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

Abstract

Background: Ethnobotanical survey in the rural villages in Nepal revealed that the fruits of Rhus chinensis Mill. have been using for the treatment of diarrhea and dysentery. Objective: To evaluate antimicrobial and antioxidant effects, and identification of chemical constituents in the fruits of R. chinensis. Materials and Methods: Phytochemical screening was performed on the hexane and 70% methanolic extracts of the sample followed by gas chromatographymass spectrometry (GC-MS). Total phenolic content (TPC) was estimated using Folin-Ciocalteu method. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical and hydrogen peroxide scavenging assays were used to evaluate the antioxidant capacity. Antibacterial effect was estabilished by the Agar well diffusion assay. Results: A total of 30 compounds belonging to phenolics, anhydrides, aldehydes, fatty acids and hydrocarbons were identified in the extracts. The TPC value of 123.52±1.29 mg GAE/g dry extract was estimated. IC50 value of 135.54±0.82 μg/mL was calculated in DPPH free radical scavenging assay. Scavenging of 42.69±0.1% DPPH free radical and 63.20±1.48% hydrogen peroxide at 100 μg/mL concentration of 70% methanolic extract were estimated. The maximum zone of inhibition (ZOI) observed was 23.00±0.57 mm against Escherichia coli at loading dose of 5 mg of the extract. Conclusion: All together 30 compounds were identified in the fruits. The extracts efficiently inhibited the growth of E. coli and Shigella dysenteriae verifying the rural knowledge. At the same time, the extracts displayed efficient antioxidant activity. The phytochemicals identified were responsible for these activities.

Keywords:Antibacterial susceptibility assay, DPPH radical scavenging assay, GC-MS, Hydrogen peroxide scavenging activity, total phenolic content

Full text

37
Original Article
Pharmacogn J. 2021; 13(1): 37-43
A Multifaceted Journal in the eld of Natural Products and Pharmacognosy
www.phcogj.com
Cite this article: Nemkul CM, Bajracharya GB, Maeda H, Shrestha I. Ethnomedicinal
Knowledge Verification for the Antidiarrheal and Antioxidant Effects of Rhus chinensis Mill.
Fruits with Identification of Thirty Constituents. Pharmacog J. 2021;13(1): 37-43.
Phcogj.com
Pharmacognosy Journal, Vol 13, Issue 1, Jan-Feb, 2021
Ethnomedicinal Knowledge Verication for the Antidiarrheal
and Antioxidant Eects of Rhus chinensis Mill. Fruits with
Identication of Thirty Constituents
Chandra Mohini Nemkul1, Gan B Bajracharya2,*, Hayato Maeda3, Ila Shrestha4
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.4-8 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 anti-
anaphylactic eects.3,10-13
Although R. chinensis has been consumed since
ancient times, the responsible phytoconstituents
for the health benets are remain to be identied.14
Isolation of gallic acid, gallicin, betulin, betulonic
acid, moronic acid, rhuscholide A, benzofuranones,
phenolics, etc. has been reported from dierent
parts (root, stem, gallarhois) of R. chinensis.9,15-16
Recently, antibacterial activity of R. chinensis against
methicillin-resistant Staphylococcus aureus,6 Shigella
species17 and Streptococcus iniae18 is reported. R.
chinensis extract was identied as the most eective
anti-inammatory and anti-photoaging agent.19 Gu
et al. have reported signicant anti-HIV-1 activity
of some constituents isolated from the stems of R.
chinensis.15 Hot water extract of the galls showed
therapeutic ecacy in mouse HSV-I infection
models.20 In several reports, anti-anaphylactic, anti-
thrombotic, antiviral, antibacterial and anti-plague
eects of the galls have been reported.12-13,21-23 ough
gall of R. chinensis leaf has been much investigated,
fruit of it is rarely explored. Zhang et al. have reported
that fruits of R. chinensis are enriched with phenolics
and the extracts have exhibited strong antioxidant
and pancreatic lipase inhibitory activities.14
In Nepal, some ethnobotanical studies have been
made on R. chinensis considering dierent ethnic
groups.4,8 During our ethnobotanical survey, we
came to know that R. chinensis fruits have been used
for the treatment of diarrhea and dysentery by the
Magar community in Hupsekot rural municipality
of Nawalpur district, Gandaki province, Nepal. To
validate the ethnomedicinal knowledge, the present
research was focused on evaluation of antibacterial
along with antioxidant activities of the fruits of R.
