Screening Study on Antioxidant Activity of Plants Grown Wildly in Mongolia

Abstract

In this study, 50% ethanol extract prepared from dry leaves of eight plants grown wildly in Mongolia were characterized for DPPH radical scavenging activity, ABTS radical cation scavenging activity, superoxide anion scavenging activity, ferric reducing power, iron chelating ability, and content of total phenol, flavonoid, and flavanol in order to find good sources of natural antioxidant from plant kingdom. Correlation coefficients between antioxidant parameters and contents of polyphenolic compounds were also calculated. Among them, leaves of Vaccinium vitis-idaeae, Paeonia anomala, Rosa acicularis, and Hippophae rhamnoides were rich in polyphenolics and exerted strong scavenging activity against DPPH radical, ABTS radical cation, and superoxide anion and reducing power.

Introduction

In Mongolia, many edible and medicinal plants have been largely harvested from the wild and used according to the theory of traditional folk medicine. Therefore, studies on biological activity of useful parts of the plants are crucial. In this study, leaves of following wild plants were investigated: black currant (BC) (Ribes nigrum), hawthorn (HT) (Crataegus sanguinea), lingonberry (LGB) (Vaccinium vitis-idaeae), peony (PE) (Paeonia anomala), rosehip (RH) (Rosa acicularis), seabuckthorn (SBT) (Hippophae rhamnoides), strawberry (SWB) (Fragaria orientalis), and tall currant (TC) (Ribes altissimum). Herbal tea prepared from these leaves is utilized as home remedy to cure certain types of diseases by the local people in Mongolia.

Leaves are a good source of bioactive compounds that may possess many functional benefits, because they are the site of synthesis of many compounds for plant functions. A number of studies have reported that functional properties of leaves are more effective than that of other plant parts such as roots, flowers, fruits, and stems. For instance, BC leaves had a higher content in phenolics and flavonol antioxidants than buds and fully ripened berries (Tabart et al., 2006). Compared with fruits, leaves of SWB were found to have higher oxygen radical absorbance capacity (Wang and Lin, 2000).

So far, SBT leaf grown in India and France were examined for 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation, superoxide anion, and nitric oxide scavenging activity, reducing power, total phenolics, total flavonoids and condensed tannin content. In the SBT leaf, eight phenolic compounds were quantified (Kumar et al., 2011; Kumar et al., 2013; Maheshwari et al., 2011; Michel et al., 2012; Upadhyay et al., 2010). The ethyl acetatesoluble fraction was rich in phenolics and gallic acid was predominant (Maheshwari et al., 2011; Kumar et al., 2013).

BC leaf had significant scavenging activity against superoxide anion and DPPH radical (Sasaki et al., 2013). Recent studies have shown that BC leaves contain several kinds of polyphenolic compounds with antioxidant potential (Raudsepp et al., 2010; Oszmianski et al., 2011; Vagiri et al., 2012; Tabart et al., 2006; Sasaki et al., 2013). The quercetin derivatives were the most predominant phenolic group and quercetin-3-O-glucosyl-6″-acetate was the most important compound (Oszmianski et al., 2011). Among 10 kinds of flavonols quantified in BC leaves as well as in the buds and berries, rutin and isoquercitrin were the most abundant (Tabart et al., 2006).

SWB leaf contained various types of polyphenolic compounds. Among them, (+)-catechin was detected as the dominant compound and along with ellagic acid moderately participated in the antioxidant capacities (Buricova et al., 2011; Mudnic et al., 2009). Among identified phenolic compounds in the leaves of SWB the most predominant compound was quercetin-3-O-rutinoside (Oszmianski et al., 2011).

To our best knowledge, there is no study on antioxidant activity of LGB leaves. However, Hokkanen et al. (2009), Ek et al. (2006), and Szakiel et al. (2012) characterized 36 and 22 phenolic compounds, and 18 triterpenoids from LGB leaves, respectively.

HT, PE, RH, and TC have not been analyzed for antioxidant properties and phytochemicals so far. Although biological activities of the roots of Paeonia species were well studied, there is no investigation focused on their leaves. Flavonol glycosides, phenolic acids, and procyanidins are the main phenolic compounds in the flowers, leaves, and fruits of many Crataegus species (Liu et al., 2011). Chemical components of leaves, flowers, and fruits of various Crataegus species have been investigated and reviewed. In case of HT, only fruits were studied (Edwards et al., 2012).

