ISSN NO 2320-5407

International Journal of Advanced Research (2013), Volume 1, Issue 7, 31-35

Journal homepage: http://www.journalijar.com

INTERNATIONAL JOURNAL
OF ADVANCED RESEARCH

RESEARCH ARTICLE
Antifungal and Cytotoxic activities of medicinal important orchid Rhynchostylis retusa Blume
B.Radhika1*, JVVSN. Murthy1, D.Nirmala Grace2
1. Department of Biotechnology, Centre for research, Dr.V.S.krishna Govt. College, Affiliated to Andhra
University, Visakhapatnam, Andhrapradesh, India.
2. Department of Microbiology, Nagarjuna University, Guntur, Andhrapradesh, India.

Manuscript Info

Abstract

Manuscript History:

Medicinal plants are distributed throughout the world and widely used in
everyday life as part of folk medicinal remedies. Rhynchostylis retusa is a
medicinal orchid, used to cure blood dysentery, Tuberculosis, epilepsy,
menstrual disorders, fewer, gout, Asthma, rheumatism, Malarial fewer etc.
The purpose of this study was to determine cytotoxic activity and antifungal
activity of medicinal orchid Rhynchostylis retusa. Hexane, Chloroform and
methanol extracts of this orchid were used to determine antifungal activity
against six phytopathogens. Among these three extracts, chloroform and
methonolic extracts shows highest zone of inhibition than the hexane extract.
Various solvent extracts of Rhynchostylis retusa was screened by the brine
shrimp lethality assay and found Methanol extract showed significant
toxicity to the brine shrimp and exhibited potent activity with LC 50. The
present study will be successful to identify antifungal and cytotoxic activity
which could be further exploited for isolation and characterization of the
novel phytochemicals in the treatment of infectious diseases especially in
light of the emergence of drug resistant microorganisms and the need to
produce more effective antimicrobial agents. Present study could be useful in
the search for new antitumor compounds from the Indian flora.

Received: 15 August 2013

Final Accepted: 24 August 2013
Published Online: September 2013

Key words:
Rhynchostylis retusa,
Agar Well Diffusion method,
Artemia saline,
cytotoxicity, Brine shrimp.

Copy Right, IJAR, 2013,. All rights reserved.

Introduction
India possesses rich floristic wealth and diversified
genetic resources of medicinal plants. Approximately
20% of the world plants have been submitted to
pharmacological or biological test, it could be
concluded that Natural products from plant origin are
important sources to discover new leads with
economical and pharmaceutical importance and great
possibilities to be developed drugs1. The use of plants
and plant extracts and pure compounds isolated from
natural sources provided the foundation of modern
pharmaceutical compounds2. Several studies have
been conducted in the past three decades that focused
on the antimicrobial properties of herbs, spices and
their derivatives such as essential oils, extracts and
decotions3. There are several reports on the presence
of antimicrobial compounds in various plants4, 5,6,7,8.
It is an evident that the use of plants for various

diseases since from Vedic period. Documentation of

the Ayurvedic system recorded by Susruta and
Charaka dates from about 1000 BC9. The commercial
availability of inexpensive brine shrimp eggs, the low
cost and easy of performing the assay make brine
shrimp lethality assay, a very useful bench top
method10. A number of studies have demonstrated the
use of the brine shrimp assay to screen the
cytotoxicity of plant extracts11, 12, 13. Lethality assay
has been successfully to biomonitor the isolation of
Cytotoxic14,
antimalarial15,
insectisidal16
and
17
antifeedent compounds from plant extracts.
Rhynchostylis retusa is a
monopodial epiphytic species that grows in the
broad-leaved forests along the lower Himalayan
ranges18. It is an epiphytic herbaceous orchid
commonly seen in the forest of Western Ghats 19. It
can tolerate a wide range of temperature from 3C 31C20. This plant leaf juice and Aerial roots were

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International Journal of Advanced Research (2013), Volume 1, Issue 7, 31-35

also used in ear pain and cleaning21,22. It also used as

emollient, throat inflammation etc23. In Bangladesh,
kurigram District, peoples were used this plant leaves
to cure Rheumatic pain24. Rhynchostylis retusa roots
were used to cure malarial fewer25. The whole plant
preparations were used to cure blood dysentery,
Tuberculosis, epilepsy, menstrual disorders, fewer,
gout, Asthma, rheumatism etc26. The purpose of this
study was to investigate antifungal activity and
cytotoxic properties of Rhynchostylis retusa. In this
paper we report the results of such studies in order to
orient future investigations towards the finding of
new, potent and safe bioactive anticancer and
antimicrobial compounds.
Materials and Methods:
The whole plant of Rhynchostylis retusa was
collected from the forest of Chinthapalli. The
specimen was identified with the help of regional
floras27, 28 and the voucher specimen was deposited at
Andhra University Herbarium (AU), Visakhapatnam,
Andhra Pradesh, India.
Preparation of plant extracts:
The collected leaves were shade dried, powered and
extracted with hexane, chloroform and methanol
using soxhlet apparatus for 8 hours. The extracts
were filtered and filtrates were concentrated under
reduced pressure at 40o C using a rotaflash
evaporator. Various concentrations of plant extracts
(500, 250, 100 mg/ml) were dissolved in DMSO (Di
methyl sulphoxide). The crude sample was subjected
to antifungal screening against the phytopathogens
and the brine shrimp lethality assay.
Collection of microorganisms:
The microbial strains Viz., Macrophomina
phaseolina(MTCC 257) , Sclerotium rolfsii(MTCC
2465),
Fusarium
oxysporium(MTCC
2087),
Altarnaria altarnata(MTCC 2723), Rhizoctonia
solani(MTCC 4634), Fusarium solani(MTCC 350)
were used and these organisms obtained from
Microbial Type Culture Collection centre, Institution