ABSTRACT
Background: Ethnobotanical survey in the rural villages in Nepal revealed that the fruits of
Rhus chinensis Mill. have been using for the treatment of diarrhea and dysentery. Objective:
To evaluate antimicrobial and antioxidant effects, and identification of chemical constituents in
the fruits of R. chinensis. Materials and Methods: Phytochemical screening was performed
on the hexane and 70% methanolic extracts of the sample followed by gas chromatography-
mass spectrometry (GC-MS). Total phenolic content (TPC) was estimated using Folin-Ciocalteu
method. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical and hydrogen peroxide scavenging
assays were used to evaluate the antioxidant capacity. Antibacterial effect was estabilished
by the Agar well diffusion assay. Results: A total of 30 compounds belonging to phenolics,
anhydrides, aldehydes, fatty acids and hydrocarbons were identified in the extracts. The TPC
value of 123.52±1.29 mg GAE/g dry extract was estimated. IC50 value of 135.54±0.82 µg/mL
was calculated in DPPH free radical scavenging assay. Scavenging of 42.69±0.1% DPPH free
radical and 63.20±1.48% hydrogen peroxide at 100 µg/mL concentration of 70% methanolic
extract were estimated. The maximum zone of inhibition (ZOI) observed was 23.00±0.57 mm
against Escherichia coli at loading dose of 5 mg of the extract. Conclusion: All together 30
compounds were identified in the fruits. The extracts efficiently inhibited the growth of E.
coli and Shigella dysenteriae verifying the rural knowledge. At the same time, the extracts
displayed efficient antioxidant activity. The phytochemicals identified were responsible for
these activities.
Key Words: Antibacterial susceptibility assay, DPPH radical scavenging assay, GC-MS,
Hydrogen peroxide scavenging activity, Total phenolic content.
Chandra Mohini Nemkul1, Gan B
Bajracharya2,*, Hayato Maeda3, Ila
Shrestha4
1Departement of Botany, Tri-Chandra
Multiple Campus, Tribhuvan University,
Ghantaghar, Kathmandu, NEPAL.
2Faculty of Science, Nepal Academy of
Science and Technology, Khumaltar, Lalitpur,
NEPAL.
3Faculty of Agriculture and Life Science,
Hirosaki University, 3-Bunkyo-cho, Hirosaki,
Aomori 036-8561, JAPAN.
4Departement of Botany, Patan Multiple
Campus, Tribhuvan University, Patandhoka,
Lalitpur, NEPAL.
Correspondence
Dr. Gan B. Bajracharya
Faculty of Science, Nepal Academy of
Science and Technology, Khumaltar,
Lalitpur, NEPAL.
Phone no: +977-1-5547368; +977-
9849636069
E-mail: ganbajracharya@yahoo.com; gan.
bajracharya@nast.gov.np
History
• Submission Date: 10-09-2020;
• Review completed: 03-11-2020;
• Accepted Date: 10-11-2020.
DOI : 10.5530/pj.2021.13.6
Article Available online
http://www.phcogj.com/v13/i1
Copyright
© 2021 Phcogj.Com. This is an open-
access article distributed under the terms
of the Creative Commons Attribution 4.0
International license.

38
Nemkul, et al.: Ethnomedicinal Knowledge Verication for the Antidiarrheal and Antioxidant Eects of Rhus chinensis Mill. Fruits with Identication of irty
Constituents
Pharmacognosy Journal, Vol 13, Issue 1, Jan-Feb, 2021
chinensis. Antibacterial activity of the fruits has never been reported
though the leaves had displayed antimicrobial activity.6,17-18 e
phytochemicals present in the plant material were also investigated by
chemical tests and GC-MS analysis to identify the biologically active
phytochemicals.
MATERIALS AND METHODS
Sampling site
e plant material was collected from Hupsekot rural municipality of
Nawalpur district, Gandaki Province, Nepal during eld 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. e fruits used for the studies were dried
in shade at room temperature.
Preparation of the extracts
Air dried fruits of R. chinensis were ground. e 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. e 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 dierent specic reagents to nd out dierent
phytoconstituents present in the fruit extracts.24 Braymer, Dragendor,
Shinoda, Liebermann-Burchard, Salkowski tests were carried out
to detect polyphenols, alkaloid, avonoids, 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 lm thickness). Helium
was used as a carrier gas at ow rate of 1.21 mL/min. e 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. e 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. e constituents were
identied 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
Briey, a solution of 70% methanolic extract of concentration 0.4 mg/
mL was prepared in distilled water. us prepared extract solution (50
μL) was treated with 25 μL of Folin-Ciocalteu reagent (Loba Chemie
Pvt. Ltd) and 100 μL of aq. Na2CO3 solution (75 g/L). Aer 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 dierent
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
e 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. Aer 30 min, the absorbance was determined at 517 nm
using an Elisa microplate reader (EPOCH2, BioTek Instruments).
