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Tripathi et al. World Journal of Pharmacy and Pharmaceutical Sciences
ANTIFUNGAL ACTIVITY OF CAPPARIS DECIDUA EXTRACTS
AGAINST SEED-BORNE PATHOGENIC FUNGI
Y.C. Tripathi1*
, Sushma Singh2, Nishat Anjum
1, K.K. Srivastava
2
1Chemistry Division, Forest Research Institute, Dehradun – 248006, India
2Forest Protection Division, Arid Forest Research Institute, Jodhpur – 342005, India
ABSTRACT
Biodeterioration of seeds due to fungal infestation cause considerable
losses in terms of their viability, chemical composition and end-use.
Control of such fungi using synthetic fungicides has limitation of
adverse health and environmental consequences. This can be overcome
through persistent exploration and promotion of eco-friendly botanical
fungicides. With this milieu, methanolic extracts of wood, bark and
seeds of Capparis decidua, traditionally acclaimed for medicinal value
were evaluated for antifungal efficacy against six seed-borne pathogenic
fungal species causing severe deterioration to economically important
seeds during storage. Methanol extracts of wood, bark and seeds of C.
decidua showed very low MIC values against all the six fungi. Results
indicated that extracts of all the three parts at all the tested concentrations were effective as
compared to negative control. However, extracts of different parts exhibited varying degrees
of inhibition. Extent of activity also differed with concentration of extracts used. Radial
growth inhibition was minimum with extracts of all the three parts at 5mg/ml concentration and
maximum at 100mg/ml. Among the treatments, bark extract was found more effective than that
of seeds and wood. Growth inhibition with bark and seed extract at concentration of 25mg/ml
was almost at par with the synthetic fungicide carbendazim. Further, growth inhibition of all
the six fungi with 50 mg/ml concentration of extracts of the three parts is significantly higher
as compared to carbendazim. The study thus revealed the promising antifungal efficacy of C.
decidua.
KEYWORDS: Biodeterioration, Seed-borne fungi, Capparis decidua, Extract, Antifungal
activity.
WWOORRLLDD JJOOUURRNNAALL OOFF PPHHAARRMMAACCYY AANNDD PPHHAARRMMAACCEEUUTTIICCAALL SSCCIIEENNCCEESS
SSJJIIFF IImmppaacctt FFaaccttoorr 55..221100
VVoolluummee 44,, IIssssuuee 1100,, 1500-1512. RReesseeaarrcchh AArrttiiccllee IISSSSNN 2278 – 4357
Article Received on
06 Aug 2015,
Revised on 27 Aug 2015,
Accepted on 18 Sep 2015
*Correspondence for
Author
Y.C. Tripathi
Chemistry Division Forest
Research Institute P.O.
New Forest, Dehradun –
248006, India
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Tripathi et al. World Journal of Pharmacy and Pharmaceutical Sciences
INTRODUCTION
Capparis decidua (Forsk.) Edgew (Syn. Capparis aphylla Roth.) belonging to the family
Capparidaceae, is a glabrous densely branched spinous shrub or small tree found in dry and
bare habitat of Sind, Baluchistan, Egypt, Socotra, Arabia, Tropical Africa, Western and
Central India and Pakistan[1]
and growing wild in Rajasthan, Gujarat, Punjab, Deccan
Peninsula and Western Ghats of India.[2]
It is commonly known as Gudhapatra, Korira, Karer
or Kair and traditionally acclaimed for its immense medicinal and livelihood importance. The
unripe fruits are considered anthelmintic and laxative and employed in the treatment of
asthma, constipation, coughs, hysteria and other psychological problems.[3]
In traditional
medicinal systems, it is regarded as laxative, astringent and vermifuge.[4]
The root bark is
prescribed for the treatment of toothache, intermittent fevers, asthma, cough, inflammations
and rheumatism.[5]
Fruits and seeds are regarded as diuretic, antidiabetic, astringent to bowels
and said to be beneficial for cardiac troubles and seed oil is used to cure skin diseases.[6]
Phytochemical studies of the plant have revealed the presence of a number of chemical
constituents including sterols,[7]
fatty acids,[8]
flavones,[9]
oxygenated heterocyclic
constituents,[10]
alkaloids[11-15]
and an isothiocyanate glucoside[16]
in different parts of the
plant. Pharmacological studies have reported its CNS sedative and depressant[17-18]
and
antimicrobial properties.[19]
Alcoholic extract of the root bark and fruit pulp is claimed to
have anthelmintic activity.[20]
Methanol and water extracts possessed hepatoprotective
activity.[21]
The effects of plant extracts on human plasma triglycerides, total lipids and
phospholipids have also been reported.[22]
The fruit has been shown to possess anti-
atherosclerotic,[23]
antidiabetic,[24-25]
anti-hypertensive[23]
and anti-hyperlipidemic
properties.[26]
Capparidisine, a spermidine alkaloid from the plant is reported to have
cardiovascular activity.[27]
Besides pharmacological efficacy, the chemical constituents and extracts of C. decidua are
reported to have varied biocidal properties.[28-29]
Our earlier work reported aphicidal properties
of different parts of C. decidua against the aphids Mysus persicae [30]
and Lipaphis erysimi.[31]
The present study reports antifungal activity of extracts of different parts of C. decidua against
certain seed-borne pathogenic fungi which are most common associates of forest seeds.
