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Pak. J. Bot., 43: 85-89, Special Issue, December, 2011 (Medicinal Plants: Conservation & Sustainable use)
ANTIOXIDANT, ANTITUMOR ACTIVITIES AND PHYTOCHEMICAL INVESTIGATION
OF HEDERA NEPALENSIS K.KOCH, AN IMPORTANT MEDICINAL
PLANT FROM PAKISTAN
SIMAB KANWAL1, NAZIF ULLAH1, IHSAN-UL-HAQ1, IMRAN AFZAL2 AND BUSHRA MIRZA1*
1Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan
2Department of Biotechnology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan
Abstract
Hedera nepalensis is a ground-creeping evergreen woody plant growing mainly in the Himalayas and Kashmir. This
plant is frequently used in folk medicines for the treatment of various ailments. The present research focused on the
pharmacological evaluation and phytochemical analysis of crude methanolic extract (CME) and three fractions, n-hexane (n-
HF), ethyl acetate (EAF) and aqueous (AQF). The biological assays used for this study included DPPH free radical
scavenging assay, DNA protection assay and potato disc antitumor assay. Maximum antioxidant activities with IC50 values
of 9.834 ppm and 14.22 ppm were shown by EAF and AQF, respectively. Crude methanolic extract (CME) and the fractions
also exhibited significant DNA protection activity in •OH induced DNA damage assay, at all the concentrations tested. Both
EAF and AQF showed well-defined tumor inhibition in the potato disc antitumor assay, with the lowest IC50 values shown
by EAF and AQF (less than 1 ppm). Phytochemical analysis showed the presence of flavonoids, steroids, tannins, terpenoids
and cardiac glycosides in the crude extract and its fractions. The present study demonstrated that EAF and AQF of Hedera
nepalensis have potent antioxidant and antitumor activity with the presence of effective phytochemicals.
Introduction
Hedera is a genus of 15 species belong to the family
Araliaceae, which includes about 70 genera and 700
species of flowering plants. Though the taxonomy and
phylogenetic relationships of Hedera species remain
problematic, the genus is well known for its economical
importance (Ackerfield & Wen, 2002). The species
selected in the present research work is H. nepalensis,
locally known as Arbambal (Shah & Khan, 2006).
Traditionally, the plant is largely used in folk medicine,
where its different parts, along with the extract, are used
for the treatment of various diseases. Studies have shown
that the leaves and berries are stimulating, diaphoretic,
cathartic, and used to treat indolent ulcers and abscesses.
A decoction of the leaves is effective against lice (Qureshi
et al., 2007). Hedera nepalensis has hypoglycemic
properties and is found to be effective against fever,
pulmonary infections and rheumatism (Shah & Khan,
2006). Hamayun et al., (2006) reported its anticancer
properties as well. Inayatullah et al., (2007) screened the
crude methanolic extract of H. nepalensis (leaves + stem)
for different biological activities such as brine shrimp
cytotoxic activity, potato disc antitumor activity and
phytotoxic activity. This plant has also been evaluated for
its antifungal activity by Xue et al., (2010).
In view of the medicinal importance of H. nepalensis,
based on traditional knowledge and surveyed literature,
the present study focused on the fractionation procedure,
antioxidant activities and phytochemical analysis of the
crude methanolic extract and fractions. Following the
work of Inayatullah et al., (2007), the potato disc
antitumor activity was also conducted on the fractions of
H. nepalensis to find out how the different
phytochemicals are separated into different fractions.
Materials and Methods
Hedera nepalensis was collected from Ayubia,
pipeline track (near Muree Pakistan), during July 2008
and was identified by Dr. Mir Ajab Khan (taxonomist,
Department of Plant Sciences, Quaid-i-Azam University,
Islamabad, Pakistan). A voucher specimen (herbarium
number HMP460) and deposited in the herbarium
collection.