e 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 Briey, 5 mM of cobalt chloride solution (Reagent A;
25 µL) and luminol solution (Reagent B; 25 µL) were mixed. en 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, ermo Fisher Scientic, Waltham, MA, USA). ereaer,
the mixture was reacted with hydrogen peroxide solution (Reagent C;
25 µL) and then luminescence of light for 120 sec was measured. e
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
e bacterial strains Staphylococcus aureus (ATCC 25923), Bacillus
subtilis (ATCC 6051), Enterococcus faecalis (ATCC 29212), Escherichia
coli (ATCC 25922), Salmonella enterica sub-sp. Entericaserovar typhi,
Klebsiella pneumoniae (ATCC 700603), Pseudomonas aeruginosa
(ATCC 27263) and Shigella dysenteriae (ATCC 13313) were used.
Agar well diusion assay28: Inoculum was prepared and standardized.
e 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. e loaded
MHA plates were incubated at 37°C for 24 h. Zone of inhibition (ZOI)
was measured in mm.
Determination of minimum inhibitory concentration (MIC) and
minimum bactericidal concentration (MBC)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. e bacterial suspension
adjusted to 1×108 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. Aer incubation for 24 h at 37°C, the MIC value was taken

39 Pharmacognosy Journal, Vol 13, Issue 1, Jan-Feb, 2021
Nemkul, et al.: Ethnomedicinal Knowledge Verication for the Antidiarrheal and Antioxidant Eects of Rhus chinensis Mill. Fruits with Identication of irty
Constituents
as the lowest concentration that inhibited the visible growth of the
tested bacteria. e MBC value was determined by plating directly the
content of wells in the MHA plate.
Statistical analysis
Statistical analysis was done using Microso excel program.
Experiments were performed in triplicates (n = 3) and the results are
presented as mean ± standard error mean (SEM).
RESULTS AND DISCUSSION
Phytoconstituents
Successive Soxhlet extractions of the fruits of R. chinensis (100 g) yielded
the hexane extract (3.67 g, 3.67%, light color) and 70% methanolic
extract (11.54 g, 11.54%, reddish brown). Phytochemical screening
of the extracts revealed that the fruits of R. chinensis constituted
terpenoids, polyphenols and avonoids.
Next, the extracts were used for the GC-MS analyses. Phytoconstituents
identied in the hexane extract by GC-MS analysis are presented in
Table 1. Sixteen compounds (accounting 94.68%) were identied in the
hexane extract with a higher percentage of phenols (47.30%) and then
fatty acids (25.25%) followed by hydrocarbons (16.90%). It has been
reported that many fatty acids possess antibacterial and antifungal
properties.30 Antibacterial potentiality of hexadecanoic acid methyl
ester, 1-heptatriacotanol and 3-pentadecylphenol that identied in the
hexane extract of the R. chinensis fruits has been reported elsewhere.31-34
All together 18 compounds, accounting 94.09%, were identied in the
70% methanolic extract of the fruits of R. chinensis by GC-MS analysis
(Table 2). e extract constituted abundance of anhydrides (52.38%) and
fatty acids (39.09%). As a avonoid fragment, the extract constituted of
0.32% of 3,5-dihydroxy-6-methyl-2,3-dihydro-4H-pyran-4-one, which
was reported as an antimicrobial and anti-inammatory ingredient.35
Hexadecanoic acid methyl ester and 3-pentadecylphenol possess
antibacterial activity.31,33-34 Antioxidant activity of hexadecanoic acid
ethyl ester and n-hexadecanoic acid is known.32,35 Linoleic acid and
oleic acid are reported as anti-inammatory, anti-androgenic, cancer
preventive, etc.32 5-(Hydroxymethyl)furan-2-carbaldehyde acts as
an antimicrobial, antioxidant and anticancer agents, and inhibits the
formation of sickled cells in the blood.36-37
No. Retention time Name of the compound Molecular formula Nature of the
compound Peak area (%)
