Evaluation of the Antioxidant Activities of Ya-hom Intajak, a Thai Herbal Formulation, and its Component Plants

Purpose: To evaluate the antioxidant effect of 80 % ethanol extract and its n-hexane and dichloromethane fractions of Ya-hom Intajak, and its 47 medicinal plants components. Methods: The 80 % ethanol extract and its n-hexane and dichloromethane fractions were investigated on human low-density lipoprotein (LDL) peroxidation by thiobarbituric acid reactive substances (TBARS) assay. Antioxidant effect was also determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity and ferric reducing antioxidant power (FRAP) assays. Results: The extract of Ya-hom Intajak exhibited moderate DPPH scavenging activity with an IC 50 value of 99.08 µ g/ml and a FRAP value of 1.12 mmol FeSO4/g. Among the individual plants, Terminalia chebula Retz., Caesalpinia sappan L., Cinnamomum bejolghota (Buch.-Ham.) and Ci. verum J. Presl, showed strong antioxidant activities. T. chebula was the most potent plant in the DPPH assay while Ca. sappan had the highest FRAP value. Among the extracts, the dichloromethane fraction showed the highest inhibitory effect on LDL peroxidation with a half maximal inhibitory concentration (IC 50 ) value comparable to that of probucol (0.82 µ g/ml). Pearson correlation analysis revealed that total phenolic content (TPC) showed high positive correlations with FRAP (r = 0.908, p < 0.01) and DPPH activities (r = 0.648, p < 0.01). Conclusion: Ya-hom Intajak is a valuable source of natural antioxidants and has a potential application as a cardiovascular protective formulation.


INTRODUCTION
The initiation and progression of early stage of atherosclerosis and the development of cardiovascular diseases have been attributed to the oxidation of low-density lipoproteins (LDL). Reduction of LDL oxidation may be one of the most important therapeutic approaches to prevent the development of atherosclerosis.
Many antioxidants have been developed to delay or inhibit the oxidation of the biomolecules by terminating the initiation or propagation of the oxidizing chain reactions by free radicals and inhibiting the foam cell formation [1]. The antioxidant activity of phenolic compounds is mainly due to their redox properties, which play an important role in adsorbing and neutralizing free radicals, quenching singlet and triplet oxygen or decomposing peroxides [2]. Many studies have indicated that there was a high correlation between the antioxidant activity of some plants and their phenolic contents [3][4][5]. The bioactive components can effectively inhibit LDL oxidation and may prevent atherosclerosis by reducing and slowing down the progression to advance stage [5].
Ya-hom is a traditional herbal formulation used widely in Thailand for the treatment of nausea, vomiting, dizziness and fainting, especially among the aged population. It is also used as a cardiotonic agent and tonic for longevity. There are many preparations of Ya-hom with most formula contain similar major ingredients but different total composition and amounts of medicinal plants [6]. There have been reports on the effects of Ya-hom on both animal and human cardiovascular function. The effect of Ya-hom on the pulse rate and pulse pressure in human has been reported [7]. The effect of the herbal preparation on blood pressure in rats and in isolated rat aortic ring and atrium have also been investigated [8][9][10]. However, the results obtained by the different studies were not in agreement and remain inconclusive, most probably due to the use of different Ya-hom formula, extraction methods and experimental models. Among those Ya-hom preparations, Yahom Intajak was listed by the National Drug Committee (Thailand) in the List of Herbal Medicine Products A.D. 2011 and declared as over the counter herbal drug. Ya-hom Inthajak is also accepted to be used as cardiotonic agent and also improving blood circulation [11]. The aim of this study was to evaluate the antioxidant effect of Ya-hom Intajak against human LDL oxidation, radical scavenging activity against 2,2diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP). The relationships between these activities and their total phenolic contents and total flavonoid content were also established.

EXPERIMENTAL Preparation of extracts
Yahum Intajak used in this study contained 47 single component plants as listed in Tables 1(a) and 1(b). The single medicinal plant components of the herbal formulation were purchased from Jaokrompur, a herbal drug store in Bangkok, Thailand and their identities were confirmed by Dr Wongsatit Chuakul of Mahidol University, Thailand. Ya-hom Intajak formula was prepared by mixing and grinding equal amount of each of the 47 component herbs, according to the formulatory provided by the List of Herbal Medicines Products A.D. 2011 [11].
The powdered mixture (100 g) was extracted with 2,000 ml of 80 % ethanol using a percolator and then the ethanol extract was successfully fractionated with n-hexane, dichloromethane and 80 % ethanol. Each plant was dried in a hot air oven at 50 °C, ground and then 30 g of the powder was macerated with 600 ml of 80 % ethanol. The extract was filtered through Whatman filter paper No.1 and the entire extraction process was repeated thrice on the marc. The extracts and filtrates were concentrated under reduced pressure.

