Antioxidant activity of aloeswood tea in vitro

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1 riginal Article Spatula DD. 2012; 2(1):43-50 Antioxidant activity of aloeswood tea in vitro Aloeswood çayının in vitro antioksidan etkinliği Weijuan Han, Xican Li School of Chinese Herbal Medicine, Guangzhou University of Chinese Medicine, Guangzhou, China SUMMARY AIM: As a typical healthcare tea, aloeswood tea becomes more popular in China now. The present study tried to systematically evaluate the antioxidant ability in vitro of aloeswood tea for the first time. METHDS: Methanolic extract of aloeswood tea (MEAT) was prepared and measured by DPPH, ABTS +, 2-, H, reducing power (Fe 3+ & Cu 2+ ), lipid peroxidation, metal-chelating assays (Fe 2+ & Cu 2+ ). Then, the total phenol content was measured by spectrophotometre method. RESULTS: The IC 50 values of MEAT were calculated as 11.63±0.16, 2.05±0.060, 7.73±0.35, 31.20±0.57, 18.56±1.60, 16.25±0.10, 0.49±0.05, 94.24±3.19, ±7.04 µg/ml, respectively, for DPPH, ABTS+, 2-, H, reducing power (Fe 3+ & Cu 2+ ), lipid peroxidation, metal-chelating assays (Fe 2+ & Cu 2+ ). The total phenol content in MEAT was found to be ±6.51 mg GAE/g. CNCLUSIN: Aloeswood tea shows excellent in vitro antioxidant activity which may contribute to its healthcare functions and pharmacological benefits. Its antioxidant ability could be attributed to phenolic compounds, especially flavonoids which may exert antioxidant action in vitro by both chelating metal ions, and scavenging free radicals via donating hydrogen atom (H ) or electron (e). Key words: Aloeswood tea, antioxidant activity, radical-scavenging, metal chelating, genkwanin, flavonoid, Aquilaria sinensis. ÖZET AMAÇ: Tipik bir sağlık koruyucu çağ olarak, aloeswood çayı şimdilerde Çin de daha popüler olmaktadır. Şimdiki çalışmamız, ilk defa olarak aloeswood çayının in vitro antioksidan yeteneklerini sistematik olarak değerlendirmeye çalışmıştır. YÖNTEM: Aleoswood çayının metanolik ekstresi (MEAT) hazırlandı ve DPPH, ABTS +, 2-, H, redükte edici güç (Fe 3+ & Cu 2+ ), lipit peroksidasyonu, metal (Fe 2+ & Cu 2+ ) şelasyon analizleri ile ölçümler yapıldı. Sonra, spektrofotometre metodu ile toplam fenol içeriği ölçüldü. BULGULAR: MEAT ın IC 50 değerleri; DPPH, ABTS +, 2-, H, redükte edici güç (Fe 3+ & Cu 2+ ), lipit peroksidasyonu, metal (Fe 2+ & Cu 2+ ) şelasyon analizleri için sırasıyla 11,63±0,16, 2,05±0,060, 7,73±0,35, 31,20±0,57, 18,56±1,60, 16,25±0,10, 0,49±0,05, 94,24±3,19 ve 134,01±7,04 µg/ml olarak hesaplandı. MEAT içindeki toplam fenol içeriğinin 157,41±6,51 mg GAE/g olduğu bulundu. SNUÇ: Aloeswood çayı, in vitro olarak, sağlığı koruma fonksiyonlarına ve farmakolojik faydalarına katkıda bulunabilecek harika bir antioksidan aktivite göstermektedir. nun antioksidan yeteneği; fenolik bileşiklere, özellikle de metal iyonları ile şelasyon yaparak ve hidrojen atomu veya elektronu kabul etmesiyle serbest radikalleri süpürerek in vitro antioksidan etki gösteren flavonoidlere bağlanabilir. Anahtar kelimler: Aloeswood çayı, antioksidan etkinlik, radikal süpürme, metal şelasyonu, genkwanin, flavonoid, Aquilaria sinensis Corresponding Author: Xican Li, No.232, Waihuan East Road, Guangzhou Higher Education Mega Center, Panyu District, Guangzhou, , China. lixican@126.com Received March 03, 2012; accepted March 31, 2012 DI /spatula Published online in ScopeMed ( Spatula DD. 2012; 2(1): Spatula DD 43