1 4.869 Furfural C5H4O2Aldehyde 0.43
2 5.502 Maleic anhydride C4H2O3Anhydride 18.16
3 8.224 3-Methylfuran-2,5-dione C5H2O3Anhydride 17.37
4 10.815 Succinic anhydride C4H4O3Anhydride 0.68
5 13.908 2,3,6,7-Tetrahydrooxepine-4-carboxylic acid, ethyl ester C9H14O3Fatty acid 0.88
6 14.470 Butanedioic acid, monomethyl ester C5H8O4Fatty acid 1.13
7 14.830 dl-Malic acid, dimethyl ester C6H10O5Fatty acid 25.84
8 15.321 3,5-Dihydroxy-6-methyl-2,3-dihydro-4H-pyran-4-one C6H8O4Pyranone 0.32
9 18.485 5-(Hydroxymethyl)furan-2-carbaldehyde C6H6O3Aldehyde 0.40
10 21.066 Phthalic anhydride C8H4O3Anhydride 16.17
11 26.117 Citric acid, trimethyl ester C9H14O7Fatty acid 0.79
12 38.446 Hexadecanoic acid, methyl ester C17H34O2Fatty acid 3.6
13 39.347 n-Hexadecanoic acid C16H32O2Fatty acid 3.79
14 42.342 Methyl (9Z,11E)-octadeca-9,11-dienoate C19H34O2Fatty acid 1.10
15 43.220 Linoleic acid C18H32O2Fatty acid 0.75
16 43.351 Oleic acid C18H34O2Fatty acid 0.86
17 43.891 Hexadecanoic acid, ethyl ester C18H36O2Fatty acid 0.35
18 50.410 3-Pentadecylphenol C21H36OPhenol 1.47
Table 2: Phytoconstituents identied by GC-MS in the 70% methanolic extract of R. chinensis fruit.
No. Retention time Name of the compound Molecular formula Nature of the
compound Peak area (%)
1 38.446 Hexadecanoic acid, methyl ester C17H34O2Fatty acid 0.61
2 39.445 n-Hexadecanoic acid C16H32O2Fatty acid 8.22
3 40.661 Octadecanal C18H36O Aldehyde 0.57
4 42.347 8,11-Octadecadienoic acid, methyl ester C19H34O2Fatty acid 2.03
5 43.411 Oleic acid C18H34O2Fatty acid 3.93
6 43.716 cis-13-Octadecenoic acid C18H34O2Fatty acid 2.48
7 43.973 Octadecanoic acid C18H36O2Fatty acid 5.86
8 48.141 Eicosanoic acid C20H40O2Fatty acid 0.38
9 50.454 (Z)-3-(Pentadec-8-en-1-yl)phenol C21H34OPhenol 26.7
10 50.623 3-Pentadecylphenol C21H36OPhenol 4.67
11 53.896 1-Heptatriacotanol C37H76OAlcohol 4.66
12 54.043 Butyl 6,9,12,15-octadecatetraenoate C22H36O2Fatty acid 1.74
13 54.349 (Z)-3-(Heptadec-10-en-1-yl)phenol C23H38OPhenol 15.93
14 54.458 Heptacosane C27H56 Hydrocarbon 6.55
15 56.444 17-Pentatriacontene C35H70 Hydrocarbon 0.53
16 57.911 Octacosane C28H58 Hydrocarbon 9.82
Table 1: Phytoconstituents identied by GC-MS in the hexane extract of R. chinensis fruit.

40
Nemkul, et al.: Ethnomedicinal Knowledge Verication for the Antidiarrheal and Antioxidant Eects of Rhus chinensis Mill. Fruits with Identication of irty
Constituents
Pharmacognosy Journal, Vol 13, Issue 1, Jan-Feb, 2021
Antioxidant property
Phenolics are potent antioxidants.38 A large number of studies
have proved that they are anti-cancer agents. And, several bioassay
techniques have been developed to estimate antioxidant capacity of
either pure compounds or plant extracts. Here, we have evaluated the
antioxidant capacity of the 70% methanolic extract of R. chinensis fruit
by estimating TPC, and measuring scavenging capacity of DPPH•
radical and hydrogen peroxide.
To estimate TPC, a linear curve of standard gallic acid (Y = 0.023x +
0.088, R2 = 0.996) was obtained from the measured absorbance values
using dierent gallic acid concentrations. e TPC content in the 70%
methanolic extract of the fruits of R. chinensis was calculated using the
regression equation and was found to be 123.52±1.29 mg GAE/g dry
extract. A few authors have reported TPC values in dierent extracts
of the fruits of R. chinensis. Fu et al. reported TPC value of 17.2±1.49
mg GAE/g wet weight in the tetrahydrofuran extract39; Sharma et al.
reported 172.84±15.33 mg GAE/g extract in the methanolic extract;40
and Heirangkhongjam and Ngaseppam reported 7.25±0.03, 42.57±0.24,
28.37±0.46 and 35.76±1.71 mg GAE/g extract in the aqueous, acetone,
ethanolic and methanolic extracts, respectively.41 ese 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, R2 = 0.998) was obtained
from the values of inhibition and the concentrations of gallic acid.