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MATERIALS AND METHODS
Plant Material
The plant materials of C. decidua, viz. bark, wood and fruits were collected from suburbs of
Jodhpur, (Rajasthan), India and got authenticated in consultation with Botany Department,
J.N.V. University, Rajasthan as well as by using the ‗Flora of Indian Desert‘.[32]
Processing of plant material
The collected plant materials i.e. bark, wood and fruits of C. decidua were surface sterilized
with 0.1% HgCl2 and washed two to three times with sterile distilled water. Properly cleaned
plant materials were dried in shade. Air dried bark and wood cuttings were powdered using
powdering mill to 50 mesh size. Seeds were separated by depulping the fruits and powdered
after proper cleaning and drying. Powdered plant materials were stored in sterile cellophane
bags in a cool dry place till further use.
Extraction of plant material
The powdered plant materials i.e. bark, wood and fruits (300gm each) were extracted with
methanol in a soxhlet extractor for 8 to 10 hours. The process was run till the decolourisation
of the solvent, after which the extract was filtered with Whitman filter paper (No.1) and the
filtrates were concentrated using rotary evaporator. Then the extracts were evaporated to
dryness over water bath and solvent free extracts of respective parts were obtained.[33]
Extracts
of wood, bark and seeds of C. decidua were weighed and kept in labelled sterile specimen
bottles.
Isolation of seed borne fungi
The isolation of seed-borne fungi was carried out by blotter test method.[34]
A pair of white
blotter papers of 8.5 cm diameter was jointly soaked in sterile distilled water and placed in
pre-sterilized petridishes of 9 cm diameter. Stored seeds of Salvadoa persica (Jal) Pongamia
pinnata (Karanj) and Azadirachta indica (Neem) were placed at equidistance on moist
blotters in autoclaved separate petridishes. All petridishes were incubated at room
temperature (28±20C) for 6 days. Altogether six dominant fungi were isolated and identified as
Aspergillus niger, A. flavus, Fusarium moniliforme, Phytophthora sp., Penicillium sp. and
Mucor sp. based on growth characteristic, mycelial morphology, spore morphology and other
important characters using standard protocol.[35-36]
Pure cultures of each of the isolated and
identified fungal species were made separately and maintained at 50C on PDA slants. These
pure cultures were used for antifungal assay.
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Preparation of test solutions
Test solutions of a series of concentrations viz., 5, 10, 25, 50 and 100 mg/ml were prepared from
methanolic extracts of wood, bark and seeds of C. decidua by dissolving extracts in Dimethyl
sulfoxide (DMSO). All test solutions were kept in refrigerator at 40C for future use.
Preparation of fungal inoculums
For antifungal assay cultured slants were used for preparing spore suspension in 0.9% saline
water. The fungal spore suspension was adjusted to give a final concentration of 1-5x105
cfu/ml.
Preparation of media
The medium was prepared by dissolving Potato dextrose agar (PDA) media (HiMedia) in
distilled water and autoclaving at 1210
C for 15 minutes. 20 ml of sterile PDA media was
poured in sterilized petridishes (9 cm diameter) and allowed to solidify which were used for
antifungal assy.
Antifungal activity assay
Antifungal activity of methanolic extracts of wood, bark and seeds of C. decidua was
determined using agar-well diffusion method.[37]
Spore suspensions (0.2ml) were applied on
the surface of the presterilized and autoclaved PDA petridishes and spread by using a sterile
glass spreader. Wells of 6mm diameter were made in centre of each of the PDA petriplates
with the help of sterilized cork borer. The wells were filled with test solutions of wood, bark
and seeds of C. decidua as prepared above with three replications for each treatment.
Carbendazim (2mg/ml) and DMSO were served as positive and negative control respectively
for each of the three extracts. All the petridishes including treatments and controls were
allowed to diffuse at room temperature for 2 hours and then incubated at room temperature
(28±20C) for 72 hours. After incubation, the antifungal activity of extracts was determined by
measuring the diameter (mm) of inhibition zones.
