Original papers
Antioxidant Activity and Phenolic Content of Mistletoe Extracts Following High-Temperature Batch Extraction
2014 Volume 20 Issue 2 Pages 201-206
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2014 Volume 20 Issue 2 Pages 201-206
Mistletoe is a traditional medicinal plant containing bioactive phenolic compounds with, for example, anti-cancer effects. We prepared extracts rich in phenolic compounds using a high-temperature batch extraction (HTBE) method. The mistletoes used in this study belonged to the genera Scurrula, Dendrophtoe and Macrosolen, which are typical plants in Indonesia. As a result of experiments regarding antioxidant activity measured by the ABTS method, along with polyphenolic profiles in each extract, we found an extract from S. oortiana showing a high antioxidant activity of 51 µmol TEAC/g extract as well as high content of phenolic compounds (289 mg GAE/g extract) among the mistletoe extracts obtained by HTBE. Candidate compounds responsible for the effect were quercetin 4′-O-β-glucopyranoside and gallic acid; other phenolic compounds such as catechin, epicatechin, rutin and quercetin partly contributed to the antioxidant effect of mistletoe extracts obtained by HTBE.
Mistletoe is a hemiparasite that grows on the branches of trees to get tree nutrients, and it also can obtain its own photosynthetic nutrients independently (Smith et al., 2001). Research interests on bioactivity of mistletoe have arisen from pharmacologists, since mistletoe has diverse compounds such as alkaloids, phenylpropanoids, triterpenes, polysaccharides, peptides, lectins, flavonoids and phytosterols (Fukunaga et al., 1987; 1988; Richter and Popp, 1992).
Mistletoe has been used as a folk medicinal source in Indonesia, since one of the typical Indonesian mistletoes, Scurrula atropurpurea, has been reported to show an anti-cancer effect (Ohashi et al., 2003). In general, mistletoe for medicinal intake is extracted with water by conventional decoction. However, the method may suffer from low yield and serious chemical degradation by heat during long extractions. In order to overcome these issues, a pressurized liquid extraction was attempted, but heat degradation of mistletoe phenolic compounds still occurred (Huie, 2002).
Recently, we have developed a high-temperature batch extraction (HTBE) method using ethanol as a solvent, in which a rapid (< 60 min) extraction to avoid any degradation of phenolic compounds was achieved even at high temperatures. Under HTBE conditions in 30% ethanol at 100°C for 10 min, the method provided higher yield of polyphenolic compounds in mistletoe (Scurrula atropurpurea) extracts as well as higher antioxidant activity than obtained by a conventional decoction method (Rahmawati and Hayashi, 2012). This finding suggested that the proposed method would be useful for the extraction of antioxidant polyphenolic compounds with less degradation during extraction. Thus, in this study, we applied the HTBE method to diverse mistletoe species to gain insight into the relationship between phenolic compounds and antioxidant activity of mistletoes.
Materials The six mistletoes used in this study (Table 1) were collected from the West and Central Java regions of Indonesia in 2011. The species used were identified at the Herbarium Bogoriense in Bogor, Indonesia. Each mistletoe was dried in an oven at 40., then ground into a powder with a 1-mm particle size by a Wiley mill (1029-A, Yoshida Seisakusho Co., Tokyo, Japan). The powdered samples were kept in an Auto Dry controlled dry cabinet (Toyo living, Tokyo, Japan) prior to use. 2, 2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) was purchased from Sigma-Aldrich Japan Co. (Tokyo, Japan). (±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) was purchased from Wako Pure Chemical Ind. (Osaka, Japan). Gallic acid, catechin, epicatechin, rutin, and quercetin were obtained from Sigma-Aldrich Japan Co. (Tokyo, Japan). Quercetin 4′-O-β-glucopyranoside (que-4-gluc) was purchased from Polyphenols Lab. (Sandnes, Norway). All other chemicals were of analytical-reagent grade and were used without further purification.
| Code | Mistletoe | Host plant | Local name | Origin |
|---|---|---|---|---|
| TP | Scurrula atropurpurea | Tea (Camellia sinensis) | Benalu teh | Puncak, West Java |
| TC | Scurrula oortiana | Tea (Camellia sinensis) | Benalu teh | Ciater, West Java |
| KE | Scurrula parasitica | Orange jessamine (Murraya paniculata) | Benalu kemuning | Sukorejo, Central Java |
| BE | Dendrophthoe pentandra | Star fruit (Averrhoa carambola) | Benalu belimbing | Sukorejo, Central Java |
| RA | Dendrophthoe pentandra | Kapok (Ceiba pentandra) | Benalu randu | Sukorejo, Central Java |
| KO | Macrosolen cochinchinensis | Coffee (Coffea arabica) | Benalu kopi | Sukorejo, Central Java |
Code based on local name of mistletoes.
