction methods did not greatly influence proanthocyanidin content from this sample. 3.1.5. Saponin content Saponins contain many valuable phytochemicals such as triterpenes, lupeol, triterpene glycosides, sterols, glycyrrhizic acid and triterpene scaffolds that display antiviral, antimicrobial,
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Fig. 2. (A) ABTS antioxidant capacity (AC); (B) DPPH radical scavenging capacity (DRSC); (C) cupric ion reducing antioxidant capacity (CUPRAC); and (D) ferric reducing antioxidant power (FRAP) of P. trimera root obtained by five solvents and three extraction methods. Different letters within the columns denotes a significant difference between groups for AC, DRSC and CUPRAC (P < 0.05) and FRAP (P < 0.005). WT: water; AT: acetonitrile, MT: methanol; EA: ethyl acetate and HX: hexane. CE: conventional extraction, UAE: ultrasound-assisted extraction and MAE: microwave-assisted extraction.
antioxidative, anti-hyperglycaemic, hepatoprotective, cardioprotective, antiherbivore/cytotoxic, immunomodulatory, antidiarrhoeal, anti-HIV, antitumour, antihepatitis B and C, antiinflammatory and anti-cancer properties (Amoros et al., 1987; Yoshikawa et al., 2003; Lacaille-Dubois, 2005; Mandal et al., 2006; Podolak et al., 2010; Tiwari et al., 2011; Osbourn et al., 2011; Doughari, 2012; Weng et al., 2014). The most efficient extraction of saponins from P. trimera was achieved using methanol (414.49 mg EE/g dried sample). This result was significantly better than the other solvents trialed, with ethyl acetate, water, hexane and acetonitrile producing yields of 221.85, 155.30, 141.31 and 114.33 mg EE/g dried sample, respectively (Fig. 1D). Extraction method produced significantly different outcomes across the three methods, with MAE (500.18 EE/g dried sample) proving most efficient, followed by CE and UAE (401.29 and 321.76 mg EE/g dried sample, respectively). Correlations between saponin content and the various antioxidant assay methods of P. trimera (Table 3) proved to be variable, very weak correlation for AC (R2 = 0.07) and modest correlations for FRAP, CUPRAC and DRSC (R2 at 0.56, 0.64 and 0.76, respectively), suggesting that saponins make a limited contribution to antioxidant capacity. Vuong et al. (2013) reported saponin content from C. papaya leaf with various solvents decreased in the order of ethanol, methanol, acetone and water (82.88, 49.14, 31.75 and 26.36 mg EE/g dried sample, respectively), while Patel et al. (2011) reported high levels of saponins and tannins (24.05 and 17.50%, respectively) to be isolated from P. amarus. Comparatively, these data indicate that the P. trimera root is extremely rich in saponins, and that both solvent type and extraction method significantly affected saponin yield.
3.1.6. Phytochemical compounds in the P. trimera extracts Generally, saponins have high molecular weight (Doughari, 2012). Fig. 3 illustrates an HPLC chromatogram of the standard compounds (Fig. 3A) and the methanolic extract (Fig. 3B) of P. trimera. Based on the retention times (tR : min) of the individual compounds in the P. trimera extract, and the commercial standard compounds used, eleven compounds were identified and quantified in the P. trimera methanolic extract (Table 2) including (1) gallic acid, (2) chlorogenic acid, (3) caffeic acid, (4) (−)-epicatechin, (5) syringic acid, (6) p-coumaric acid, (7) rutin, (8) myricetin, (9) quercetin, (10) (+)-catechin and (11) kaempferol. In which, kaempferol content was the highest (16.72 mg/g dried sample), followed by chlorogenic acid, gallic acid, rutin and p-coumaric acid (7.72, 2.79, 2.27 and 2.14 mg/g dried sample, respectively), and the least content was caffeic acid (0.34 mg/g dried sample). However, many other major compounds in the P. trimera extracts have yet to be identified and quantified in this study, highlighting the need for further research. Pham et al. (2013) reported that ostruthin is a major constituent in the P. trimera roots and stems. These results show that P. trimera root is a very rich source of phytochemicals, and therefore warrants further investigation for their potential application in the nutraceutical and pharmaceutical industries. 3.2. Effects of solvents and extraction methods on antioxidant capacity 3.2.1. ABTS antioxidant capacity (AC) Fig. 2A shows the AC of P. trimera extracts obtained by five various solvents and three different extraction methods. Of the five solvents tested, methanol and water produced the highest AC
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Fig. 3. HPLC chromatograms of commercial standard compounds (A) and methanolic extract of P. trimera root (B), with UV–vis detector was set at 210 nm.