e IC50 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 IC50 value calculated was 135.54±0.82 µg/
mL. Heirangkhongjam and Ngaseppam also reported IC50 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 aect 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 showing ZOI. e 70%
methanolic extract showed strong antibacterial activity against resistant
strain of P. aeruginosa, K. pneumoniae and B. subtilis. is extract
displayed similar antibacterial potentiality as standard antibiotics used
against E. faecalis, a causal agent of urinary tract infection. e 70%
methanolic extract was found more eective against S. aureus than
gentamicin. e growth of E. coli and S. dysenteriae was also eectively
inhibited, which are casual agents of diarrhea and dysentery. e hexane
extract was also found eective to inhibit the growth of S. aureus, B.
subtilis, E. coli and P. aeruginosa. Perhaps the antibacterial activity
observed was mainly due to the presence of fatty acids. Noteworthy
is that both the extracts were ineective towards S. typhi. You et al.
have reported antimicrobial activity of the ethanolic extract of R.
chinensis leaf against methicillin-resistant S. aureus, and mentioned its
bactericidal eect on the bacterial strain.6 Ya ng et al. have also reported
antimicrobial activity of the ethanolic extract of R. chinensis against
S. dysenteriae;17 however, to the best of our knowledge, antimicrobial
activity of the fruits of R. chinensis is not evaluated yet.
MIC and MBC values obtained are tabulated in Table 4. e 70%
methanolic extract displayed bactericidal eect on S. aureus, E. faecalis,
E. coli and P. aeruginosa while it was bacteriostatic to B. subtilis, K.
pneumoniae and S. dysenteriae. e bacteria were killed at higher
concentrations of the extract in a dose-dependent manner. e hexane
extract was found signicantly eective against S. aureus, B. subtilis, E.
coli and P. aeruginosa.
e result of the antimicrobial susceptibility assay presented above
supports the ethnomedicinal use of R. chinensis fruit by magars of
Sample
Diameter of ZOI ± SEM (in mm)
Gram positive bacteria Gram negative bacteria
Sa Bs Ef Ec St Kp Pa Sd
Hexane extract 14.00 ± 0.57 15.66 ± 0.33 – 16.00 ± 0.33 – – 12.33 ± 0.33 –
70% Methanolic
extract 20.25 ± 0.75 20.66 ± 0.33 17.83 ± 0.16 23.00 ± 0.57 – 14.66 ± 0.33 16.33 ± 0.33 14.66 ± 0.33
Ampicillin 32.50 ± 0.50 8.50 ± 0.50 17.75 ± 0.25 25.00 ± 1.00 15.50 ± 0.50 8.50 ± 0.50 – 23.75 ± 0.25
Gentamicin 16.75 ± 0.25 15.50 ± 0.50 18.5 ± 0.50 17.50 ± 0.50 12.66 ± 0.33 11.33 ± 0.88 14.66 ± 0.33 18.66 ± 0.66
DMSO––––––––
Sa = S. aureus, Bs = B. subtilis, Ef = E. faecalis, Ec = E. coli, St = S. typhi, Kp = K. pneumoniae, Pa = P. aeruginosa, Sd = S. dysenteriae. (–) sign indicates no ZOI was
observed.
Table 3: Antibacterial activity of the extracts of R. chinensis fruit.
No. Bacterial strain Hexane extract 70% Methanolic extract
MIC (mg/mL) MBC (mg/mL) MIC (mg/mL) MBC (mg/mL)
1S. aureus 6.25 50 3.12 3.12
2B. subtilis 12.5 25 25 50
3E. faecalis – – 25 25
4E. coli 1.56 6.25 25 25
5S. typhi – – – –
6K. pneumoniae – – 6.25 25
7P. aeruginosa 12.5 50 12.5 12.5
8S. dysenteriae – – 12.5 25
Table 4: MIC and MBC values of the extracts of R. chinensis fruit.

41 Pharmacognosy Journal, Vol 13, Issue 1, Jan-Feb, 2021
Nemkul, et al.: Ethnomedicinal Knowledge Verication for the Antidiarrheal and Antioxidant Eects of Rhus chinensis Mill. Fruits with Identication of irty
Constituents
the study site for curing of diarrhea and dysentery. From the result,
it can also be concluded that the fruits of R. chinensis are ecacious
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
e 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. is work showed
an ecient antibacterial activity of the fruits of R. chinensis against
E.coli and S. dysenteriae in the support of the traditional knowledge. e
growth of S. aureus, B. subtilis and P. aeruginosa were also inhibited by the
extracts indicating the antibacterial ecacy 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. anks to the National
Herbarium and Plant Laboratories, Godavari, Lalitpur for the plant
identication.