2 Antioxidant activity of aloeswood tea INTRDUCTIN Chinese healthcare tea is developed under the direction of the basic theory of traditional Chinese medicine (TCM), and has been used for thousands years in China. Healthcare tea has been playing the more and more important role in healthcare medicine nowadays, as it mainly derives from natural plants not chemical synthesis. It was reported that there were more than 350 kinds of healthcare teas in China [1]. As a typical healthcare tea, aloeswood tea ( 沉香茶 in Chinese) becomes more and popular in China now. Aloeswood (Aquilaria sinensis (Lour.) Gilg.) is a fragrant wood and one of the valuable non-timer products in Asian tropical forest which results from the action of damages on Aquilaria plants and then infections by fungi such as fusarium spp [2]. It has been widely used in the treatment of various kinds of pain, cough and anaphylaxis for hundreds of years in Asia, especially in China [3]. The leaves of A. sinensis which widely cultivated in Guangdong, Hainan and Taiwan provinces in China are orally reported to be used locally as the best beverage for long term health of people [3], it is so-called aloeswood tea in the study. The extract of leaves of A. sinensis was proved to have notable analgesic, mild cathartic, antitumor and hypnotize activities [2,4,5]. According to free radical biology&medicine, all the pharmacological effects mentioned above have been related to antioxidant activity [6]. However, there was no report on its antioxidant evaluation. Thus, the objective of the present study was to systematically evaluate its antioxidant effects in vitro then analyze its possible mechanism. MATERIALS AND METHDS Chemicals reagents DPPH (1,1-diphenyl-2-picrylhydrazyl radical), pyrogallol, linoleic acid, Trolox (±-6-hydroxyl- 2,5,7,8-tetramethlyhromane-2-carboxylic acid), Ferrozine [3-(2- pyridyl) -5,6-bis(4- phenylsulfonicacid)-1,2,4-triazine], neocuproine (2,9-dimethyl- 1,10-phenanthroline), BHA (butylated hydroxyanisole), and murexide (5,5 - nitrilodibarbituric acid monoammonium salt) were purchased from Sigma Co. (Sigmaaldrich Trading Co., Shanghai, China); ABTS diammonium salt [2,2 -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid diammonium salt)], and D-2-deoxyribose were obtained from Amresco Co.; All other chemicals were of analytic grade. Plant material The aloeswood tea (Leaves Type) was obtained from Junyuan Pharmaceutical Co., LTD in Guangdong province, China. Product standard code: Q/MJY Preparation of methanol extract of aloeswood tea (MEAT) Aloeswood tea was extracted by methanol in Soxhlet extractor for 12 hours, and then concentrated in a rotary evaporator under reduced pressure to obtain dried methanolic extract of aloeswood tea (MEAT). It was stored at 4 until used. DPPH scavenging activity DPPH radical-scavenging activity of MEAT was measured by the method [7]. Briefly, 1 ml of DPPH solution (0.1 mmol/l) was mixed with 0.5 ml of various concentrations of sample methanolic solutions. The mixture was kept at room temperature for 30 min, and then the absorbance at 519 nm was measured on a spectrophotometer (Unico 2100, Shanghai, China), using methanol as the blank. Lower absorbance of the reaction mixture indicates higher radical scavenging activity. Trolox and BHA were used as the positive controls, and the percentage DPPH inhibition of the test samples was calculated as: Inhibition % = (1 A s /A 0 ) 100 Where A s is the absorbance in the presence of MEAT or positive controls, while A 0 is the absorbance in the absence of MEAT and positive controls. ABTS + scavenging activity The scavenging activity of ABTS + was determined as described [7]. The ABTS + was produced by the reaction between 0.35 ml of ABTS diammonium salt (7.4 mmol/l) and 0.35 ml of potassium persulfate (2.6 mmol/l), stored in the dark at room temperature for 12 h to allow completion of radical generation. Before usage, the mixture was diluted with 95% ethanol (about 1:50) to get an absorbance of 0.70±0.02 at 734 nm on a spectrophotometer (Unico 2100, Shanghai, China). To determine the scavenging activity, 1.2 ml of ABTS + reagent was mixed with 0.3 ml of sample or negative control (methanol), and the absorbance at 734 nm was measured 6 min after the initial mixing, using 95% ethanol as the blank. The 44 Spatula DD