Extraction Powdered mistletoe samples were extracted by a HTBE method according to our previous report (Rahmawati and Hayashi, 2012). Namely, 5 mL of 30% ethanol was added to a 6-mL batch reactor (1/2-inch SUS316 stainless tube) containing 0.5 g of sample. After mixing, nitrogen gas at 0.2 MPa was fed into the reactor, followed by shaking at 100°C. Under these conditions, the solvent temperature rapidly (within 1.5 min) reached the desired temperature of 100 ± 0.3°C, and the temperature was then maintained for 10 min for extraction. After the 10-min extraction, the reactor was immediately cooled down to room temperature within 1 min. The solution was centrifuged at 10,000 x g at 4°C for 15 min. The supernatant was then concentrated by an Eyela N-N series rotary evaporator (Tokyo Rikakikai Co., Tokyo, Japan). The concentrate was then lyophilized. The powered extract was dissolved in ultrapure water at a concentration of 1 mg/mL for further experiments.
Measurement of total phenol content Total phenol content (TPC) was measured according to the method of Javanmardi et al. (2003). To an aliquot (50 µL) of sample solution (1 mg/mL), 2.5 mL 1/10-fold diluted Folin-Ciocalteu reagent and 2 mL 7.5% (w/v) Na2CO3 were added. After the incubation at 45°C for 15 min, absorbance at 765 nm was monitored for measuring TPC. Gallic acid was used as a standard to calculate TPC. The calculated TPC was represented as mg-gallic acid equivalent (GAE) per gram of extract.
Measurement of antioxidant activity Antioxidant activity was measured by an ABTS assay (Re et al., 1999) with a slight modification. Briefly, ABTS solution dissolved in water was reacted with potassium persulfate so as to produce ABTS cation radicals (ABTS· +). The resulting solution was then allowed to stand in the dark at room temperature for 12 – 16 h prior to the use in the antioxidant assay. The ABTS· + solution was diluted with ethanol to adjust it to an appropriate radical concentration, corresponding to an absorbance of 0.70 ± 0.02 at 734 nm. Antioxidant activity was determined by monitoring the absorbance at 734 nm using a V-530 UV-Vis spectrophotometer (Jasco, Tokyo, Japan) after incubating 1 mL of the ABTS· + solution with 10 µL of sample solution or standard solution for 6 min at 30°C. The Trolox equivalent antioxidant capacity (TEAC) in µmoles per gram extract was used as an index of antioxidant activity.
Measurement of phenolic compounds in mistletoe extracts by high performance liquid chromatography (HPLC) HPLC on an LC-6A series system (Shimadzu Co., Kyoto, Japan) was performed using a TSK gel ODS-80Ts column (4.6 mm x 250 mm, Tosoh Co., Kyoto, Japan) to measure phenolic compounds in mistletoe. Stepwise gradient elution was performed using a mobile phase of acetonitrile containing 0.1% formic acid at 40°C at a flow rate of 0.6 mL/min at 280 nm: 10% to 20% acetonitrile (10 min), 20% to 40% (10 min), 40% to 80% (10 min), and back to 10% acetonitrile (3 min). The measurement of phenolic compounds in mistletoe was performed on the basis of retention time of standard phenolic compounds (gallic acid, catechin, epicatechin, rutin, que-4-gluc, quercetin). The quantification of each compound in mistletoe was made by calibration curves of standard solutions ranging from 5 to 100 µg/mL (Table 2).