The aim of this screening study was to investigate antioxidant properties with different analytical methods and phenolic contents of the selected plant leaves in order to find good sources of natural antioxidant from plant kingdom.

Materials and Methods

Plant materials    Fresh leaves of the selected plants were collected from Tuv province of Mongolia in the beginning of August, 2011. These plants were identified in the Institute of Biology, Mongolian Academy of Science. Well-grown and healthy leaves were collected without its petiole, air-dried at room temperature in the shade to constant weights and powdered.

Chemicals    All chemicals and solvents purchased from local suppliers in Japan were the highest commercial grade and were used without further purification.

Extraction    Two grams of each sample were extracted with 40 mL of 50% ethanol on a magnetic stirrer for 2 h at room temperature and centrifuged at 5000 rpm for 10 min at 4°C. The supernatant was filtered with No.5C paper (Advantec Toyo, Tokyo, Japan) and decanted into 100 mL volumetric flask. The pellet was extracted again under identical condition. The supernatants were combined, diluted to 100 mL with 50% ethanol and stored at −20°C until use.

DPPH radical scavenging assay    Effect of the leaves on DPPH radical was determined as described by Adedapo et al. (2009) with a minor modification. Two mL of 0.135 mM DPPH (Wako Pure Chemical Industries, Osaka, Japan) in methanol were mixed with 100 µL of extract, diluted with methanol. After 30 min in the dark, absorbance was measured at 517 nm (UV-Vis spectrophotometer UV mini 1240, Shimadzu, Kyoto, Japan). Results were expressed as µmol of 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox, Sigma-Aldrich, St Louis, MO) equivalents (TE) per gram of dry leaf by using a calibration curve of Trolox (0 – 1.5 mM).

ABTS radical cation scavenging assay    In this study, ABTS radical cation assay (Chang et al., 2007) was adopted with a little modification. An aqueous solution of 7 mM ABTS (Roche Diagnostic K.K., Tokyo, Japan) was oxidized using 2.45 mM K₂S₂O₈ for 16 h in the dark at room temperature. Afterward, the ABTS radical solution was diluted with methanol to an absorbance of 0.75 ± 0.05 at 734 nm. Each extract (10 µL) was mixed with 1 mL of ABTS radical solution and absorbance was read at 734 nm exactly after 7 min. Results were expressed as µmol of Trolox equivalents (TE) per gram of dry leaf by reference to the calibration curve of Trolox.

Superoxide anion scavenging assay    Superoxide anion scavenging activity (SOSA) was evaluated by SOD Assay Kit-WST from Dojindo Laboratories (Kumamoto, Japan). WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) produces a water-soluble formazan dye which can be determined spectrophotometrically at 450 nm (Sunrise Rainbow RC; Tecan Group Ltd, Männedorf, Switzerland), upon reduction with superoxide anion. In this kit, for the production of superoxide anion, which is a substrate of SOD (superoxide dismutase), a xanthine-xanthine oxidase reaction was used. Fifty percent inhibition of the reaction by the leaf extracts and the enzyme SOD (2260 U/mg protein, Sigma-Aldrich, St Louis, MO) was used for the activity determination (IC₅₀). To calculate SOSA (units/mg dry leaf), IC₅₀ (units/mL) of SOD was compared with IC₅₀ (mg/mL) of leaf extract.

Ferric reducing antioxidant power assay    Ferric reducing antioxidant power (FRAP) assay was performed as previously described (Benzei and Strain, 1996) with a slight modification. FRAP reagent containing 5 mL of 2,4,6-tripyridyl-S-triazine (TPTZ, 10 mM) in 40 mM HCl, 5 mL of FeCl₃ × 6H₂O (20 mM), and 50 mL of acetate buffer (300 mM, pH 3.6) was freshly prepared and warmed at 37°C before use. Sample extracts (100 µL) were allowed to react with 3 mL of the FRAP reagent for 30 min in the dark. Readings of the colored product were taken at 593 nm. Aqueous solutions of FeSO₄ × 7H₂O (0.1 – 1 mM) were used for calibration (r² = 0.999). The values were expressed as the concentration of ascorbic acid, which is most effective natural antioxidant, having a ferric reducing ability equivalent to that of 1 mM FeSO₄.