3 Han, Li percentage inhibition of the samples was calculated as: Inhibition % = (1 A s /A 0 ) 100 Where A 0 is the absorbance at 734 nm of the negative control, A s is the absorbance at 734 nm of the mixture with sample. Trolox and BHA, with a final concentration range of µg/ml, were prepared as positive controls. Ferric ion (Fe 3+ ) reducing power Ferric cyanide (Fe 3+ ) reducing power was analyzed by the method of yaizu [8]. Different volumes of MEAT (x µl) were mixed with Na 2 HP 4 /KH 2 P 4 buffer (350-x µl, 0.2 mol/l, ph 6.6) and K 3 Fe(CN) 6 (250 µl, 1 g/100 ml). The mixture was incubated at 50 ºC for 20 min, 250 µl trichloroacetic acid (10 g/100 ml) was added, and the mixture was centrifuged at 3500 g for 10 min. The upper layer of solution (400 µl) was mixed with distilled water (400 µl) and FeCl 3 (400 µl, 0.1 g/100 ml) and placed immediately into the spectrophotometer (Unico 2100, Shanghai, China), and the timer was started. The absorbance at 700 nm was measured at 90 s. Samples were analyzed in groups of three, and when the analysis of one group has finished, the next group of three samples were mixed with FeCl 3 to avoid oxidization by air. Trolox and BHA were used as the positive controls, and an increased absorbance reading indicated increased reducing power. The percentage reducing power of the sample as compared to the maximum absorbance tested which appeared in BHA at µg/ml was calculated by using the formula: (A s /A m ) 100. Here, A m = absorbance of maximum absorbance tested and A s = absorbance of sample. Cupric ion (Cu 2+ ) reducing power The cupric ions (Cu 2+ ) reducing power capacity was determined by the method [9] with slight modification. Briefly, 125 µl CuS 4 aqueous solution (10 mmol/l), 125 µl neocuproine ethanolic solution (7.5 mmol/l) and 500 µl CH 3 CNH 4 buffer solution (100 mmol/l, ph 7.0) were added to test tubes containing different volumes of MEAT sample. Then, the total volume was adjusted with the buffer to 1 ml and mixed vigorously. Absorbance against a buffer blank was measured at 450 nm after 30 min. Increased absorbance of the reaction mixture indicates an increase of reduction capability. Trolox and BHA were used as the positive controls. The percentage reducing power of the sample as compared to the maximum absorbance tested which appeared in BHA at 25 µg/ml was calculated by using the formula: (A s /A m ) 100. Here, A m = absorbance of maximum absorbance tested and A s = absorbance of sample. Superoxide anion ( 2 - ) scavenging assay Measurement of superoxide anion scavenging activity of MEAT was based on the pyrogallol method [10]. Briefly, samples were dissolved in methanol at concentration of 1 mg/ml. The sample solution (x µl, where x = 30, 60, 90, 120, 80, 150 µl) was mixed with Tris-HCl buffer ( x µl, 0.05 mol/l, ph 8.2) containing EDTA (1 mmol/l) and pyrogallol (80 µl, 6 mmol/l), then shaken rapidly at room temperature. The absorbance at 325 nm of the mixture was measured (Unico 2100, Shanghai, China) against the Tris-HCl buffer as blank every 30 s for 5 min. Trolox and BHA were used as the positive controls. The slope of the correlation of absorbance with time was calculated. The reaction mixture without sample was used as the control. The 2 - scavenging ability was calculated as: (1 Slope of sample/slope of control) 100 Hydroxyl radical ( H) scavenging assay The scavenging activity on the hydroxyl radical ( H) was performed by the improved deoxyribose method as described by Wang [11]. In brief, all test samples were firstly dissolved in methanol (1 mg/ml), and µl sample solution was taken into mini tubes, the methanol solvent was then removed at 80 to eliminate its interference. The reactions were performed in 0.2 mol/l phosphate buffer (ph 7.4), containing 2.8 mmol/l deoxyribose, 2.8 mmol/l H 2 2, 25 µmol/l FeCl 3, 80 µmol/l Na 2 EDTA, and the test sample (15-75 µg). The reaction was started by adding ascorbic acid to a final concentration of 100 µmol/l and the reaction mixture (600 µl in total) was incubated for 20 min at 50 in a water bath. After incubation, the color was developed by addition of 0.5 ml 2- thiobarbituric acid (1 g/100 ml) followed by 0.5 ml trichloroacetic acid (5 g/100 ml) and heating in a boiling water bath for 15 min. The absorbance of mixture was measured at 532 nm against buffer (as blank). The reaction mixture without sample was used as control. The scavenging activity on hydroxyl radicals was expressed as: Inhibition % = (1 - A s /A 0 ) 100 Where A 0 is the absorbance at 532 nm of control (without sample), and A s is the absorbance at 532 nm of the reaction mixture containing sample. Trolox and BHA were taken as positive controls. Spatula DD 45