Extraction and fractionation procedure: Fresh aerial
parts of the plant were collected from the field, rinsed
with distilled water, cut into small pieces and shade dried
at room temperature. Extraction from the aerial part was
carried out following a maceration procedure. A total of
671 g of plant material was ground in 3.0 l of methanol
using a kitchen blender. The poorly homogenized mixture
was kept for four weeks at room temperature (25°C ±
2°C) in an extraction bottle. After four weeks, the mixture
was filtered by squeezing the plant material in gauze cloth
before filtering with Whatman #1 filter paper. The filtrate
was then concentrated in a rotary evaporator at 45°C
under low pressure and dried to a constant weight of 15.0
g. This crude methanolic extract (CME) was subjected to
different biological assays for pre-screening of potential
pharmacological activities. CME was also investigated for
the presence of some important phytochemicals, including
steroids, alkaloids, cardiac glycosides and saponins. The
crude methanolic extract (15 g) was then suspended in
250 ml distilled H2O. This aqueous suspension was then
fractionated by solvent-solvent extraction, first with n-
hexane and then with ethyl acetate, using a separating
funnel (Pyrex, England). Three fractions, namely n-
hexane fraction (n-HF), ethyl acetate fraction (EAF) and
aqueous fraction (AQF), were obtained. All the fractions
were concentrated in a rotary evaporator and dried to a
constant weight in a vacuum oven at 45ºC. These
fractions were then subjected to the same biological
assays and phytochemical analysis used with the CME.
The extracts were kept at 4ºC when not in use.
*E-mail: bushramirza@qau.edu.pk
SIMAB KANWAL ET AL.,
86
Bioassays
Procedures for the selected bench top bioassays are
given below in detail.
1. Antioxidant assays: In order to evaluate the
antioxidant potentials, the 2,2-diphenyl-1-picrylhydrazyl
(DPPH) free radical scavenging assay and DNA
protection assay were used.
a. DPPH free radical scavenging assay: The DPPH
assay was performed according to the procedure described
by Kulisic et al., (2004) and modified by Obeid et al.,
(2005). In a glass vial, 2800 µl of 0.1 mM DPPH solution
(in 82% methanol) was mixed with 200 µl of test sample
(in methanol). This stock solution was serially diluted
with methanol to obtain final concentrations of 100 ppm,
50 ppm, 25 ppm, 10 ppm, 5 ppm, 2 ppm and 1 ppm. Each
concentration was assayed in triplicate. The vials were
capped, shaken well and kept in the dark at 37ºC for one
hour. After incubation, the change in colour (from deep-
violet to light-yellow) of DPPH free radical was then
measured by taking the absorbance of the reaction
mixtures at 517 nm on a UV/Visible spectrophotometer
(DAD Agilent 8453). A mixture of 2800 µl of 82%
methanol and 200 µl of methanol was used as a blank
while ascorbic acid was used as a positive control. The
percentage scavenging of DPPH free radical for each
concentration of test sample was calculated by using the
following formula:
% Scavenging = [(A-B)/A] × 100
where
A = Absorbance of negative control
B = Absorbance of test sample
IC50 values were calculated by a table curve using 2D v4
(AISN software)
b. DNA protection assay: Antioxidant and pro-oxidant
activities of CME and its fractions were evaluated by
conducting a DNA protection assay according to the
method reported by Tian & Hua (2005). Plasmid DNA
(pBR322 Fermentas) with a concentration of 0.5 μg/3 μl
was treated with three different concentrations of plant
extracts (30 ppm, 300 ppm and 3000 ppm dissolved in
methanol) in the final reaction volume of 15 µl. Fenton
reaction was induced by addition of 30% H2O2 (4 µl) and
2 mM FeSO4 (3 µl) into the reaction mixture. Four
controls (untreated DNA, DNA treated with 2 mM FeSO4,
DNA treated with 30% H2O2, DNA treated with 2 mM
FeSO4 and 30% H2O2) were run simultaneously. Each
mixture was incubated at 37ºC for one hour. After
incubation, 3 μl of bromophenol blue (loading dye) was
added to each reaction mixture, the samples were loaded
on a 1% agarose gel containing TBE (Tris, boric acid,
EDTA) buffer and ethidium bromide, and visualized with
Doc-IT (VWR). Evaluations of antioxidant or pro-oxidant
effects on DNA were based on the increase or loss
percentage of a super-coiled monomer, compared with the
control value. To avoid the effects of photo excitation of
samples, experiments were done in the dark.
2. Antitumor potato disc assay: CME and its fractions
were screened for their antitumor activities by using the
antitumor potato disc assay as reported by McLaughlin &
Rogers (1998). A 48-hour-old bacterial culture of
Agrobacterium tumefaciens (At 10) was used in this
experiment. Inocula with five concentrations of test
samples (1000, 100, 10, 5 and 1 ppm) were prepared with
1.5 ml autoclaved distilled water, 2.0 ml of bacterial
culture (1 x 108 CFU/ml) and 0.5 ml sample solution in
DMSO. A negative control was prepared by replacing the
sample solution with 0.5 ml DMSO. Red-skinned potatoes
were surface sterilized using a 0.1% HgCl2 solution.
