Ethnomedicinal Knowledge Verification for the Antidiarrheal and Antioxidant Effects of Rhus chinensis Mill. Fruits with Identification of Thirty Constituents

Belonging to the family Anacardiaceae, over 250 species of genus Rhus are distributed worldwide.1 Species Rhus chinensis Mill. (synonyms: R. javanica var. chinensis (Mill.) T. Yamaz., R. semialata Murray) is known as Chinese sumac.2 In Nepal, it is called as Bhaki-amilo (in Nepali) and Muruk (in Magar language). R. chinensis has been used by folk medicine practitioners for long time in Asia.3 Fruits are used in stomachache, profuse bleeding in menstruation, bloody dysentery, diarrhea, gastrointestinal disorders, and foot and mouth diseases of animals.4-8 Roots have been used in folk medicines as antitussive, and for the treatments of anasarca, jaundice and snake bite.9 Gallarhois on the leaf of R. chinensis has been used for treating diarrhea, seminal emission, excessive sweating, bleeding, chronic cough and polyuria; and possesses anti-thrombotic and antianaphylactic effects.3,10-13


INTRODUCTION
Belonging to the family Anacardiaceae, over 250 species of genus Rhus are distributed worldwide. 1 Species Rhus chinensis Mill. (synonyms: R. javanica var. chinensis (Mill.) T. Yamaz., R. semialata Murray) is known as Chinese sumac. 2 In Nepal, it is called as Bhaki-amilo (in Nepali) and Muruk (in Magar language). R. chinensis has been used by folk medicine practitioners for long time in Asia. 3 Fruits are used in stomachache, profuse bleeding in menstruation, bloody dysentery, diarrhea, gastrointestinal disorders, and foot and mouth diseases of animals. [4][5][6][7][8] Roots have been used in folk medicines as antitussive, and for the treatments of anasarca, jaundice and snake bite. 9 Gallarhois on the leaf of R. chinensis has been used for treating diarrhea, seminal emission, excessive sweating, bleeding, chronic cough and polyuria; and possesses anti-thrombotic and antianaphylactic effects. 3,[10][11][12][13] Although R. chinensis has been consumed since ancient times, the responsible phytoconstituents for the health benefits are remain to be identified. 14 Isolation of gallic acid, gallicin, betulin, betulonic acid, moronic acid, rhuscholide A, benzofuranones, phenolics, etc. has been reported from different parts (root, stem, gallarhois) of R. chinensis. 9,[15][16] Recently, antibacterial activity of R. chinensis against methicillin-resistant Staphylococcus aureus, 6 Shigella species 17 and Streptococcus iniae 18 is reported. R. chinensis extract was identified as the most effective anti-inflammatory and anti-photoaging agent. 19 Gu et al. have reported significant anti-HIV-1 activity of some constituents isolated from the stems of R. chinensis. 15 Hot water extract of the galls showed therapeutic efficacy in mouse HSV-I infection models. 20 In several reports, anti-anaphylactic, antithrombotic, antiviral, antibacterial and anti-plague effects of the galls have been reported. [12][13][21][22][23]  chinensis. Antibacterial activity of the fruits has never been reported though the leaves had displayed antimicrobial activity. 6,[17][18] The phytochemicals present in the plant material were also investigated by chemical tests and GC-MS analysis to identify the biologically active phytochemicals.

Sampling site
The plant material was collected from Hupsekot rural municipality of Nawalpur district, Gandaki Province, Nepal during field visit in April 2017. Ethnomedicinal data of R. chinensis in the Magar community was collected through questionnaires, structural and un-structural interviews among healers and knowledgeable people. Fruits were collected from the study area. The fruits used for the studies were dried in shade at room temperature.

Preparation of the extracts
Air dried fruits of R. chinensis were ground. The ground material (100 g) was successively extracted with hexane (800 mL) and 70% methanol in water (800 mL) using a Soxhlet extractor until a clean solution was noticed. The extracts were concentrated using a rotary evaporator, vacuum dried and then stored in a refrigerator at 4ºC until further use.

Phytochemical screening
Phytochemical screening of the hexane and 70% methanolic extracts was performed using different specific reagents to find out different phytoconstituents present in the fruit extracts. 24 Braymer, Dragendorff, Shinoda, Liebermann-Burchard, Salkowski tests were carried out to detect polyphenols, alkaloid, flavonoids, steroids and terpenoids, respectively.