CONFLICTS OF INTEREST
No conicts of interest has been declared by any of the authors.
ABBREVIATIONS
DPPH: 2,2-diphenyl-1-picrylhydrazyl; GAE = gallic acid equivalents;
GC-MS: gas chromatography-mass spectrometry; HPLC =
high performance liquid chromatography; IC50: 50% inhibition
concentration; MBC: minimum bactericidal concentration; MHA =
Mueller-Hinton agar; MHB = Mueller-Hinton broth; MIC: minimum
inhibitory concentration; TPC = total phenolic content; ZOI: zone of
inhibition.
REFERENCES
1. Rayne S, Mazza G. Biological activities of extracts from sumac (Rhus spp.): a
review. Plant Foods Human Nutr. 2007;62:165-75.
2. Wagstaff DJ. International poisonous plants checklist: an evidence-based
reference. Boca Raton: CRC Press, Taylor and Francis; 2008.
3. Djakpo O, Yao W. Rhus chinensis and Galla chinensis - folklore to modern
evidence: review. Phytother Res. 2010;24:1739-47.
4. Parajuli SP. Ethnobotanical study at Khandbari municipality of Sankhuwasabha
district, Nepal. Banko Janakari. 2000;10:29-34. Available from: https://www.
nepjol.info/index.php/BANKO/article/view/17600
5. Pradhan BK, Badola HK. Ethnomedicinal plant use by Lepcha tribe of Dzongu
valley, bordering Khangchendzonga, Biosphere Reserve, in north Sikkim, India.
J Ethnobiol Ethnomed. 2008;4:22. Doi: 10.1186/1746-4269-4-22.
6. You YO, Choi NY, Kang SY, Kim KJ. Antibacterial activity of Rhus javanica against
methicillin-resistant Staphylococcus aureus. Evid Based Complement Alternat
Med. 2013. http://dx.doi.org/10.1155/2013/549207.
7. Tangpu V, Yadav AK. Antidiarrhoeal activity of Rhus javanica ripen fruit extract in
albino mice. Fitopterapia. 2004;75:39-44.
8. Manandhar NP. Plants and people of Nepal. Portland Oregon, USA: Timber
Press; 2002.
9. Lee TH, Chiou JL, Lee CK, Kuo YH. Separation and determination of chemical
constituents in the roots of Rhus javanica L. var. roxburghiana. J Chinese Chem
Soc. 2005;52:833-41.
10. Lee SM, Lee DW, Park JD, Kim JI. Study on formation and development of gall
in Rhus javanica. Korean J Appl Entomology. 1997;36:83-7.
11. Song GY, Park BJ, Kim SH. Antithrombotic effect of galla rhois. Korean J
Pharmacog. 2002;33:120-3.
12. Kim SH, Park HH, Lee S, Jun CD, Choi BJ, Kim SY, et al. The anti-anaphylactic
effect of the gall of Rhus javanica is mediated through inhibition of histamine
release and inflammatory cytokine secretion. Int Immunopharmacol.
2005;5:1820-9.
13. Shim YJ, Doo HK, Ahn SY, Kim YS, Seong JK, Park IS, et al. Inhibitory effect of
aqueous extract from the gall of Rhus chinensis on alpha-glucosidase activity
and postprandial blood glucose. J Ethnopharmacol. 2003;85:283-7.
14. Zhang C, Ma Y, Zhao Y, Hong Y, Cai S, Pang M. Phenolic composition,
antioxidant and pancreatic lipase inhibitory activities of Chinese sumac (Rhus
chinensis Mill.) fruits extracted by different solvents and interaction between
myricetin‐3‐O‐rhamnoside and quercetin‐3‐O‐rhamnoside. Int J Food
Sci Technol. 2018;53:1045-53.
15. Gu Q, Wang RR, Zhang XM, Wang YH, Zheng YT, Zhou J, et al. A new
benzofuranone and anti-HIV constituents from the stems of Rhus chinensis.
Planta Med. 2007;73:279-82.
16. Wang RR, Gu Q, Wang YH, Zhang XM, Yang LM, Zhou J, et al. Anti-HIV-1
activities of compounds isolated from the medicinal plant Rhus chinensis. J
Ethnopharm. 2008;117:249-56.
1 7. Yang SK, Kim HJ, Oh SW. Antimicrobial activities of ethanol extracts of Rhus
javanica and Terminalia chebula against Shigella species. J Food Safety.