Determination of Minimum inhibitory concentration
The minimum inhibitory concentration (MIC) was determined through the broth dilution
method.[38-39]
Fungi were first grown in the potato dextrose broth for 24 hrs and then the
inoculums were diluted for five times (10-5
dilution) to control its vigorous growth. Then each
test tube was added with 1.8 ml of potato dextrose broth and different concentrations (1-10
mg/ml) of wood, bark and seed extracts separately followed by inoculation of 0.2 ml of
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respective fungi and kept at 28°C for 48 hrs. The tubes were examined for visual turbidity.
Lowest concentrations of the extracts showing no turbidity (without microbial growth) were
considered as the minimal inhibitory concentration.
RESULTS AND DISCUSSION
The antifungal activity of wood, bark and seeds of C. decidua obtained by the agar-well
diffusion method is shown in Table 1.
Table 1: Antifungal activity of Capparis decidua extracts against Seed-borne Pathogenic
Fungi
Plant
Parts
Conc.
(mg/ml)
Zone of Inhibition in mm
A. niger A. flavus F. moniliforme Phytophthora sp Penicillium sp Mucor sp
Wood
5 6.61±0.41 6.23±0.53 4.59±0.33 5.29±0.41 6.16±0.47 6.96±0.23
10 11.50±0.65 10.87±0.31 8.64±0.25 9.89±0.33 10.51±0.31 11.19±0.43
25 18.23±0.33 16.93±0.51 15.71±0.23 16.66±0.43 17.53±0.33 17.83±0.41
50 33.53±0.31 31.87±0.33 29.63±0.25 30.69±0.31 32.66±0.43 31.36±0.35
100 48.41±0.19 46.26±0.31 46.81±0.43 44.93±0.53 45.53±0.31 46.29±0.09
Bark
5 8.89±0.23 7.13±0.41 5.99±0.31 6.61±0.41 7.33±0.43 7.26±0.26
10 15.53±0.41 13.83±0.33 12.31±0.25 12.29±0.33 14.51±0.33 15.29±0.13
25 24.46±0.23 22.91±0.33 21.63±0.53 22.33±0.41 23.21±0.35 23.63±0.51
50 35.83±0.36 34.29±0.31 32.64±0.33 32.81±0.33 33.56±0.43 32.86±0.53
100 51.41±0.29 49.23±0.53 49.83±0.31 48.33±0.33 49.23±0.31 48.63±0.16
Seed
5 11.61±0.33 10.43±0.36 9.63±0.45 10.53±0.55 10.23±0.46 11.13±0.38
10 19.13±0.45 17.73±0.46 18.31±0.24 18.21±0.30 19.51±0.73 18.51±0.45
25 30.41±0.56 28.91±0.33 28.23±0.54 29.63±0.68 29.11±0.56 28.23±0.27
50 41.83±0.47 34.29±0.17 32.64±0.53 32.81±0.27 33.56±0.38 32.86±0.35
100 63.41±0.38 61.23±0.62 59.83±0.28 58.33±0.45 59.23±0.46 58.63±0.32
Carbendazim [
Control] 20.00±0.07 19.67±1.08 21.33±0.41 23.46±0.71 22.67±0.71 24.25±0.82
DMSO [ Control]
Methanolic extracts of wood, bark and seeds exhibited varying degrees of antifungal activity
against six fungal species A. niger, A. flavus, Fusarium moniliforme, Phytophthora sp.,
Penicillium sp. and Mucor sp. isolated from Salvadoa persica, Pongamia pinnata and
Azadirachta indica seeds. The data presented in Table-1 clearly indicate significant reduction
in growth of seed borne fungi with different concentrations of methanolic extract. Different
parts i.e. wood, bark and seeds showed significant differences in their efficacy. Methanolic
extracts of bark showed the maximum inhibition of mycelia growth of all species whereas
minimum inhibition is recorded with wood extract. From the result it is also evident that
growth inhibition of all the fungi increased with increase in concentration of extracts. Of the
concentrations of different parts, it is observed that inhibition of radial growth at concentration
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of 5 and 10 mg/ml is less than that of positive control. However, 25mg/ml and above
concentrations of extracts of all the three parts of C. decidua are more effective on growth
inhibition of all the tested fungi. Growth inhabitation impact of bark extract at 25 mg/ml
concentration is better than the positive control whereas impact of the same concentration of
seed extract is almost at par with positive control. Maximum growth inhibition of all the test
fungi is evident at concentration of 100 mg/ml of all the extracts followed by 50 mg/ml
concentration (Fig. 1). The mean radial growth inhibition of A. niger, A. flavus, Fusarium
moniliforme, Phytophthora sp., Penicillium sp. and Mucor sp. with various concentrations of
extracts of all the three parts of the plant ranged between 6.61- 63.41, 6.23 - 61.23, 4.59 - 59.83,
5.29 - 58.33, 6.16 - 59.23 and 6.96 - 58.63 mm respectively.