4 Antioxidant activity of aloeswood tea Chelating activity on Fe 2+ The chelating activity on Fe 2+ of MEAT was estimated by the method as described by Gülçin [12]. Briefly, samples (100, 200, 300, 400, and 500 µg/ml) in 200 µl was added to a solution of 250 µmol/l FeCl 2 (100 µl). The reaction was initiated by the addition of 1 mmol/l Ferrozine (150 µl) and total volume of the systems was adjusted to 1000 µl with methanol. Then, the mixture was shaken vigorously and stood at room temperature for 10 min. Absorbance of the solution was then measured spectrophotometrically at 562 nm. The percentage of chelating effect was calculated by using the formula given bellow: Ferrous chelating effect (% ) = (1- A s /A 0 ) 100 Where A s is the absorbance in the presence of the sample MEAT or positive control, while A 0 is the absorbance in the absence of the sample MEAT or positive control sodium citrate. Chelating activity on Cu 2+ The chelating activity on Cu 2+ of MEAT and positive controls was measured by a complexometric method using murexide [13]. Briefly, 60 µl CuS 4 (20 mmol/l) was added to hexamine HCl buffer (ph 5.0, 30 mmol/l) containing 30 mmol/l KCl and 0.3 µmol murexide. After incubation for 1 min at room temperature, the reaction was initiated by the addition of 1 mg/ml MEAT methanol solution (40, 80, 120, 160, and 200 µl). The total volume of the systems was adjusted to 1.5 ml with methanol. Then, the mixture was shaken vigorously and left at room temperature for 10 min. Absorbance of the solution was then measured spectrophotometrically at 485 and 520 nm. The absorbance ratio (A 485 /A 520 ) reflected the free Cu 2+ content. Therefore, the percentage of cupric chelating effect was defined as follows: Cupric chelating effect (% ) = [(A 485 /A 520 ) max - (A 485 /A 520 )] /( A 485 /A 520 ) max 100 Where (A 485 /A 520 ) is the absorbance ratio in the presence of the samples, while (A 485 /A 520 ) max is the maximum absorbance ratio without any samples. Sodium citrate was used as the positive control. Lipid peroxidation assay The effect of MEAT on anti-lipidperoxidation was investigated in linoleic acid emulsion [11]. The linoleic acid emulsion was prepared by mixing and homogenizing mg of linoleic acid, 78.2 mg of Tween-20 as emulsifier, and 30 ml of 30% ethanol (v/v), 0.1 ml of various concentrations of samples were added to 1.5 ml of linoleic acid emulsion and 0.4 ml distilled water. The reaction mixtures (2 ml) were incubated at room temperature in glass bottles. The degree of oxidation was measured when the absorbance of the control reached its maximum. To 0.15 ml of sample solution, 3.65 ml of 75% ethanol (v/v), 0.1 ml of ammonium thiocyanate (30%, m/v), and 0.1 ml of ferrous chloride (0.02 mol/l in 3.6% HCl) were added. The peroxide value was measured by monitoring absorbance at 500 nm in a spectrophotometer (Unico 2100, Shanghai, China). The peroxides formed during linoleic acid peroxidation oxidize Fe 2+ to Fe 3+, and Fe 3+ forms a complex with thiocyanate that has a maximum absorbance at 500 nm. The solution without MEAT was used as blank. Linoleic acid mixture without sample was used as positive control. The percentage of inhibition of LP in linoleic acid emulsion was calculated by the equation: Inhibition % = (1 A s /A 0 ) 100 Where A s is the absorbance in the presence of the sample or positive controls, while A 0 is the absorbance in the absence of the sample and positive controls. Determination of total phenolics Total phenolic contents were determined according to the Folin-Ciocalteu method [14]. Briefly, 