2014;34:95-102.
18. Choi HS, Kim JS, Jang DS, Yu YB, Kim YC, Lee JS. Antibacterial activities of galla
rhois extracts against fish pathogenic bacteria. J Fish Pathol. 2005;18:239-45.
19. Ha JS, Lee J, Kim H, Song KM, Lee NH, Kim YE, et al. Preventive effect of
Rhus javanica extract on UVB-induced skin inflammation and photoaging. J
Functional Foods. 2016;27:589-99.
20. Kurokawa M, Ochiai H, Nagasaka K, Neki M, Xu H, Kadota S, et al. Antiviral
traditional medicines against herpes simplex virus (HSV-1), poliovirus, and
measles virus in vitro and their therapeutic efficacies for HSV-1 infection in
mice. Antiviral Res. 1993;22:175-88.
21. Kurokawa M, Sato H, Ohyama H, Hozumi T, Namba T, Kawana T, et al. Effects
of traditional herbal medicines against herpes simplex virus (HSV) type 2 and
acyclovir-resistant HSV type1 in vitro and in vivo. J Tradit Med. 1995;12:187-94.
22. Nakano M, Kurokawa M, Hozumi T, Saito A, Ida M, Morohashi M, et al.
Suppression of recurrent genital herpes simplex virus type 2 infection by Rhus
javanica in guinea pigs. Antiviral Res. 1998;39:25-33.
23. Cho MJ, Hong SJ, Choi CH, Jeong SS. Effects of dentifrice containing extract
of galla rhois or Psoralea corylifolia on inhibition of plaque formation. J Korean
Acad Dental Health. 2005;29:141-52.
24. Ciulei I. Practical manual on the industrial utilisation of medicinal and aromatic
plants 1. Methodology for analysis of vegetable drugs. Bucharest, Romania;
1982.
25. Singleton VL, Orthofer R, Lamuela-Raventós RM. Analysis of total phenols
and other oxidation substrates and antioxidants by means of Folin-Ciocalteu
reagent. Methods Enzymol. 1999;299:152-78.
26. Brand-Williams W, Cuvelier ME, Berset C. Use of a free radical method to
evaluate antioxidant activity. LWT - Food Sci Technol. 1995;28:25-30.
27. Maeda H, Fukuda S, Izumi H, Saga N. Anti-oxidant and fucoxanthin contents
of brown alga Ishimozuku (Sphaerotrichia divaricata) from the west coast of
Aomori, Japan. Marine Drugs. 2018;16:255. Doi: 10.3390/md16080255.
28. Perez C, Pauli M, Bazerque P. An antibiotic assay by the agar-well diffusion
method. Acta Biologiae et Medecine Experimentalis. 1990;15:113-5.
29. Wiegand I, Hilpert K, Hancock REW. Agar and broth dilution methods to
determine the minimal inhibitory concentration (MIC) of antimicrobial
substances. Nature protocols. 2008;3:163-75.
30. Knapp HR, Melly MA. Bactericidal effects of polyunsaturated fatty acids. J
Infectious Diseases. 1986;154:84-94.
31. Rukshana MS, Doss A, Kumari PRTP. Phytochemical screening and GC-MS
analysis of leaf extract of Pergularia daemia (Forssk) Chiov. Asian J Plant Sci
Res. 2017;7:9-15.
32. Alagammal M, Tresina SP, Mohan VR. Chemical investigations of Polygala
chinensis L. by GC-MS. Sci Res Reporter. 2011;1:49-52.
33. Nemkul CM, Bajracharya GB, Shrestha I. Phytochemical, antibacterial and
DPPH free radical scavenging evaluations of the barks of Aegle marmelos (L.)
Correa. J Pharmacog Phytochem. 2018;7:1637-41.
34. Devakumar J, Keerthana V, Sudha SS. Identification of bioactive compounds
by gas chromatography-mass spectrometry analysis of Syzygium jambos (L)
collected from Western Ghats region Coimbatore, Tamil Nadu. Asian J Pharma
Clin Res. 2017;10:364-9.
35. Kumar PP, Kumaravel S, Lalitha C. Screening of antioxidant activity, total
phenolics and GC-MS study of Vitex negundo. African J Biochem Res.
2010;4:191-5.
36. Jadhav V, Kalase V, Patil P. GC-MS analysis of bioactive compounds in methanolic
extract of Holigarnagrahamii (wight) Kurz. Int J Herbal Med. 2014;2:35-9.