of Microbial Technology (IMTECH), Chandigarh,

India.
Antifungal activity:Antifungal activity of extracts was determined by
well diffusion method on Potato Dextrose Agar
(PDA) medium. PDA medium29 was prepared,
sterilized and 0.2 ml of 24 hrs broth culture was
mixed in the PDA agar medium and poured in
petriplates. After solidifying wells (6mm diameter)
are made in agar plates using cork borer 30, 31, 32.
Different concentrations (500, 250, 100 mg/ml) of
different solvent extracts (hexane, chloroform and
methanol) were poured in wells and incubated at 30 0
C for 72 hrs. The antifungal activity was evaluated by
measuring the zone of inhibition around the well. The
experiment was done in triplicate and the mean
diameter of the inhibition zone was calculated33.
Brine Shrimp Lethality bioassay:
Brine Shrimp Lethality bioassay was carried out to
investigate the cytotoxicity of extracts of medicinal
plants of India. Brine shrimp (Artemia saline) were
hatched using Brine shrimp eggs in a conical shaped
vessel (1L), filled with sterile artificial sea water
(prepared using sea salt 38 g l-1 and adjusted to pH
8.5 using 1 N NaOH) under constant aeration for 48
h. after hatching, active nauplii free from egg shells
were collected from brighter portion of the hatching
chamber and used for the assay. Ten nauplii were
drawn through a glass capillary and placed in each
vial containing 4.5 ml of brine solution. In each
experiment, 0.5 ml of plant extract was added to 4.5
ml of brine solution and maintained at room
temperature for 24 hr under the light and surviving
larvae were counted. Experiments were conducted
along with control34,35
Statistic analysis:
The percentage lethality was calculated from the
Mean survival larvae of extracts treated tubes and
controls. LC50 values were obtained by best-fit line
method.

Table - 1. Antifungal activity of leaf extract of Rhynchostylis retusa

S.No.

1
2
3
4
5
6

Name of organism

Macrophomina phaseolina
Sclerotium rolfsii
Fusarium oxysporium
Altarnaria altarnata
Rhizoctonia solani
Fusarium solani

Hexane extract mg/ml

500
250
100
12
10
10
11
10
9
11
10
8
11
9
8
-

Zone of inhibition(in mm)

Cholorofom extract mg/ml
500
250
100
14
13
13
14
13
12
14
12
10
8
7
8
7
7
11
9
8

Methanol extract mg/ml

500
250
100
10
9
9
7
7
7
7
8
8
7
9
8
7
12
11
11

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International Journal of Advanced Research (2013), Volume 1, Issue 7, 31-35

The maximum zone of inhibition was obtained for

Macrophomina phaseolina
at a concentration of
500 mg/ml (Fig 1). The chloroform extract was more
effective on test pathogens showed significant
inhibition zones ranged from 7-14 mm. Hexane
extract doesnt show any antifungal activity against
these test pathogens except Macrophomina
phaseolina, Sclerotium rolfsii, Fusarium oxysporium
and Rhizoctonia solani. Rhynchostylis retusa
chloroform extract shows more effective on test
organisms than the methanol and hexane extracts.
The results of antifungal assay of three
concentrations (100, 250,500 mg/ml) of different
solvent plant extracts like chloroform and methanol
exhibited most effective antifungal activity in Table 1.
Rhynchostylis
retusa
contains
several
phytochemical compounds, which are very much
necessary to control the growth of microorganisms.

Table 2. LC50 values of different solvent extracts

of Rhynchostylis retusa
S.No. Name of the solvent extract
LC 50, 24 h
1
2
3
4

Hexane
Chloroform
Methanol
Podophyllotoxin

86.41
30.50
18.52
2.82

LC50 values of the brine shrimp obtained for extracts

of this plant and that of the positive control,
Podophylotoxin, have been presented in Table 2 and
Fig 2. In the evaluation for general toxicity using
brine shrimp, maximum mortalities took place at a
concentration of 1000 g/ml where as: least mortality
was at 10 g/ml concentration and the lethality (LC50)
values were 86.41, 30.50 and 18.52 g/ml respectively
(Table 2). The LC50 values of plant extracts (24 h)
were obtained by a plot of percentage of the shrimps
killed against the concentrations of the extracts and
the best-fit line was obtained from the data by means
of regression analysis. LC50 was obtained from the
best-fit line method. This method allows the use of
smaller quantity of the extracts and permits larger
number of samples and dilutions within shorter time
than using the original test vials36. Further clinical
studies need to be carried out for isolation of
potential chemicals, which can augment the cancer
fighting ability of modern medicine.
Conclusion:
From the preliminary screening, we have identified
that the Methanol extract out of 3 different solvent
extracts screened for toxicity against brine shrimp
had less LC50 values less than 100 g ml-1 in
interesting and lend supporting to the traditional use
of this plant extract. Although the brine shrimp
lethality assay is rather inadequate the elucidation of
the mechanism of action, it is very useful to assess
the bioactivity of the plant extract. The brine shrimp
lethality assay results suggest that the plant can be a
promising source of anticancer compound. Based on
the possible relationship between brine shrimp
lethality and plant bioactivity, this work could serve
for further ehanobotanical and phytochemical
research. From this study, suggesting a need to
isolate and evaluate active constituents responsible
for the exhibited biological activities.

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