0.5 ml of MEAT methanol solution (1 mg/ml) was mixed with 0.5 ml of 0.25 mol/l Folin- Ciocalteu reagent. The mixture was kept for 3 min, followed by the addition of 1.0 ml of 20% Na 2 C 3. After 30 min of incubation at room temperature, the mixture was centrifuged for 3 min (3,500 g/min). The absorbance of the supernatant was measured at 760 nm (Unico 2100, Shanghai, China). The results were expressed as gallic acid equivalents (GAE) in milligrams per gram of MEAT. Each test was repeated three times, and the results were averaged. Statistical analysis The measurements were performed in triplicate and the data were recorded as mean ± SD. The IC 50 value was defined as the final concentration of 50% free radical inhibition (or reducing power) and was calculated by linear regression analysis. All linear regression was analyzed by rigin 6.0 professional software in this paper. A value of P < 0.05 was considered to be significant. The statistical analysis was performed by SPSS (SPSS Inc., Chicago, IL). RESULTS AND DISCUSSIN DPPH and ABTS + assays have been widely used to determine the free radical-scavenging 46 Spatula DD

5 Han, Li activity of various pure compounds or extracts. Both DPPH and ABTS + are stable free radicals which dissolve in methanol or ethanol, and their colors show characteristic absorptions at 519 nm or 734 nm. When an antioxidant scavenges the free radicals, the colors in the DPPH and ABTS + assay solutions become lighter. As presented in Figure 1A, MEAT had effective DPPH radical scavenging activity in a concentration-dependent manner ( µg/ml). The scavenging effect of MEAT and the positive controls on the DPPH radical decreased in the order BHA > Trolox > MEAT. As shown in Figure1B, the ABTS + inhibition percentage values of MEAT and the positive controls were also dosedependent in the range of the tested concentration, and decreased in the order of BHA > MEAT > Trolox. The DPPH and ABTS + IC 50 values (IC 50 value is the concentration of the sample required to inhibit 50% of radical) of MEAT, Trolox and BHA were calculated and listed in Table 1. Many studies have revealed that the phenolic contents in plants are related to their antioxidant activities [15]. According to the linear regression equation y = c (R=0.9995,y for A 760 nm, c for amount of gallic acid), total phenolic contents in MEAT was calculated as ±6.51 mg GAE/g. Generally speaking, total phenolics in plants include flavonoid, phenolic acid, anthocyanidin, tannins and so on. Previous studies suggested [16] that the main phenolics compounds in A. sinensis was flavonoid, including genkwanin, luteolin, 7, 3, 4 -tri--methylluteolin, luteolin-7,4 - dimethylethers, hydroxygenkwanin and apigenin- 7,4 -dimethylethers. Since the content of genkwanin far exceeded that of others [16], here we took genkwanin for example to analyze the antioxidant mechanism of aloeswood tea. It is proposed that DPPH may be scavenged by an antioxidant through donation of hydrogen atom (H ) to form a stable DPPH-H molecule which does not absorb at 519 nm. According to this hypothesis and previous study, the reaction between DPPH and genkwanin could be explained by the following mechanism [17]: N N 2 N N 2 + CH 3 Homolysis H 2 N DPPH H Intramolecular hgdrogen bond Genkwanin N H N 2 N N 2 + CH 3 2 N DPPH-H H However, the scavenging of ABTS + is assumed to be an electron transfer process [17]: ABTS + + e ABTS Figure 1. Radical-scavenging abilities of MEAT, Trolox and BHA: (A) DPPH assay, (B) ABTS + assay. Each value is expressed as mean ± standard deviation, n =3. MEAT: methanolic extract of aloeswood tea. ABTS + can be previously produced by the reaction between ABTS diammonium salt and potassium persulfate: Spatula DD 47