Determination of DPPH radical scavenging activity
DPPH scavenging activity of the extracts was carried out according to the method described by Kordali et al with slight modification [12]. Briefly, 2 ml of each extract at various concentrations (100, 80, 60, 40 and 20 µg/µl) were respectively added to 2 ml of freshly prepared DPPH methanol solution (4 mg/100 ml). The mixtures were vortexed vigorously with a votex mixer and allowed to stand in the dark for 30 min at room temperature for any radical-antioxidant reaction to occur. Finally, the absorbance of these mixtures was measured by using a Shimadzu UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan) at 517 nm. The difference in absorbance between the sample and the control was calculated as percentage (%) inhibition of DPPH activity. The IC 50 values, i.e. concentration of sample providing 50 % of radical scavenging activity was obtained through interpolation of linear regression analysis. Ascorbic acid in methanol was used as a positive control and the assay was conducted in triplicate for each sample concentration.

Ferric reducing ability power assay (FRAP)
The FRAP assay was performed according to the procedure described by Benzie and Szeto [13]. The FRAP reagent was prepared from 300 mM sodium acetate buffer (pH 3.6), 10 mM TPTZ dissolved by 40 mM HCl and 20 mM ferric chloride in the volume ratio of 10:1:1. The different concentrations (400-800 µg/ml) of sample was dissolved using methanol as a solvent. 25 µl of each concentration of the sample was added to 175 µl of FRAP reagent. Blank experiment contained 25 µl of sample and 175 µl of sodium acetate buffer. The absorbance of the mixtures was measured at 593 nm using UV-spectrophotometer (DKSH). Ascorbic acid in methanol was used as a positive control and the experiment was conducted in triplicate. The standard curve was plotted using FeSO 4 solutions and the result is expressed as mM of FeSO 4 equivalents.

Evaluation of inhibitory effect on human low density lipoprotein peroxidation
The assay was performed following the method of Dillon with slight modification [14]. The procedure included LDL isolation, LDL oxidation and TBARS assay. The use of human whole blood in this study was approved by the Ethics Committee of the Universiti Kebangsaan Malaysia (approval no. FF-120-2007), following the International Ethical Guidelines for Biomedical Research Involving Human Subjects (CIOMS and WHO) [15]. Whole blood was drawn from the vein of healthy volunteers aged between 24 and 70 years who were normolipidemic, nonsmoking, had not taken any medications and supplements within the last two weeks and also claimed they fasted for 8 h, prior to blood withdrawal.
For LDL isolation, 9 ml of the blood was added into 1 ml of 3 Germany) solution as an anticoagulant, then centrifuged at 2,000 g for 20 min to separate plasma. The plasma (3.2 ml) was mixed well with 0.8 ml of OptiprepTM (60 % iodixanol) (Sigma Chemical Co.) as the density gradient medium to give a final iodixanol concentration of 12 % (v/v). This mixture was added in to 8.9 ml OptisealTM tube, then carefully added 4 ml of 6 % iodixanol in saline, after that topped up with 0.9 ml of saline. The tube was ultracentrifuged at 402,000 times gravity (g), 16 °C for 3 h 10 min using the Ti.70.1 rotor. The subfractions of lipoprotein: highdensity lipoprotein (HDL), low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) was obtained. LDL was characterized by measuring the amount of protein by using Bovine serum albumin (Protein kit Sigma Chemical Co.) as a standard. The purity of LDL was measured by using electrophoresis. Agarose gel (Bio-Rad, USA) was used as solid support in electrophoresis. Samples were electrophoresed at a constant 45 mA/gel for 45 min, then oven dried at 85 °C for 20 min and stained with Sudan Black (Sigma,USA). The band was visually observed under UV detector. Then, LDL was diluted with phosphate buffer saline (PBS) (pH 7.4) to a final concentration of 200 µg protein/ml before oxidation testing.
For oxidation of LDL, the reaction was initiated by adding freshly prepared 10 µM CuSO 4 (Sigma, USA) solution. The samples were dissolved in DMSO to obtain serial concentrations of 1.25, 0.63, 0.31, 0.16 and 0.08 µg/µl. Five µl of the samples were added to a cuvete containing 945 µl of LDL, 50 µl of 10 µM CuSO 4 and incubated at 37 °C for 5 h. The final concentrations of the extracts in the mixture were 6.25, 3.13, 1.56, 0.78 and 0.39 µg/ml. 0.5 % DMSO was used as control and blank while probucol (Sigma, USA) was used as the positive control. The oxidation of LDL was terminated by rapid freezing and kept at -20 °C with no longer than 24 h.
For thiobarbituric reactive substances (TBARS) assay, 100 µl of sodium dodecyl sulphate (SDS) (Zeptometrix Co., USA) and 2.5 ml of thiobarbituric acid (TBA) (Zeptometrix Co., USA) were added to the mixture. Then, it was incubated at 95 °C for 1 h to promote peroxidation. The mixture was put on ice for 10 min to cool down and stop the peroxidation process. The precipitate formed was removed by centrifugation at 3000 rpm, 15 min. The absorbance was measured at 532 nm. Then, the malondialdehyde (MDA) in the supernatant was calculated and expressed as nmoles of MDA/mg LDL protein. Standard MDA was used as a reference and plotted the standard curve.