Ferrous ions chelation assay    The chelating activity on ferrous ions (Fe²⁺) was measured as described by Tang et al. (2002). Briefly, 1 mL of different concentrations of each extract was mixed with 3.7 mL of distilled water and then was reacted with 0.1 mL of 2 mM FeCl₂ × 4H₂O and 0.2 mL of 5 mM 3-(2-pyridyl)-5,6-bis(4-phenyl-sulfonic acid)-1,2,4-triazine (ferrozine, Wako Pure Chemical Industries, Osaka, Japan). After 10 min, absorbance of the Fe²⁺-ferrozine complex was measured at 562 nm. The amount of Fe²⁺ chelated was calculated as percentage of the total amount of Fe²⁺. Na₂EDTA × 2H₂O (Wako Pure Chemical Industries, Osaka, Japan) was used as a positive control.

Determination of total phenolics    Total content of phenolic substances was evaluated using the Folin-Ciocalteu method (Singleton et al., 1999). Briefly, each 20 µL of sample solution was mixed with 1.58 mL of distilled water and 100 µL of the Folin-Ciocalteu's reagent (1.8 N, Nacalai Tesque, Kyoto, Japan) and allowed to stand for 5 min. After that 300 µL of 20% Na₂CO₃ was added and shaken vigorously. After 2 h of incubation at room temperature in the dark, absorbance was measured at 765 nm. The calibration curve (r² = 0.998) was established with gallic acid (0 – 500 µg/mL). Results were expressed as mg of gallic acid equivalents (GAE) per gram of dry leaf.

Determination of total flavonoids    Total flavonoid content was measured by AlCl₃ assay (Adedapo et al., 2009). Equal volume of sample solution and 2% AlCl₃ in ethanol were mixed well and after 1 h at room temperature absorbance was measured at 420 nm. The interference background of the extract was corrected by preparing the test without AlCl₃. The results were expressed as mg of quercetin equivalents (QE) per gram of dry leaf by using a standard curve (r² = 0.998) of quercetin (0 – 100 µg/mL).

Determination of total flavanols    The total flavanol content was determined using p-dimethylaminocinnamaldehyde (DMACA, Nacalai Tesque, Kyoto, Japan) method (Arnous et al., 2001). This method has a great advantage over the widely used vanillin method since there is no interference by anthocyanins (Li et al., 1996). The DMACA produces a blue coloration on reaction with flavanols/condensed tannins under acidified conditions. The sample solution diluted with methanol (0.2 mL) was mixed well with 1 mL of DMACA solution (0.1% in 1 N HCl in methanol) and allowed to react for 10 min. The absorbance at 640 nm was then read against a blank prepared similarly without DMACA. The content of total flavanols was estimated from a calibration curve (r² = 0.999) plotted at 640 nm with catechin (0 – 20 µg/mL) and expressed as mg of catechin equivalents (CE) per gram of dry leaf.

Statistics    All results are expressed as mean ± standard deviation (SD) of at least three determinations. A p-value < 0.05 was considered as statistically significant.

Results and Discussion

There are approximately 2000 edible and medical plants in Mongolia. However, scientific research into biological activities and phytochemicals of Mongolian plants is rarely carried out. In this study, the antioxidant potential of 50% ethanol extract prepared by eight plant leaves were evaluated in vitro with five different analytical methods. To obtain more detailed understandings of antioxidant properties, contents of polyphenolics were also estimated by biochemical methods. The selected plants belong to different families such as Paeoniaceae (PE), Elaeagnaceae (SBT), Ericaceae (LGB), Grossulariaceae (BC and TC) and Rosaceae (RH, HT, and SWB). Seven of them, except for PE, are berry plants. The berries are used freshly and processed into juice, jam, and wine. Herbal tea prepared from these plant leaves have been widely used as folk medicine by the local people since ancient times.

The choice of 50% ethanol in water, which has moderate polarity, as extraction solvent is based on its possibility to utilize in food and drug industry and on its capability to extract both hydrophilic and hydrophobic bioactive compounds.