42
Nemkul, et al.: Ethnomedicinal Knowledge Verication for the Antidiarrheal and Antioxidant Eects of Rhus chinensis Mill. Fruits with Identication of irty
Constituents
Pharmacognosy Journal, Vol 13, Issue 1, Jan-Feb, 2021
37. Al-Marzoqi AH, Hadi MY, Hameed IH. Determination of metabolites products by
Cassia angustifolia and evaluate antimicrobial activity. J Pharmacog Phytother.
2016;8:25-48.
38. Zeb A. Concept, mechanism, and applications of phenolic antioxidants in foods.
J Food Biochem. 2020; e13394. Doi: 10.1111/jfbc.13394
39. Fu L, Xu BT, Xu XR, Qin XS, Gan RY, Li HB. Antioxidant capacities and total
phenolic contents of 56 wild fruits from South China. Molecules. 2010;15:8602-17.
40. Sharma PB, Handique PJ, Devi HS. Antioxidant properties, physico-chemical
characteristics and proximate composition of five wild fruits of Manipur, India.
J Food Sci Technol. 2015;52:894-902.
41. Heirangkhongjam MD, Ngaseppam IS. Nutritional evaluation of Rhus chinensis
Mill. (Heimang) and development of value added products. Indian J Tradit
Knowledge. 2019;18:16-24.
GRAPHICAL ABSTRACT
SUMMARY
• Thirty phytoconstituents present in the fruit of Rhus chinensis Mill. are identified. (Z)-3-(Pentadec-8-en-1-yl)phenol,
(Z)-3-(heptadec-10-en-1-yl)phenol, maleic anhydride, phthalic anhydride, 3-methylfuran-2,5-dione and dl-malic acid,
dimethyl ester are the major constituents.
• The fruit possesses antidiarrheal property as the extracts could inhibit the growth of Escherichia coli and/or Shigella
dysenteriae.
• The fruit contains abundant amounts of phenolics and antioxidants hence considered to be efficacious in cancer
prevention.
ABOUT AUTHORS
Chandra Mohini Nemkul is currently working in the capacity of Associate professor at Department of Botany,
Tri-Chandra Multiple Campus, Tribhuvan University, Nepal. She has been involving in research focusing on
ethnomedicinal plants used by indigenous people, evaluating indigenous knowledge scientifically as well as
isolation and identification of bioactive compounds. Previously, she had successfully conducted studies on
ethnomedicinal knowledge of Newar communities residing in Nepal, a project granted by University Grants
Commission (UGC), Nepal.

43 Pharmacognosy Journal, Vol 13, Issue 1, Jan-Feb, 2021
Nemkul, et al.: Ethnomedicinal Knowledge Verication for the Antidiarrheal and Antioxidant Eects of Rhus chinensis Mill. Fruits with Identication of irty
Constituents
Dr. Gan B. Bajracharya is a Senior Scientific Officer at Nepal Academy of Science and Technology, Nepal.
He received his Doctor of Science degree in 2004 from Tohoku University, Japan working under Prof. Dr.
Yoshinori Yamamoto. He carried out post-doctoral researches at Osaka University, Japan (with Prof. Dr. Hiroaki
Sasai); University of Houston, Texas (with Prof. Dr. Olafs Daugulis); and HEJ Research Institute of Chemistry,
University of Karachi, Pakistan (with Prof. Dr. Muhammad Iqbal Choudhary). He has published about 50
research articles in international and national peer-reviewed journals. His research interest is focused on the
development of catalytic reactions and natural product chemistry.
Dr. Hayato Maeda is Associate Professor in Faculty of Agriculture and Life Science at Hirosaki University, Japan.
He analyzes chemicals in foods using high performance liquid chromatography and gas chromatography.
He is affiliated with Japan Society for Bioscience, Biotechnology, and Agrochemistry; Japanese Society of
Nutrition and Food Science; the Japanese Society for Food Science and Technology; Japanese Society for
Food Factors; etc.
Dr. Ila Shrestha (Pradhan) is Professor at Department of Botany, Patan Multiple Campus, Tribhuvan University,
Nepal. Her research is focused on ethnobotanical study of different geographic areas and social communities
with application of Geographic Information System and Remote Sensing. She had involved in herbal farming
in Dhading district, Nepal. She is affiliated with Ethnobotanical Society of Nepal.
Cite this article: Nemkul CM, Bajracharya GB, Maeda H, Shrestha I. Ethnomedicinal Knowledge Verification for the Antidiarrheal
and Antioxidant Effects of Rhus chinensis Mill. Fruits with Identification of Thirty Constituents. Pharmacog J. 2021;13(1): 37-43.