Results indicated that all the treatments were effective as compared to negative control (Fig. 1).
Radial growth inhibition was minimum at 5mg/ml concentration and maximum at 100mg/ml
concentration of extracts of all the three parts. Among the treatments bark extract was found
more effective than extracts of seeds and wood. Individual effect of extracts on radial growth
followed the descending order as Bark > Seed > Wood.
Fig. 1: Growth inhibition (in mm) of tested fungi under different treatments
Minimum inhibitory concentrations (MIC) of wood, bark and seed extracts of C. decidua were
found in the range of 3.25–3.75, 2.75–3.25 and 3.00–3.50 mg/ml respectively (Table 2).
0
10
20
30
40
50
60
70
A.niger A.flavus F.moniliforme Phytophthora sp. Penicillium Mucor
Wood Ext. Seed Ext.Bark Ext.
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Table 2: Minimum Inhibitory concentration of wood, bark and seed extracts of C. decidua
Fungal species Minimum inhibitory concentration (mg/ml)
Wood Extract Bark Extract Seed Extract
Aspergillus niger 3.50 2.75 3.00
Aspergillus flavus 3.25 3.00 3.25
Fusarium moniliforme 3.75 3.25 3.50
Phytophthora sp 3.25 3.00 3.50
Penicillium sp 3.25 3.00 3.25
It is finally confirmed by the results that extracts of different parts of C. decidua have very
low MIC values against fungi. Extracts of bark and seeds of the plant at concentration of
25mg/ml have shown growth inhibition almost at par with synthetic fungicide Carbendazim
(positive control). Further, growth inhibition of all the six fungi at 50 mg/ml concentration of
extract of the three parts is significantly higher as compared to positive control. Maximum
inhibition however is recoded at 100 mg/ml concentration of extracts of all the three parts
(Fig. 1). The antifungal potency of different parts of C. decidua may be due to presence
chemical constituents which are too complex in their structure and show diverse action
mechanisms.
Biodeterioration of seeds due to fungal diseases cause considerable losses in terms of seed
germination capacity and their chemical composition.[40-44]
Treatment of seeds to control
seed-borne fungal diseases using synthetic chemical fungicides has limitation of pesticide
toxicity and serious environmental consequences.[45]
In recent years, large number of
synthetic fungicides has been banned in the western world because of their undesirable
attributes. In addition, many pathogenic microorganisms have developed resistance against
chemical fungicides.[46]
Plant based biocides proved to be one of the better alternatives owing
to their negligible health and environmental hazard in contrast to synthetic chemicals.[47-48]
Extracts of many higher plants have been reported to exhibit antifungal properties.[49-57]
However, a few seed borne fungi remain constantly posing serious problems by deteriorating
seed viability and vigour. In view of these, the present investigation was undertaken to
examine the extracts of different parts of C. decidua for their antifungal potency against
phytopathogenic seed borne fungi causing significant deterioration of forest tree seeds during
storage.
CONCLUSION
Capparis decidua has a wide range of biological activity due to the presence of different
types of bioactive phytochemicals like alkaloids, terpenoids, glycosides, flavonoids, fatty
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acids, etc in good quantity. Recent studies on this plant showed that it may be used as
antibacterial agent.[20,28,58-59]
Based on these facts, it was decided to evaluate different parts of
the plants for their fungicidal action against harmful seed-borne fungi. The efficacy of wood,
bark and seed extracts of C. decidua in inhibiting the growth of some detrimental seed-borne
fungi as found in the present study is suggestive of its promising antifungal activity which has
not been reported so far. The study further revealed that the antifungal activity of the extracts
enhanced by an increase in the concentration of the extract. In vitro evaluation of plants and
plant products for antifungal property is the first step towards achieving the goal for
developing ecofriendly management of fungi. Demand for herbal biocides is increasing in
order to find effective alternatives to synthetic fungicides. In this context, antifungal efficacy
of methanolic extracts of tested parts of C. decidua is interesting. Therefore, it may be
concluded that C. decidua is a significant addition in the existing record of phytofungicides that
could be exploited as the source of effective herbal fungicide which can be an eco-friendly
option for the management of seed borne fungi and development of antifungal commercial
formulation.
ACKNOWLEDGEMENTS
The authors are highly thankful to the Director, Forest Research Institute, Dehradun and the
Director, Arid Forest Research Institute, Jodhpur (India) for providing necessary facilities for
carrying out this work.
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