Potato discs of 8 mm x 4 mm were prepared. Autoclaved
plain agar (1.5%) was poured in small petri plates and
allowed to solidify. Ten discs were placed on the agar
surface of each plate, and then 50 μl of inoculum was
poured on the surface of each disc. The plates were sealed
with Parafilm and incubated at 28°C in dark. The
experiment was carried out in triplicate. After 21 days of
incubation, potato discs were stained with Lugol’s
solution (10% KI, 5% I2), and tumors were counted.
Percentage tumor inhibition was calculated with the
following formula:
Percentage inhibition = (1- Ns/Nc) x 100
where
Ns = Average number of tumors in sample
Nc = Average number of tumors in negative control
More than 20% tumor inhibition was considered
significant (McLaughlin & Rogers, 1998).
Phytochemical analysis: The crude methanolic extract of
H. nepalensis and its fractions were qualitatively analyzed
for the presence or absence of certain phytochemicals by
using standard methods of analysis (Harborne, 1993;
Sofowara, 1993; Trease & Evans, 2002; Edeoga et al.,
2005; Parekh & Sumitra, 2007) as described by Parekh
and Sumitra, (2007). For alkaloids, 400 mg plant material
was mixed with 20 ml methanol and filtered. A mixture of
2 ml filtrate + 1% HCl + was steamed and filtered, and 1
ml filtrate + 6 drops of Mayer’s reagent, Wagner’s reagent
or Dragendorff reagent was checked for precipitates. A
creamish precipitate, a brownish-red precipitate or an
orange precipitate indicated the presence of alkaloids
using the respective reagents. The presence of
carbohydrates was determined using Bradford's and
Fehling's tests. In Bradford’s test, 3 ml of filtrate + 2 ml of
Bradford's reagent were mixed, heated for 7-12 minutes in
a water bath, and a red precipitate indicated the presence
of disaccharides. In Fehling’s test, 0.5 g of plant material
was dissolved in 5 ml distilled water and filtered, 1 ml
each of Fehling's A and B were mixed, boiled for 2
minutes on a spirit lamp, 1 ml of filtrate was added, and
the mixture was boiled again for 1 minute. A red
precipitate indicated the presence of carbohydrates
(reducing sugars). For tannins, 100 mg of sample was
dissolved in 5 ml distilled water and filtered, and 2 ml
filtrate + 2 ml FeCL3 was checked for a blue-black
precipitate that indicated the presence of tannins. The
presence of phlobatannins was determined by mixing 0.5
g plant material in 5 ml distilled water, boiling and
filtering, and adding add 1% aq. HCl to the filtrate; a red
precipitate indicated the presence of phlobatannins. For
ANTIOXIDANT ACTIVITIES AND PHYTOCHEMICAL INVESTIGATION OF HEDERA NEPALENSIS K.KOCH
87
saponins, the frothing test and emulsion test were used. In
the frothing test (0.5 ml filtrate + 5 ml of distilled water,
shaken well), frothing persistence indicated the presence
of saponins. In the emulsion test, 0.1 g plant material in 5
ml distilled water was filtered, 2 ml filtrate + few drops of
olive oil were shaken and checked for the presence of an
emulsion, which indicated the presence of saponins. For
cardiac glycosides, the Keller-Kiliani test was used (2 ml
filtrate + 1 ml glacial acetic acid + FeCl3 + concentrated
H2SO4); a green-blue color indicted the presence of
cardiac glycosides. For the identification of steroids, the
Liebermann-Burchard reaction was used. For this 100 mg
plant materials in 5 ml chloroform were mixed and
filtered, then 2 ml filtrate + 2 ml acetic anhydride +
concentrated H2SO4 was checked for color. Steroids
appeared as green to pink and terpenoids as pink to
purple. For flavonoids, 100 mg plant material in 5 ml
ethanol was mixed and filtered. A 2 ml filtrate +
concentrated HCl + magnesium ribbon will produce pink-
tomato red color if flavonoids are present.