Gas chromatography-mass spectrometry (GC-MS)
GC-MS analyses of the hexane and 70% methanolic extracts of R. chinensis fruits were performed using an Agilent 7890A GC system coupled with an Agilent 5975 C mass selective detector, equipped with a HP-5MS GC column (5% phenyl methyl siloxane, Agilent 19091S-433, 30 m × 250 μm internal diameter, 0.25 μm film thickness). Helium was used as a carrier gas at flow rate of 1.21 mL/min. The instrument was operated in the electron impact (EI) mode at 70 eV and ion source temperature 230°C in the scan range of 50-500 m/z. The initial column temperature was set at 40°C held for 2 min, ramped at a rate of 4°C/ min to 270°C and held for 5.5 min (total run time 65 min). Dilute sample solutions of the extracts were prepared in HPLC grade hexane or methanol, and a volume of 2 μL was injected. The constituents were identified by comparing the mass spectra available in a MS database of National Institute Standard and Technology (NIST 08).

Total phenolic content (TPC)
TPC value was estimated by using the Folin-Ciocalteu method. 25 Briefly, a solution of 70% methanolic extract of concentration 0.4 mg/ mL was prepared in distilled water. Thus prepared extract solution (50 μL) was treated with 25 μL of Folin-Ciocalteu reagent (Loba Chemie Pvt. Ltd) and 100 μL of aq. Na 2 CO 3 solution (75 g/L). After 1 h, absorbance at 760 nm was measured using an Elisa microplate reader (EPOCH2, BioTek Instruments). Distilled water was taken as a blank.
To obtain a calibration curve, standard gallic acid solutions of different concentrations 100, 50, 25, 12.5, 6.2, 3.1 and 1.6 μg/mL prepared in distilled water were used. TPC value was expressed as mg gallic acid equivalents (GAE) per g dry extract, which was calculated by using the formula: C = cV/m, where c = concentration of gallic acid obtained from the calibration curve in mg/mL, V = volume of the extract in mL, and m = mass of the extract in g.

2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay
The 70% methanolic extract was used to evaluate antioxidant capacity using the DPPH radical scavenging assay. 26 DPPH• radical solution of concentration 0.1 mM was prepared by overnight stirring of DPPH (3.94 mg; Sigma-Aldrich) in methanol (100 mL) at 0ºC. A stock methanolic solution of the extract was prepared (concentration 2000 μg/mL). In a microplate, appropriate amounts of the stock solution were diluted with methanol to obtain 1500, 1000, 750, 500, 250, 100, 50 and 25 μg/mL concentrations (total volume = 50 μL), and then treated with 250 μL of DPPH• radical solution. To obtain linear curve of a positive control, 50 μL of gallic acid solutions of concentrations 20, 10 and 5 μg/mL were used. Methanol was used as a blank. A mixture of DPPH• radical solution (250 μL) and methanol (50 μL) was used as a control. After 30 min, the absorbance was determined at 517 nm using an Elisa microplate reader (EPOCH2, BioTek Instruments). The DPPH• radical scavenging ability was calculated according to the equation given below:

Hydrogen peroxide scavenging activity
Hydrogen peroxide scavenging activity was measured according to the instructions for a commercial kit (Radical catch; Hitachi Ltd., Tokyo, Japan). 27 Briefly, 5 mM of cobalt chloride solution (Reagent A; 25 µL) and luminol solution (Reagent B; 25 µL) were mixed. Then 10 µL of a methanolic solution of 70% methanolic extract of 100 µg/mL concentration was added. Subsequently, the content was incubated at 37°C for 5 min in an incubator (Varioskan LUX Multimode Microplate Reader, Thermo Fisher Scientific, Waltham, MA, USA). Thereafter, the mixture was reacted with hydrogen peroxide solution (Reagent C; 25 µL) and then luminescence of light for 120 sec was measured. The luminescence was observed to subtract an amount of 120 to 80 sec. Control was measured using methanol. Hydrogen peroxide scavenging activity was calculated following the equation below: Antibacterial susceptibility assay Agar well diffusion assay 28 : Inoculum was prepared and standardized. The bacterial inoculums were swabbed on sterile Mueller-Hinton agar (MHA) plates. Both the hexane and 70% methanolic extracts were dissolved in dimethyl sulfoxide (DMSO) to prepare sample solutions of 0.1 g/mL concentration. Wells of 6 mm diameter were bored on the MHA plates and were loaded with 50 μL of the samples prepared. Ampicillin and gentamicin of 10 μg per disc (Mast Diagnostics) were used as standards. DMSO was used as a negative control. The loaded MHA plates were incubated at 37°C for 24 h. Zone of inhibition (ZOI) was measured in mm. 29 : In a microplate, the extract solutions of 0.1 g/mL concentration prepared above in DMSO (50 µL) were mixed with Mueller-Hinton broth (MHB) (50 µL) and then the content was serially double diluted. The bacterial suspension adjusted to 1×10 8 cfu mL -l (equivalent of McFarland 0.5) was further diluted to 1:100 using MHB and then 50 µL of the suspension was inoculated. After incubation for 24 h at 37°C, the MIC value was taken Nemkul, et

Statistical analysis
Statistical analysis was done using Microsoft excel program. Experiments were performed in triplicates (n = 3) and the results are presented as mean ± standard error mean (SEM).

Antioxidant property
Phenolics are potent antioxidants. 38 41 These data clearly indicate that the value of TPC varies with the extractive solvents and our sample constituted comparably a higher amount of phenolics.
In DPPH free radical scavenging activity assay, a linear curve of standard gallic acid (Y = 3.043x + 12.03, R 2 = 0.998) was obtained from the values of inhibition and the concentrations of gallic acid. The IC 50 value of gallic acid in the assay was found to be 12.47 µg/mL. We found that the 70% methanolic extract of the fruits of R. chinensis scavenged 42.69±0.1% DPPH• radical at 100 µg/mL concentration and maximum of 87.24±0.14% of DPPH• radical scavenged at 750 µg/ mL concentration, and the IC 50 value calculated was 135.54±0.82 µg/ mL. Heirangkhongjam and Ngaseppam also reported IC 50 values of 86.54±0.64, 10.35±0.13, 11.19±0.22 and 12.27±0.04 µg/mL using the aqueous, acetone, ethanolic and methanolic extracts of R. chinensis fruits respectively in the DPPH assay. 41 Similar antioxidant activity was also reported by Sharma et al. 40 Next, we found that the 70% methanolic extract scavenged 63.20±1.48% of hydrogen peroxide at 100 µg/mL concentration. From these results, it was considered that the fruits of R. chinensis contained phenolic compounds abundantly hence suitable for consumption to affect cancer chemo-prevention.

Antidiarrheal (antibacterial) activity
Results of the antibacterial susceptibility assay of both the hexane and 70% methanolic extracts are given in Table 3    the study site for curing of diarrhea and dysentery. From the result, it can also be concluded that the fruits of R. chinensis are efficacious against other infectious diseases, such as urinary tract infection and pneumonia, as the fruit extracts exhibited antimicrobial activity against the related bacteria of the diseases.

CONCLUSION
The Magar community of Hupsekot rural municipality, Nawalpur district, Gandaki Province, Nepal uses fruits of R. chinensis (with yogurt) for the treatment of diarrhea and dysentery. This work showed an efficient antibacterial activity of the fruits of R. chinensis against E.coli and S. dysenteriae in the support of the traditional knowledge. The growth of S. aureus, B. subtilis and P. aeruginosa were also inhibited by the extracts indicating the antibacterial efficacy of the plant material in the treatment of other infectious diseases. Evaluation of TPC, and DPPH• radical and hydrogen peroxide scavenging activities has indicated that the fruits of R. chinensis constituted potential antioxidants.

ACKNOWLEDGEMENT
We thank the University Grants Commission (UGC), Nepal for providing a research grant. For laboratories facilities, Nepal Academy of Science and Technology (NAST) is highly acknowledged. We thank local villagers, healers and informants of Hupsekot rural municipality of Nawalpur district for their cooperation. Thanks to the National Herbarium and Plant Laboratories, Godavari, Lalitpur for the plant identification.

CONFLICTS OF INTEREST
No conflicts of interest has been declared by any of the authors.