(174.31 mg and 171.42 mg TE/g dried sample, respectively), followed by acetonitrile, ethyl acetate and hexane extracts (129.12, 123.95 and 45.51 mg TE/g dried sample, respectively). The three extraction methods were found to be significantly different, with AC from CE (196.72 mg TE/g dried sample) being greater than both UAE and MAE (162.62 and 91.08 mg TE/g dried sample, respectively). Correlations between AC and the respective compound groups (TPC, TFC, proanthocyanidins and saponins) was found to be uniformly weak, with R2 values of 0.31, 0.14, 0.27 and 0.07, respectively (Table 3). Dhanani et al. (2013) indicated that the AC of an ethanolic extract from W. somnifera was the highest as compared to extracts from a mix of water and ethanol (9:1 v/v) and water, while the AC of the extract from MAE with ethanol was the best, followed by CE and UAE. Kamonwannasit et al. (2013) indicated the aqueous extract of Aquilaria crassna leaves exhibited substantial AC as compared to BHT (IC50 = 218.93 and 83.09 g/ml, respectively). These results illustrate that both solvent type and extraction method significantly affected the AC of P. trimera. 3.2.2. DPPH radical scavenging capacity (DRSC) The highest level of DRSC was obtained from the methanol extract (66.99 mg TE/g dried sample), which was significantly
higher than acetonitrile, ethyl acetate, water and hexane extracts (35.59, 29.81, 26.82 and 0.43 mg TE/g dried sample, respectively; Fig. 2B). Negligible DRSC activity in the hexane extract was indicative of the absence of lipophilic radical scavengers in P. trimera. No significant differences in DRSC were observed among extracts from the three extraction methods, with 73.17 mg, 71.66 and 66.72 mg TE/g dried sample recorded for CE, MAE and UAE, respectively. Table 3 indicates strong correlations between the TPC, TFC, proanthocyanidins and saponins and DRSC of P. trimera (R2 = 0.83, 0.77, 0.77 and 0.76, respectively), suggesting a similar in vitro radical scavenging capacity for each compound class. This result is similar to the findings by Vijayakumar et al. (2013), who showed the DRSC of various extracts of Illicium griffithii seeds reduced in order by methanol, ethyl acetate and hexane, and Shahriar et al. (2013) indicated chloroform, petroleum ether and methanolic extracts from W. somnifera root possessed maximum DRSC, whereas ethanol and n-hexane extracts produced the least DRSC. The DRSC of P. amarus Schum and Thonn extracts decreased in order by methanol, ethanol and petroleum ether as found by Sen and Batra (2013). Maity et al. (2013) indicated the DRSC of P. amarus root extracts reduced in the order ethyl acetate, water, crude and petroleum ether with IC50 values of 18.43, 29.52, 35.48 and 96.05 g/mL, respectively. Dhanani et al. (2013) reported that
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the ethanolic extract of W. somnifera by MAE possessed the maximal DRSC, followed by CE and least by UAE. The finding illustrates the DRSC of P. trimera strongly depended on solvents but it was weakly affected by extraction methods. 3.2.3. Cupric ion reducing antioxidant capacity (CUPRAC) Fig. 2C indicates the CUPRAC of P. trimera extracts were significantly different between five extraction solvents (18.72, 14.10, 10.22, 7.47 and 1.93 mg TE/g dried sample for methanol, acetonitrile, water, ethyl acetate and hexane, respectively). Between three different extraction methods, the MAE possessed the highest CUPRAC (21.61 mg TE/g dried sample), but was not significantly different to CE or UAE (19.45 and 19.21 mg TE/g dried sample, respectively). Correlation between CUPRAC and TPC of P. trimera was strong (R2 = 0.91; Table 3), but was less so for TFC, saponins and proanthocyanidins (R2 = 0.83, 0.64, and 0.63, respectively). Vuong et al. (2013) reported higher CUPRAC in polar extracts of Carica papaya leaf varying in the order by water, methanol, acetone and ethanol (166.66, 158.91, 133.18 and 122.47 g TE/g dried sample, respectively). By contrast, Shahriar et al. (2013) found higher CUPRAC in less polar extracts of W. somnifera root, with the chloroform extract being comparable to ascorbic acid, BHA and BHT, followed by petroleum ether, methanol, ethanol and n-hexane. The results for P. trimera extracts indicate that solvent type greatly affected the CUPRAC of P. trimera but was only marginally improved by altering the extraction method. 