The presence or absence of these phytochemicals is
represented in Table 3 using plus (+) and minus (-) signs
respectively, as displayed by Parekh & Sumitra, (2007)
and Usman et al., (2007).
Results and Discussion
Bench top bioassays have proved to be good tools for
biological evaluation of plant extracts. According to Jerry
et al., (1998) crude botanical extracts are comprised of
very effective mixtures of bioactive compounds, and it is
quite possible to sort out which activities are due to which
components with the help of simple bioassay procedures
and various separation techniques. Antioxidant activity of
H. nepalensis crude extract and its fractions was
determined by using two assays. These assays showed
dose-dependent free radical scavenging capacity and a
protective effect on DNA cleavage. DPPH assay is
considered to be a simple, convenient and rapid method
for screening of plant extract/compounds for their
antioxidant potential by measuring the reducing ability of
tested compounds. All three fractions were tested for their
DPPH scavenging activity. A well-known antioxidant,
ascorbic acid, was used as positive control. The lowest
IC50 values were observed for EAF (9.83) and AQF
(14.2). DPPH scavenging patterns for CME and its three
fractions, along with IC50 values, are presented in Table 1.
Table 1. DPPH free radical scavenging activities of CME and its fractions along with IC50 values.
% Scavenging at different concentrations (ppm)
No. Test samples 100 50 25 10 5 2 1
IC50 (ppm)
1. CME 80.4 71.8 35.2 0 0 0 0 35.29
2. n-HF 58 23 23 0 0 0 0 89
3. EAF 82 82 81 59 24 4 0 9.834
4. AQF 81 80 81 30 10 0.4 0 14.22
5. Asc. acid 95.04 94.8 90.0 86.4 44.7 17.6 1.77 7.02
CME = crude methanolic extract, n-HF = n-hexane fraction, EAF = ethyl acetate fraction, AQF = aqueous fraction, Asc. Acid =
ascorbic acid
Antioxidant effects of CME and its fractions were
also investigated in vitro by using a free radical-induced
oxidative plasmid (pBR322) DNA break system. All the
fractions, along with the crude extract, exhibited
significant DNA protection activity, while no obvious
DNA damage was observed in any of the test samples as
presented in Fig. 1(a, b). Despite its low DPPH free
radical scavenging activity, n-HF exhibited pronounced
DNA protection activity, indicating that while n-HF may
not possess free radical scavenging potential, it might
have some effect on the Fenton reaction. The nature of
DNA protection is ambiguous because there are several
potential inhibition pathways. The antioxidants can inhibit
the reaction by reacting directly with H2O2 or reacting
with intermediates formed from enzymes and H2O2
(Martinez et al., 2001). DNA protection was variable for
all three concentrations of the extract and fractions tested.
Previous research indicated that the leaves of H.
nepalensis have anticancer properties (Hamayun et al.,
2006). Inayatullah et al., (2007) examined the crude
methanolic extract of H. nepalensis (leaves + stem), so we
decided to check all the fractions for antitumor activity.
Our results showed significant tumor inhibition. The n-HF
and EAF exhibited 95% and 90% tumor inhibition at 1000
ppm, respectively. Tumor inhibition was observed in a
concentration-dependent mode: tumor inhibition increased
with increase in concentration of the test samples (Table
2). Unlike the fractions, the crude extract did not exhibit a
pronounced level of tumor inhibition, which might be due
to low concentration or the antagonistic effects of the
compounds present in the crude extract. The effect of
crude extract of H. nepalensis on viability of A.
tumefaciens was found to be quite insignificant
(Inayatullah et al., 2007), indicating that the
extract/fractions are not involved in killing the bacterium
(A. tumefaciens) that causes tumors, but rather inhibit
tumors by other means.
Table 2. Inhibition of tumor formation by CME and its fractions along with IC50 values.
% Inhibition at different concentrations (ppm)
No. Extract/Fraction 1000 100 10 5 1
IC50 (ppm)
1. CME 67 63 60 60 36 2.96
2. n-HF 90 78 66 58 45 3.6
3. EAF 95 90 80 85 61 <1 ppm
4. AQF 84 85 71 74 66 <1 ppm
CME = crude methanolic extract, n-HF = n-hexane fraction, EAF = ethyl acetate fraction, AQF = aqueous fraction
SIMAB KANWAL ET AL.,
88
Fig. 1(a). DNA protection assay with three different concentrations of CME
1. 1 kb DNA Ladder, 2. Plasmid DNA (pBR322), 3. Plasmid DNA treated with 2 mM FeSO4, 4. Plasmid DNA treated with 30% H2O2,
5. Plasmid DNA treated with 2 mM FeSO4 + 30% H2O2, 6. CME (1000 ppm) + plasmid DNA treated with FeSO4 + H2O2, 7. CME
(100 ppm) + plasmid DNA treated with FeSO4 + H2O2, 8. CME (10 ppm) + plasmid DNA treated with FeSO4+H2O2.