6 Antioxidant activity of aloeswood tea Table 1. The values of IC 50 (µg/ml) Assays MEAT Trolox BHA DPPH 11.63±0.16 *, ** 4.86± ±0.30 ABTS ±0.06 *, ** 1.40± ± ±0.57* ± ±77.33 H 7.73±0.59* 9.13± ±0.35 Reducing power (Fe 3+ ) 18.56±1.60*, ** 7.38± ±0.02 Reducing power (Cu 2+ ) 16.25±0.10 *, ** 4.97± ±0.02 Chelating Fe ±3.19* 7.22±0.83 a --- Chelating Cu ± ±2.45 a --- LP 0.49±0.05*, ** 0.02± ±0.00 a The positive control was sodium citrate, instead of Trolox. All values were mean ± SD (n =3). Results were analyzed by ANVA. Value with * significantly different from Trolox or sodium citrate (P < 0.05); Value with ** significantly different from BHA (P < 0.05). MEAT: methanol extract from aloeswood tea. ---: Cannot be detected. ur results indicating that MEAT can effectively inhibit DPPH and ABTS +, suggested that aloeswood tea exerted radical-scavenging action by donating hydrogen atom (H ) and electron (e). Although a reductant is not necessarily an antioxidant, an antioxidant is commonly a reductant. The reducing power of a compound may therefore serve as a significant indicator of its potential antioxidant activity [17]. It can be seen that the reducing power percentage values of MEAT and the positive controls were concentration related in the range of the tested concentration (Figure 2). According to the IC 50 values (Table 1), their relative reducing powers on Fe 3+ and Cu 2+ were both as follows: BHA > Trolox > MEAT. In this assay, the superoxide radical was generated by the pyrogallol system at ph 8.2. Figure 3A indicated that MEAT and the positive controls Trolox and BHA demonstrated an ability to inhibit superoxide anion in a dose-dependent manner. However, MEAT exhibited a much higher scavenging level than the positive controls BHA and Trolox. Hydroxyl radical ( H) is one of the most important free radicals in living cells. In the study, the degree of deoxyribose degradationwas used for determining the hydroxyl radical scavenging capacity of MEAT. The data in Figure 3B and Table 1 suggested MEAT possessed the similar scavenging activity to Trolox and BHA. As we know, the production of both superoxide and hydroxyl radicals is catalyzed by transition metal iron (mainly Fe 2+ & Cu 2+ ) through Feton reaction (Fe 2+ + H 2 2 Fe 3+ + H + H ) or Haber-Weiss reaction ( H 2 2 H + H ) [17]. Figure 2. Reducing power assays of MEAT, Trolox and BHA: (A) Fe 3+ Fe 2+ ; (B) Cu 2+ Cu +. Each value is expressed as mean ± standard deviation, n =3. MEAT: methanolic extract of aloeswood tea. 48 Spatula DD

7 Han, Li Figure 3. Superoxide anion (A) and hydroxyl (B) radical scavenging activity of MEAT, Trolox and BHA. Each value is expressed as mean ± standard deviation, n =3. MEAT: methanolic extract of aloeswood tea. As shown in Figure 4A&B, the metal chelating activities of MEAT and positive control sodium citrate increased dose-dependently within the tested concentrations. The IC 50 values of Fe 2+ & Cu 2+ chelating abilities of MEAT were calculated as 94.24±3.19 µg/ml and ±7.04 µg/ml, respectively. Hence, MEAT possessed an effective metal chelating ability. Generally, compounds with structures containing two or more of the following functional groups: H, SH, CH, P 3 H 2, C=, NR 2, S and in a favorable structure function configuration will present chelating activity [12]. Therefore, some flavonoids with the functional groups above can be responsible for the metal chelating ability of MEAT. For example, the chelating reaction of genkwanin can be explained by the following mechanism [18] Figure 4. Metal chelating ability of MEAT and sodium citrate: (A) Fe 2+, (B) Cu 2+.Each value is expressed as mean ± standard deviation, n =3. MEAT: methanolic extract of aloeswood tea. Figure 5 showed that MEAT exhibited effective antioxidant activity in the linoleic acid emulsion system in a concentration-dependent manner ( mg/ml) and the effects of MEAT and positive controls on lipid peroxidation decreased in the order BHA > Trolox > MEAT (Table 1). CH 3 H CH 3 H H Genkwanin + Fe 2+ Fe Figure 5. Inhibition of lipid peroxidation at different concentrations of MEAT, Trolox and BHA. Each value is expressed as mean ± standard deviation, n =3. MEAT: methanolic extract of aloeswood tea. Spatula DD 49

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