3.2.4. Ferric reducing antioxidant power (FRAP) The FRAP antioxidant assay (Fig. 2D) displayed a similar profile to the CUPRAC assay, save for higher relative activity in polar extracts, suggesting a greater sensitivity towards polar solventsoluble compounds. Methanol again processed the highest CUPRAC (71.82 mg TE/g dried sample) while the hexane extract (7.12 mg TE/g dried sample), as with the other assay methods, again proved to be the least active extract. There were no significant differences in FRAP between hexane and ethyl acetate extracts (16.34 mg TE/g dried sample). MAE generated a marginally improvement in the FRAP of the P. trimera methanolic extract (88.98 mg TE/g dried sample) over both UAE and CE (80.61 and 79.80 mg TE/g dried sample, respectively). Correlations between the FRAP assay and the phytochemical profile of the P. trimera extracts showed significant variability, ranging from R2 = 0.98 for TPC to R2 = 0.40 in the case of proanthocyanidins. Vijayakumar et al. (2013) reported the methanolic extracts of I. griffithii seeds had the highest FRAP (0.297 mM Fe(II)/g) as compared to ethyl acetate and hexane (0.142 and 0.108 mM Fe(II)/g, respectively), but they were much lower than the known antioxidant standards of BHT and ascorbic acid (2.312 and 2.270 mM Fe(II)/g, respectively) at 300 g/ml. Kalia et al. (2008) indicated that the FRAP of Potentilla atrosanguinea was gradually reduced depending on the extraction methods: soxhlet, microwave, ultrasound and maceration (41.43, 26.88, 19.41 and 13.64 mg of TE/g dried sample, respectively). Of these, the solvent type greatly affected the FRAP of P. trimera and methanol was the best solvent for retaining the FRAP of P. trimera, while the MAE was a superior extraction method in retaining the FRAP of P. trimera. 4. Conclusion This study demonstrates that solvent type and extraction method had great effects on the phytochemical yield and antioxidant activity of P. trimera root extracts. Of these, methanol and microwave-assisted extraction are the best selections for the extraction of biologically active phytochemicals from P. trimera root. Investigation of extraction conditions is of great interest to those researchers, not just interested in the biological activity of P.
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trimera, but also those interested in the preparation of any natural product extracts and their potential application for the functional food and pharmaceutical industries. In further study, we optimize the microwave-assisted extraction parameters for saponins and antioxidant capacity from P. trimera root.
Conflict of Interest The authors declare no conflicts of interest.
Acknowledgments We sincerely acknowledge the following funding support: Ramaciotti Foundation (ES2012/0104); Cancer Australia and Cure Cancer Australia Foundation (1033781). The authors kindly thank the Vietnamese Government through the Vietnam International Education Development-Ministry of Education and Training (Project 911) and the University of Newcastle for awarding a VIEDTUIT scholarship to Van Tang Nguyen. The authors also would like to thank Mr Duy An Nguyen for kindly providing the P. trimera roots and Mr Thanh Son Le for the supporting references from the National Institute of Medicinal Materials, Vietnam.
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