Among the selected eight plant leaves, LGB, PE, RH, and SBT had significant antioxidant potential and were rich in polyphenolics. To the best of our knowledge, this may be first investigation on antioxidant activities of LGB, PE, and RH leaves. The genus Paeonia is widespread all over the world, and many species of this genus have been used in traditional folk medicine. There are about 35 species in Paeonia, only 13 species have been investigated so far (Wu et al., 2010). PE and some other species of this genus have received no or only little attention. Moreover, most of the literature reports have focused on chemical constituents and bioactivities of the roots for Paeonia species and almost no work has been devoted to the leaves. According to ancient Mongolian prescription, leaves and roots of PE are used for alleviating diuretic effects and decreasing high blood pressure. Although several phenolic compounds detected in LGB leaf (Hokkanen et al., 2009; Ek et al., 2006; and Szakiel et al., 2012), antioxidant activity of which has not been reported to date. Cho et al. (2003) evaluated DPPH radical scavenging activity of methanol extracts of 11 kinds of Rosaceae and 12 other kinds of Korean medicinal plants. Among them, roots of Rosa rugosa and Rosa davurica showed most potent scavenging activity with IC₅₀ values below 2 µg/mL, whereas RH (leaf and stem) as well as Rosa davurica (leaf) and Rosa wichuraiana (leaf and stem) exerted strong scavenging effects with IC₅₀ values below 3 µg/mL.

DPPH radical scavenging activity    DPPH radical scavenging assay is commonly employed in evaluating the ability of antioxidants to scavenge free radicals. All plant leaves exhibited a remarkable activity to scavenge DPPH radical and calibration curve of Trolox (r² = 0.999) was used for calculation. The activities of the selected plant leaves varied from 260 to 1544 µmol TE/g and were in the following decreasing order: PE > RH > LGB > SBT > SWB > TC > BC > HT (Table 1). DPPH radical scavenging activity of 50% ethanol extract prepared from SBT leaf grown in Mongolia was 797 ± 4 µmol TE/g dry leaf corresponding to 199 mg TE/g dry leaf. Thus, 50% ethanol extract of SBT leaf evaluated in this study was more effective to scavenge DPPH free radicals, as the scavenging activity for water and 70% ethanol extract of SBT leaf from India was 109 and 143 mg TE/g dry leaf, respectively (Upadhyay et al., 2013). It seems that 50% ethanol may be suitable to extract antioxidative compounds from SBT leaves. In addition, there was higher quercetin-3-O-galactoside and quercetin-3-O-glucoside content in the water extract of SBT leaves, whereas higher kaempferol and isorhamnetin content was observed in 70% ethanol extract (Upadhyay et al., 2013).

Table 1. Scavenging activity against DPPH radical, ABTS radical, and superoxide anion

Plant leaf	Scavenging activitya (mol TE/g dry leaf)		Superoxide anion scavenging activityb (units/mg dry leaf)
	DPPH assay	ABTS assay
BC (Ribes nigrum)	324 ± 5	309 ± 5	9
HT (Crataegus sanguinea)	260 ± 1	198 ± 4	4
LGB (Vaccinium vitis-idaeae)	923 ± 8	510 ± 8	5
PE (Paeonia anomala)	1544 ± 44	992 ± 16	16
RH (Rosa acicularis)	1300 ± 19	910 ± 7	15
SBT (Hippophae rhamnoides)	797 ± 4	695 ± 10	13
SWB (Fragaria orientalis)	561 ± 4	428 ± 4	10
TC (Ribes altissimum)	405 ± 2	290 ± 4	6

a  Each value represents means ± SD (n = 5).

b  Data presented are average of three measurements.

ABTS radical cation scavenging activity    The selected plant leaves scavenged ABTS radical and the activities ranged from 198 to 992 µmol TE/g. The calibration curve was linear between 0.2 – 2 mM Trolox with r² value of 0.998. PE had the greatest activity to quench ABTS radical followed by RH, SBT, LGB, SWB, BC, TC, and HT (Table 1). ABTS radical cation scavenging activity of 50% ethanol extract from SBT leaf assessed in this paper was 695 ± 10 µmol TE/g dry leaf, which corresponds to 174 mg TE/g dry leaf. Upadhyay et al. (2013) reported that scavenging activity against ABTS radical cation for water and 70% ethanol extract from SBT leaf grown in India was 120 and 167 mg TE/g dry leaf, respectively. The scavenging activity of the 70% ethanol extract was comparable with the result in this study, whereas the water extract had less scavenging potential.

