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All content in this area was uploaded by Kaladhar SVGK Dowluru on Sep 27, 2016
Content may be subject to copyright.
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ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF RHIZOME
EXTRACTS OF Kaempferia galanga
Narasinga Rao V, DSVGK Kaladhar*
Dept. of Biochemistry, GIS, GITAM University, Vishakhapatnam, AP, India
ABSTRACT
Several Zingiberaceae family members show active principles in
treatment of various human ailments. An experiment on antioxidant
and antimicrobial activity in rhizome extracts of Kaempferia galanga
has been conducted. The Kaempferia galanga rhizome extracts shown
antioxidant activity of the tested plant extracts (IC50: 490µg/ml to
720µg/ml) is less compared to the ascorbic acid (IC50: 230µg/ml).
Methanol, ethylacetate, ethanol and aqueous rhizome extracts has
shown good antimicrobial activity against tested microbes (9.5 to
22mm zone diameter). The extracts was shown good antimicrobial
activity against fungi (11 to 22mm) when compared with the gram negative bacteria (9.5 to
15mm) and gram positive bacteria (9.5 to 13). Hence Kaempferia galanga rhizome extracts
was shown good antioxidant and antimicrobial activities.
Key words: Kaempferia galanga, solvent extraction, antioxidant and antimicrobial studies
INTRODUCTION
Plants have a precious medicinal source of natural products for maintain high-quality of
human health with added intensive studies for natural therapies [1]. The use of plant
metabolites for pharmaceutical implements has progressively increased in India [2].
Microorganisms comprise the genetic capability to transmit and attain resist to antibiotics,
which are used as therapeutic agents [3]. In the present years, microbial resistance is
developing and applying of antimicrobial agents in the prospect is still uncertain [4]. Thus,
the therapeutic actions must be taken to diminish these problems and to supervise the use of
antibiotics. Highly developed research strategies can make available improved understanding
in the genetic mechanisms of resistance in microbes. The crucial aim is to produce a suitable
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Article R
eceived on
06 March 2014,
Revised on 20 March
2014,
Accepted on 24 April 2014
*Correspondence for Author
Dr. Kaladhar Dowluru
Dept. of Biochemistry, GIS,
GITAM University,
Vishakhapatnam, AP, India
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and efficient antimicrobial drug to the patients [5].
About 75% of individuals use traditional medicine, which are resultant from medicinal plants
in a variety of developed countries [6]. Hence, the search of different phyto-components from
medicinal plants should be to recognize the properties and efficiency with possible treatment
against diseases [7]. The family Zingiberaceae has enormous number of medicinal plants and
is well known for use in ethnomedicine. The wisdom of Zingiberaceae members indicates
systematically analyzes compounds for the management and prevention of various human
ailments from India. The phytochemical assessments from the plants complete humans for
studies in biodiversity and its protection for future pharmacological studies [8]. A number of
medicinal plants have good sources with nutrient and non nutrient molecules, lots of which
have anti-fungal, anti-bacterial, anti-oxidant and anti-viral properties that can guard the
human system against cellular oxidation reactions and pathogens [9].
Essential oils have good anti-fungal, anti-viral, insecticidal, anti-bacterial and antioxidant
properties. Several oils are used in various skin disorders [10]. Essential oils are also having
industrial applications like fragrance, food preservation and aromatotherapy [11]. The
essential oils of aromatic plant extracts are rich sources of biologically vigorous compounds
which are enhanced interest in looking at anti-microbial properties [12]. So, it is sensible to
anticipate a diversity of plant compounds in these oils with the appropriate general antibiotic
potential and antimicrobial activity [13].
The severe infections caused by various disease causing microorganisms enhanced and an
important cause of morbidity and mortality patients in worldwide [14]. Nearly three quarters
of the population in developing countries used in their traditional medicinal system depends
upon plant based on measures and as the basic requirements for human primary health care
[15]. Hence, several medicinal plants need to be necessary possible antimicrobial activity
evaluation and to get admirable remedy for a microbial origin from a variety of ailments [16].
The living organisms protected from several antioxidants and prevent scratches caused by
uninhibited formation of reactive oxygen species; concurrent lipid per oxidation, protein
damage and DNA strand breakage [17]. Antioxidant that can decrease free radicals activity
can prevent the oxidation of various molecules and may possibly have health promoting
special effects in the interference of degenerative diseases [18]. In accumulation there is a
contrary relationship between the frequency of human diseases and dietary intake of
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antioxidant rich food [19]. The natural antioxidants had shown a extensive range of
biochemical activities, like inhibition of Reactive Oxygen Species (ROS) making, through
direct or indirect scavenge of free radicals, and differences of intra cellular redox potential
[20].