Determination of total phenolic content
The total phenolic content (TPC) of extract was determined using the Folin-Ciocalteau method with some modification [16]. Briefly, 33 µl of each sample was mixed with 83 µl of Folin-Ciocalteau reagent (Sigma, USA) which was diluted (1:10) with water. Then 133 µl of 7.5 % w/v sodium bicarbonate was added in to the mixture. The absorbance of the mixture was measured at 765 nm after for 30 min standing at room temperature. Gallic acid (5-35 µg/ml) (Sigma, USA) was used as standard and plotted standard curve. The total phenolic content was reported as gallic acid equivalents (GAE) per gram of sample.

Determination of total flavonoid content
The total flavonoid content (TFC) was determined using the Dowd method [17]. Briefly, 100 µl of 2 % AlCl 3 in methanol was mixed with 100 µl of the sample solution. The absorbance of the mixture was measured at 415 nm using Pelkin Elmer UV-VIS lambda 25 spectrophotometer after 10 min against a blank sample consisting of methanol. The TFC was determined using a standard curve of quercetin (5 -30 µg/ml). The mean of three readings was used and expressed as milligrams of quercetin equivalents (QE)/l g of extract.

Statistical analysis
All the data are presented as standard error of the mean ± (SEM) from triplicate experiments and were analysed using Statistically Package for Social Sciences (SPSS) software version 17.0. A one-way analysis of variance (ANOVA) was used for multiple comparison. The concentration of the compounds required to inhibit 50 % oxidation (IC 50 ) for active extract was determined using probit programme. The correlation between TPC and TFC in the extracts and antioxidant properties was described by the Pearson product-movement correlation coefficient (r). P < 0.05 was considered to be statistically significant. The TPC and TFC of the extracts were determined and their values varied considerably, ranging from 6.73 to 268.11 mg gallic acid equivalents per gram (mg GAE/g) and from 0.80 to 87.33 mg QE/g, respectively [Tables 1(a) and (b)]. Statistical correlations have been studied between TPC and TFC of the extracts of the 47 plants and their DPPH scavenging capacity and FRAP. To access the degree and the direction of the linear relationship between TPC, TFC and antioxidant activity, a bivariate Pearson's product-movement correlation coefficient (r) was calculated. The Pearson correlation analysis indicates that the DPPH assay showed positive correlation with TPC (r = 0.647, p < 0.01) and TFC (r = 0.285, p < 0.05) and the FRAP assay showed strong positive correlation with both phenolic contents (r = 0.908, p < 0.01) and flavonoid contents (r = 0.508, p < 0.01). The high correlation between the antioxidant activity and their TPC and TFC indicated that phenolic compounds might be a major contributor of antioxidant activities of these plants.

In
The ethanol extract of Ya-hom Intajak and its fractions were also investigated for their ability to inhibit copper-mediated oxidation on isolated human LDL. The human LDL was isolated by ultracentrifugation method and its purity was evaluated by using UV spectrophotometer and agarose gel electrophoresis. LDL was incubated with copper ions which catalyzed a lipid peroxidation process, in the presence or absence (negative control) of each of the extract. The level of in vitro oxidative modification of LDL oxidation was measured quantitatively by the TBARS method based on malondialdehyde (MDA) production. Probucol was used as the positive control. The inhibitory effect (IC 50 ) of the extracts on LDL oxidation are shown in Table 2.