Superoxide anion scavenging activity    All sample exhibited SOSA which was concentration-dependent. The SOSA was calculated by using the IC₅₀ of the selected plant leaves and the IC₅₀ of enzyme SOD (2.1 ± 0.1 units/mL). The SOSA was in the following decreasing order: PE > RH > SBT > SWB > BC >TC > LGB > HT (Table 1). Sasaki et al. (2013) reported that the 70% ethanol extract prepared from air-dried leaves of BC had significant SOSA compared with DPPH scavenging activity. In addition, among the identified 14 compounds, the following seven compounds showed potent SOSA with IC₅₀ values ranging from 1.12 to 6.09 µM, which were relatively higher than those of the positive control, butylated hydroxyanisole (BHA, IC₅₀ = 17.02 ± 0.95 µM) : ribesin B, ribesin C, ribesin D, 3,3'-didemethoxynectandrin B, ribesin G, ribesin H and kaempferol. From these seven compounds, only ribesin D and kaempferol showed moderate DPPH radical scavenging activity, which were comparable to those of BHA. According to the present results, SOSA activity of BC leaf was higher than those of TC, LGB, and HT leaves. However, BC leaf was more effective than only HT and less effective than other plant leaves to scavenge DPPH free radicals. This tendency was consistent with the above report of Sasaki et al. (2013).

Ferric reducing antioxidant power    The results of FRAP assay are reported in Table 2. All plant leaves assayed in this study showed considerable reducing power from 569 to 3309 µmol Fe²⁺/g, equivalent from 41 to 240 mg ascorbic acid/g. The PE had the highest reducing power, followed by RH, LGB, SBT, SWB, BC, TC, and HT. SWB leaf was one of the five most active plants with ferric reducing power among 70 medical plants (Katalinic et al., 2006). Ferric reducing antioxidant power as well as total phenolics and total flavonoids of wild SWB leaves were higher than those of HT leaves with flowers (Mudnic et al., 2009).

Table 2. Fe³⁺ reducing power of the selected plant leaves

Plant leaf	Reducing powera
	µmol Fe2+/g dry leaf	mg ascorbic acid/g dry leaf
BC (Ribes nigrum)	911 ± 3	67 ± 0
HT (Crataegus sanguinea)	569 ± 6	41 ± 1
LGB (Vaccinium vitis-idaeae)	1839 ± 8	137 ± 1
PE (Paeonia anomala)	3309 ± 56	240 ± 4
RH (Rosa acicularis)	2209 ± 21	166 ± 2
SBT (Hippophae rhamnoides)	1809 ± 16	135 ± 1
SWB (Fragaria orientalis)	1269 ± 10	94 ± 1
TC (Ribes altissimum)	785 ± 9	57 ± 1

a  Each value represents means ± SD (n = 5).

Chelating activity on Fe²⁺    Fe²⁺ are the most effective pro-oxidants in food systems, their good chelating effect are beneficial. However, the plant leaves showed relatively weak ability to chelate Fe²⁺. Only RH exhibited just more than 50% chelation at 5 mg/mL. For SBT and SWB, more than 40% chelation was observed and more than 30% chelation was recognized for TC. The other leaves exerted less than 30% chelation at 5 mg/mL. The weakest activity for iron bonding was observed for LGB leaf. In this assay condition, EDTA exerted very strong chelating activity with IC₅₀ of 36 ± 0.2 µg/mL.

Phenolic contents    Polyphenols have been reported to be responsible for the antioxidant activities of plants. Therefore, total phenol, flavonoid, and flavanol content were measured by Folin-Ciocalteu, AlCl₃, and DMACA assays, respectively. The results are given in Table 3. The selected plant leaves contained a considerable amount of phenolics from 175 mg GAE/g with LGB to 58 mg GAE/g with HT. One gram of air-dried SWB leaf wildly grown in Mongolia contained the total phenols equivalent to 100 ± 1 mg gallic acid. According to previous studies, total phenol content of SWB leaf was 62 ± 1 (Buricova et al., 2011) and 55 ± 4 mg GAE/g of dry matter (Wang and Lin, 2000). Total phenol content of wild SWB leaf infusion was among the highest in the study comparing 70 plants (Katalinic et al., 2006). Total phenol content of BC leaves was 89 – 97 mg GAE/g of dry weight when it was extracted with 30 – 70% ethanol containing 0.05 M H₃PO₄ (Vagiri et al., 2012).