| Standard | RT* (min) | Linear regression | r2 |
|---|---|---|---|
| Gallic acid | 8.2 | y = 0.00138x − 0.308 | 0.996 |
| Catechin | 18.6 | y = 0.00444x − 0.925 | 0.986 |
| Epicatechin | 21.6 | y = 0.00415x − 1.356 | 0.975 |
| Rutin | 24.8 | y = 0.00286x + 1.118 | 0.999 |
| Que-4-gluc | 27.7 | y = 0.00164x + 3.367 | 0.966 |
| Quercetin | 32.3 | y = 0.00184x + 1.159 | 0.999 |
*RT, retention time on an TSK gel ODS-80Ts column; y, concentration [µg/mL]; x, detected peak area
Statistical analysis Results represent the mean±standard deviation (SD), based on triplicates and under the same experimental conditions. Statistical differences between groups were analyzed by a simple one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison using an SPSS software version 17. Differences were considered significantly different if p < 0.05 (α = 0.05).
Measurement of polyphenolic compounds in mistletoe extracts Fig. 1 shows HPLC chromatograms of mistletoe extracts obtained by HTBE, together with 6 phenolic standards. By comparison of the elution profiles of each standard with those of sample mistletoe extracts, we found five phenolic compounds, i.e., gallic acid, catechin, epicatechin, rutin and que-4-gluc were found in TP mistletoe extract as naturally occurring compounds. In TC mistletoe, we found gallic acid, catechin, rutin and que-4-gluc; in KE, we found gallic acid, catechin, rutin, que-4-gluc and quercetin; and in RA mistletoe, we found gallic acid, epicatechin, rutin and que-4-gluc. In contrast, we found only gallic acid and que-4-gluc for BE mistletoe extracts and gallic acid, que-4-gluc and rutin for KO mistletoe.
Typical HPLC chromatogram of mistletoe extracts by the HTBE method. “a” to “f” correspond to standard polyphenolic compounds: gallic acid, catechin, epicatechin, rutin, que-4-gluc and quercetin, respectively. HPLC separation of mistletoe extracts was performed on an TSK gel ODS-80Ts column at 0.6 mL/min at 40°C, while monitoring at 280 nm. Abbreviations for mistletoe extracts are the same as in Table 1.
Yield following extraction by HTBE The RA extract showed the highest yield of 300 ± 12 mg/g mistletoe material by the HTBE method among the 6 mistletoe extracts, with yield decreasing in the order RA > BE > KE > TC > KO > TP (Fig. 2). This indicates that extraction by the HTBE method was greatly affected by mistletoe characteristics or phenolic contents in each mistletoe.
Results for yield of mistletoe extracts by HTBE method. Six mistletoes were extracted by HTBE method using 30% ethanol under 0.2 MPa pressure at 100°C for 10 min. Data represent the mean ± SD (n=3). Bars with different letters are significantly different (P < 0.05). Abbreviations for mistletoe extracts are the same as in Table 1.
TPC in mistletoe extracts In order to get information on polyphenolic content in mistletoe extracts, the TPC of each mistletoe was determined by the Folin-Ciocalteu method. As shown in Figure 3, TC had the highest polyphenolic content in the HTBE extract (280 ± 24 mg GAE/g extract), indicating that most compounds present in the TC extract obtained by HTBE were polyphenolic compounds. Although the TPC values of BE and RA were lower than other extracts, the TPC in the extracts was estimated to be over 35%. Therefore, the HTBE method under the present extraction conditions with 30% ethanol seems useful for extracting natural polyphenolic compounds from mistletoe.
Results for total phenolic content of mistletoe extracts obtained by the HTBE method. Six mistletoes were extracted by HTBE using 30% ethanol under 0.2 MPa pressure at 100°C for 10 min. Total phenolic content (mg GAE equivalent/g extract) is represented by the mean ± SD (n = 3). Bars with different letters are significantly different (P < 0.05). Abbreviations for mistletoe extracts are the same as in Table 1.