DISCUSSION
The antioxidant activity of botanical materials has been measured by various methods such as DPPH radical scavenging activity assay, ABTS radical cation scavenging activity assay, superoxide anion radical scavenging activity assay, oxygen radical absorbance capacity (ORAC) assay, ferric reducing/antioxidant power (FRAP) assay and metal chelating activity assay. The need to use different methods of antioxidant capacity measurement is due to these various mechanisms of antioxidant action. Determination of the antioxidant activity of plant extracts and compounds often gave different results as the methods used are based on different reaction mechanisms [18].
The ethanol extract of Ya-hom showed moderate DPPH scavenging activity (99.08 µg/ml). The IC 50 values of the 47 single plants ranged from 15.35 to 1261.84 µg/ml with a difference of 83fold between the least and most active extract. Of all the plants tested, ten plants showed greater DPPH scavenging activity than the whole Ya-hom formula, and Terminalia chebula was the most potent with IC 50 value (15.35 µg/ml), comparable to that of ascorbic acid, the positive control used. In a previous study, gallic acid from the gall of T. chebula has showed in vitro antiaging activities on DPPH radical scavenging and stimulation index on normal human fibroblast proliferation [19].   [20].
The ethanol extract of Ya-hom showed the highest antioxidant capacity (1.12 mmol Fe(II)/g) while its fractions exhibited lower activity. The FRAP values of the 47 plants varied from 0.18 to 2.77 mmol Fe(II)/g with a difference of 15-fold between the lowest and highest activity. Among the plants tested, seven plants showed higher FRAP value than the whole Ya-hom formula and Caesalpinia sappan had the highest FRAP value (2.77 mmol Fe(II)/g) which was comparable to that of the positive control, ascorbic acid. C. sappan alcohol extract has been reported to exhibit IC 50 values on DPPH and nitric oxide method comparable to those of ascorbic acid and rutin. Moreover, hematein, a compound isolated from C. sappan wood was able to reduce fatty streak lesion of cholesterol-fed rabbits and has a potential to be used as an anti-atherogenic agent [21]. Other plants with strong FRAP activity were Terminalia chebula, Cinnamomum bejolghota and C. verum.
The results showed that the methanol extracts of these plants contained high levels of phenolic contents. Among all the plant extracts, the highest TPC and TFC was observed in the extract of Terminalia chebula while the lowest TPC was in the extract of Nigella sativa and the lowest TF was in the extract of Myristica fragrans. Phenolic compounds have been reported to be the major contributor to the antioxidant activities of grain, vegetables and other botanical materials [18]. Flavonoid is one of the most important natural phenolics which some of them became to be widely known as potent antioxidants such as quercetin and rutin.
The extracts and its fractions showed significant antioxidant activity on LDL oxidation as at 6.25 µg/ml, all samples exhibited greater than 70 % inhibition of LDL oxidation. The most active sample was dichloromethane fraction which showed an IC 50 value (0.82 µg/ml) comparable to that of probucol, a potent inhibitor of coppercatalysed LDL peroxidation ( Table 2). The results demonstrated that the extracts inhibited the copper-mediated oxidation of LDL in a dosedependent manner, as the concentration of the extract increased the percentage inhibition increased. The results indicate that the extract of Ya-hom contained compounds that are relatively strong inhibitors of LDL peroxidation. The antioxidant effect is due to the ability of the compounds in the plants extracts to chelate Cu 2+ ion and thus may inhibit the initiation of LDL oxidation and free radical formation at the lipoprotein. The extracts may also form chelating complexes with transition metals to reduce their availability as catalysts for free radical generation [20].

CONCLUSION
This study indicates that the two types of antioxidant capacity measurements, DPPH and FRAP, of the plant extracts provide broadly similar order of antioxidant activity. The results imply that the antioxidants in these plants are capable of scavenging free radicals and reducing oxidants. The high correlation between the antioxidant and activities and their TPC and TF indicates that phenolic compounds are the major contributor of antioxidant capacities of these plants. The extract of Ya-hom Intajak and its fractions possesses significant antioxidant activity on LDL oxidation, indicating that the extract contained compounds that were relatively strong inhibitors of LDL peroxidation. The plants are valuable sources of natural antioxidants.