Fig. 1(b). DNA protection assay with all the fractions of CME at three different concentrations
1. 1 kb DNA ladder, 2. Plasmid DNA (pBR322), 3. Plasmid DNA treated with 2 mM FeSO4, 4. Plasmid DNA treated with 30% H2O2,
5. Plasmid DNA treated with 2 mM FeSO4 + 30% H2O2, 6. n-HF (1000 ppm) + plasmid DNA treated with FeSO4 + H2O2, 7. n-HF
(100 ppm) + plasmid DNA treated with FeSO4 + H2O2, 8. n-HF (10 ppm) + plasmid DNA treated with FeSO4 + H2O2, 9. EAF (1000
ppm) + plasmid DNA treated with FeSO4 + H2O2, 10. EAF (100 ppm) plasmid DNA treated with FeSO4 + H2O2, 11. EAF (10 ppm)
plasmid DNA treated with FeSO4 + H2O2, 12. AQF (1000 ppm) + plasmid DNA treated with FeSO4 + H2O2, 13. AQF (100 ppm) +
plasmid DNA treated with FeSO4 + H2O2, 14. AQF (10 ppm) + plasmid DNA treated with FeSO4 + H2O2.
The preliminary phytochemical studies demonstrated
the presence of alkaloids, flavonoids, steroids, tannins and
terpenoids it varying concentrations. Terpenoids were
detected at high concentrations in all the extract/fractions
except n-HF, as shown in Table 3. Tannins were found at
moderate concentrations, while phlobatannins were
completely absent. Terpenoids, flavonoids, alkaloids and
tannins are considered to possess high antioxidant
activities, which prevent or can be used in the treatment of
many diseases, including cancer (Madhuri & Pandey,
2009). Therefore, the presence of appreciable to moderate
amounts of these phytochemicals can be correlated with
the possible significant medicinal potential of the plant.
Conclusion
Crude methanolic extract of Hedera nepalensis and
its ethyl acetate and aqueous fractions showed significant
antioxidant activity in a DPPH free radical scavenging
activity assay and DNA protection assay. The results for
the potato disc antitumor assay showed that this plant has
highly potent antitumor agents. Further isolation and
purification of bioactive compounds from EAF and AQF
in the future may reveal the presence of potent novel
antioxidants and anticancer agents from H. nepalensis.
Acknowledgments
The authors are thankful to the Higher Education
Commission (HEC) Islamabad, for providing a grant to
accomplish this study and also to Prof. Dr. Mir Ajab
Khan, Plant Sciences Department, Quaid–i-Azam
University, Islamabad, Pakistan for plant identification.
ANTIOXIDANT ACTIVITIES AND PHYTOCHEMICAL INVESTIGATION OF HEDERA NEPALENSIS K.KOCH
89
Table 3. Phytochemical analysis of CME and its fractions.
Extract/fractions
No. Constituents/test CME n-HF EAF AQF
Alkaloids
Dragendorff's - - - -
Mayer’s ++ - ++ -
1.
Wagner’s + - ++ -
Carbohydrates
Bradford’s + + - -
2.
Fehling’s + + + +
Flavonoids
3. Harborne ++ - + +
Steroids
4. Liebermann-Burchard reaction ++ ++ - -
Saponins
Frothing Test - - + +
5.
Emulsion Test - + + -
Tannins
6. FeCl3 Test ++ + - -
7. Phlobatannins - - - -
8. Terpenoids +++ - +++ +++
Cardiac Glycosides
9. Keller-Kiliani Test + - ++ ++
Key: CME = Crude methanolic extract, n-HF = n-hexane fraction, EAF = Ethyl acetate fraction, AQF = Aqueous
fraction, +++ = Good amount, ++ = Moderate amount, + = Trace amount, - = Completely absent
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(Received for publication 20 October 2011)