Polyphenolic HPLC profiles in mistletoe extracts The HTBE-aided mistletoe extracts were separated by reversed phase-HPLC to clarify the natural compounds responsible for the high TPC values in the extracts (Fig. 3). As shown in Fig. 1, we detected polyphenolic compounds in each extract aided by their standard elution on a TSK gel ODS-80Ts column, similar to other reports (Ohashi et al., 2003; Vicas et al., 2011). Fig. 4 summarizes the content of polyphenolic compounds detected by HPLC. Que-4-gluc was commonly and predominantly present in the 6 mistletoe extracts used in this study; RA (38 mg/g extract) as well as TC (24 mg/g extract) and KO (26 mg/g extract) had the highest content of que-4-gluc. In contrast, the content of other polyphenolic compounds such as gallic acid, catechin, epicatechin, rutin and quercetin differed among mistletoe species, suggesting that the nutritional composition of mistletoe may be a factor distinct from their growing conditions (Łuczkiewicz et al., 2001).
Profiles of phenolic content in six mistletoe extracts by HTBE method. Abbreviations for mistletoe extracts are the same as in Table 1.
Antioxidant activity Considering the high content of polyphenolic compounds in mistletoe extracts obtained by HTBE, we next evaluated their antioxidant activity by the ABTS method. As shown in Fig. 5A, TP, KE, and TC extracts had a high ABTS radical scavenging activity of >40 µmol TEAC/g extract, similar to values for antioxidant activity per gram of starting mistletoe material, except for TP (Fig. 5B). The lower antioxidant activity of TP mistletoe material (5.4 ± 1.0 µmol TEAC/g mistletoe material) indicated that TP contains many more inactive contaminants than other mistletoe species. In order to clarify the contribution of antioxidant activity of the detected polyphenolic compounds to total activity of mistletoe extracts, we evaluated the total antioxidant activity of each polyphenolic compound based on its content in extracts. As summarized in Table 3, TP and RA extracts showed the highest total antioxidant activity (22 µmol TEAC/g extract) of the polyphenolic compounds, which decreased in the order TP = RA > KE > KO > TC > BE. This indicates that the antioxidant effect of RA extracts might result from the contribution of the polyphenolic compounds listed in Table 3. In contrast, the lower contribution of polyphenolic compounds to the antioxidant activities of other extracts, in particular the TC extract with a ca. 20% contribution ratio, suggested that other antioxidant compounds must be present in mistletoe extracts, as reported by Haas et al. (2003), who demonstrated the presence of phytochemicals such as triterpenoids in mistletoe. Therefore, further experiments for identification of other antioxidants in mistletoe extracts obtained by HTBE are now in progress.
Results of antioxidant activities of mistletoe extracts by HTBE method (µmol TEAC/g extract) (A) and the activities of mistletoe material (µmol TEAC/g mistletoe material) (B). Data represent the mean ± SD (n = 3). Bars with different letters are significantly different (P < 0.05). Abbreviations for mistletoe extracts are the same as in Table 1.
| Phenolic standards | Antioxidant activity (µmol TEAC/mg for standard or µmol TEAC/g for extract) | ||||||
|---|---|---|---|---|---|---|---|
| Standard | TP | TC | KE | BE | RA | KO | |
| Gallic acid | 1.5 ± 0.001 | 3 | 2 | 8 | 3 | 5 | 4 |
| Catechin | 1.0 ± 0.005 | 12 | 1 | 2 | * | * | * |
| Epicatechin | 1.0 ± 0.005 | 1 | * | * | * | 1 | * |
| Rutin | 0.3 ± 0.025 | 1 | 2 | 1 | * | 1 | 1 |
| Que-4-gluc | 0.4 ± 0.061 | 5 | 9 | 7 | 2 | 15 | 10 |
| Quercetin | 0.8 ± 0.023 | * | * | 1 | * | * | * |
| Total | 22 | 14 | 19 | 5 | 22 | 15 | |
*: not available. Abbreviations for mistletoe extracts are the same as in Table 1.
In this study, we clarified candidates responsible for the antioxidant effect of mistletoe by the aid of HTBE. The predominant antioxidant in the extracts was que-4-gluc, even though other antioxidant polyphenolic compounds including gallic acid, catechin, epicatechin, rutin, and quercetin were also found. Among the 6 mistletoe species used in this study, the TP as well as the KE, TC and KO extracts possessed high antioxidant activity of >40 µmol TEAC/g extract, together with high total phenolic content. Taken together, this information demonstrates that the HTBE method could be applicable for the extraction of antioxidant compounds from mistletoe, and that the differing antioxidant activity of mistletoes apparently is based on species differences.