Table 3. Polyphenolic composition of the selected plant leaves

Plant leaf	Total phenolicsa (mg GAE/g dry leaf)	Total flavonoidsa (mg QE/g dry leaf)	Total flavanolsa (mg CE/g dry leaf)
BC (Ribes nigrum)	62 ± 2	5 ± 0	10 ± 0
HT (Crataegus sanguinea)	58 ± 1	5 ± 0	6 ± 0
LGB (Vaccinium vitis-idaeae)	175 ± 2	9 ± 0	32 ± 1
PE (Paeonia anomala)	121 ± 2	5 ± 0	0
RH (Rosa acicularis)	126 ± 2	8 ± 0	1 ± 0
SBT (Hippophae rhamnoides)	96 ± 1	7 ± 0	3 ± 0
SWB (Fragaria orientalis)	100 ± 1	12 ± 0	15 ± 0
TC (Ribes altissimum)	64 ± 0	11 ± 0	6 ± 0

a  Each value represents means ± SD (n = 5).

According to previous reports, a highly positive relationship between total phenols and antioxidant activity appears to be the trend in many plant species. Thus, we also examined the relationship between total phenol content and antioxidant parameters. Plotting of total phenol content of the selected plant leaves against DPPH radical scavenging activity, ABTS radical cation scavenging activity, SOSA, reducing power, and iron chelating ability gave r of 0.494, 0.335, 0.433, 0.082, and 0.038, respectively. On the other word, moderate relationships were observed between total phenol content and DPPH radical scavenging activity, ABTS radical cation scavenging activity and SOSA. The LGB leaf were exceptions as it was the richest in phenolics but the scavenging activity against DPPH radical and Fe³⁺ reducing power were as not effective as those of PE and RH leaves. Among the selected plant leaves, the LGB leaf was ranked by its ABTS radical cation scavenging activity in fourth place after PE, RH, and SBT leaves. Furthermore, the LGB leaf showed very weak SOSA and chelating activity on Fe²⁺. Thus, when data for LGB leaf was omitted, the corresponding correlation coefficients increased up to 0.856, 0.877, 0.778, 0.880, and 0.437, respectively. These results suggested that total phenolics present in the selected plant leaves except for LGB contributed significantly to their antioxidant properties. The LGB leaf may contain phenolic compounds with biological activity other than antioxidant.

The quantities of total flavonoids ranged from 5 to 12 mg QE/g. Total flavonoids of the plant leaves increased in the order, SWB > TC > LGB > RH > SBT > BC > PE > HT (Table 3). No correlation was found between total flavonoid content of the selected plant leaves and their antioxidant properties, as the corresponding coefficients varied from 0.007 to 0.063 (n = 5). In contrast to total phenolics, much low positive and negative correlations have been observed between antioxidant activity and flavonoid content in numerous studies. As AlCl₃ method is specific for flavones and flavonols, the content of total flavonoids could be underestimated (Prasad et al., 2009).

High level of flavanols was recognized in LGB, SWB, and BC, while low level was noted in TC, HT, SBT, and RH. Interestingly, the PE leaf which had no flavanol content and the RH leaf contained very small amount of flavanols exerted excellent antioxidant activities. The correlation coefficients (r) between total flavanol content and DPPH radical scavenging activity, ABTS radical cation scavenging activity, SOSA, reducing power and Fe²⁺ chelating ability were 0.042, 0.130, 0.041, 0.308 and 0.478 (n = 5), respectively. These results indicated that the total flavanols may be not related to the DPPH and ABTS radicals scavenging activity, and reducing power. A rather moderate correlation of total flavanols with SOSA and Fe²⁺ chelating ability suggested that flavanols in the leaves were partly responsible for these two antioxidant parameters.

Conclusion

Eight plant leaves screened in this study showed a variety of antioxidant activities and phytochemical compounds. Among them, LGB, PE, RH, and SBT leaves can be observed as a potent antioxidant sources for further investigation and development into value-added foods, beverages and neutraceuticals that can replace import products to Mongolia.

Since phytochemicals present in PE and RH leaves have not been well studied so far, more research is needed to identify their antioxidative constituents. Furthermore, the LGB leaf which was very rich in polyphenolics should be studied for biological activity other than antioxidant.

Acknowledgments    This work was supported by JSPS RONPAKU (Dissertation PhD) Program. This work was financially supported by Japan Society for the Promotion of Science (113209).

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