The ROS are free radicals such as superoxide anion radicals (O2-), hydroxyl radicals (OH-)
and other non-free-radical species such as H2O2 and singlet oxygen (1O2) are several forms of
activated oxygen [21]. The importance of free radicals provided increasing attention over the
decades in understanding antioxidant activity. The cellular injury and in ageing process these
molecules are exacerbating factors [22]. ROS have important interest between scientist and
their extensive range of property on biological and medicinal systems studied in many
experimental investigations [23]. In living organisms, several ROS can form signaling
networks in knowing different components used in different traditions. Normal aerobic
respiration stimulates macrophages, peroxisomes and polymorph nuclear leukocytes that
emerge to be the chief endogenous sources of mainly the oxidants formed by cells. The
exogenous or external sources of ROS include certain pesticides, tobacco smoke, pollutants
and organic solvents [24].
Ageing is one of the unique features in all living organisms. The impaired functions of many
systems characterize aging. The impairments occur in the brain causes susceptibility to
neurodegenerative diseases that amplifies significantly. The free radical presumption of aging
posits the functional impairments in brains that are due to the attack on serious cellular
mechanism by reactive nitrogen species, free radicals and ROS produced during normal
metabolism [25].
MATERIALS AND METHODS
Collection of Plant Material
Fresh plants consisting of rhizomes of Kaempferia galanga (K. galanga or KG) were
collected from Visakhapatnam District, Andhra Pradesh and some regions of Kerala during
July and August 2011. The plant is authenticated by Dr. P.V. Arjun Rao, Ethanobotanist,
Dept. of Botany, Phytopharma Technology Laboratory, Visakhapatnam (No. Res/1 dated 21-
09-2010). K. galanga is confirmed based on details given in “Flora of the Presidency of
Madras” by J.S. Gamble, Vol. iii, page numbers 1483-1484, Bishen Singh Mahendra Pal
Singh Publishers, India (2004). Fully matured rhizomes were washed systematically and
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dried out in sunlight. The desiccated plant rhizomes were grinded to powder and about
150gms of the dehydrated plant material were used for the extraction.
Extraction Process
Nearly 30g of air dried powder were taken in 200ml of ethyl acetate, methanol, ethanol and
aqueous separately, plugged with cotton wool and then kept on orbital shaker for 48 hours
with 150rpm at room temperature. The extracts were filtered with whatmann no 1 filter paper
and collect the supernatant. Then solvent evaporated through rotavapour and make the final
volume one-fourth of the original volume and stored at the 4oC in air tight containers.
Antioxidant Activity with DPPH
Chemicals & Reagents
Standard: Ascorbic acid
a. 100µM of DPPH (1,1-diphenyl-2-picrylhydrazyl): Nearly 3.9432 mg of DPPH was
dissolved in 3ml of methanol and made up to 100 ml to obtain a final concentration of
100µM.
b.Stock solutions of test items: 3mg/ml stock solutions were prepared in DMSO.
c. Test items preparation: Appropriate dilutions (0 to 1000µg/ml) of test items were prepared.
The reaction mixture was made by addition of 20µl of test items and 280µl of DPPH reagent
to reach a final volume of 300µl and kept incubation in dark for 50 minutes and then
absorbance was read at 517 nm using spectrophotometer. An IC50 value was determined as
the concentration that elicits the half maximal response.
Antimicrobial Activity
The antimicrobial activity was conducted based on zone method.
Microorganisms
Microbes from ATCC (American Type Culture Collection), USA have been used in the
present study. Several bacteria used in the current research work are Bacillus subtilis (ATCC
6051) and Klebsiella pneumonia (ATCC 13883) belongs to gram positive bacteria, and
Serratia marcescens (ATCC 14756), Pseudomonas aeruginosa (ATCC 15442), Enterobacter
aerogenes (ATCC 13048) and Escherichia coli (ATCC 25922) belong to gram negative
bacteria. Fungi used in the work are Aspergillus niger (ATCC 6275) and Candida albicans
(ATCC 10231).
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Antimicrobial Activity Using Zone Method
Antimicrobial activity has been conducted based on zone method. The medium used in this
experimentation are Muller-Hinton Agar (Hi Media Pvt. Ltd., Mumbai., India) for bacteria
grown at 370C for 24 hours and fungi in Sabourand Dextrose Agar (Hi Media Pvt. Ltd.,
Mumbai., India) at 250C for 48 hours. The freshly prepared nutrient broth has been prepared
and inoculated the broth cultures. The broth was inoculated into growth medium. The growth
medium has prepared in the petriplates. Wells of 8mm size were made in the growth media
with sterile borer. Nearly 50µl of extracts were added to the wells of growth media. The
MHA (Muller-Hinton Agar) plates of bacteria were incubated at 370C for 24hrs. The
Sabourand Dextrose Agar plates were incubated using micro pipette at 250C for 72 hours for
fungi. After incubation diameter of zones of inhibition was measured using Hi Media zone
reader in mm.
RESULTS AND DISCUSSION
India has rich sources of medicinal plants shows important part of life from ancient times. An
important part of this indigenous knowledge was consistent from the past into the organized
systems of medicines such as Sidha, Yunani and Ayurveda or other systems (Chetna and
Anoop, 2009). Caffeic acid (3, 4-dihydroxycinnamic acid) is a powerful antioxidant isolated
from plants (Gülçin, 2006).
One such plant Kaempferia galanga belongs to Zingiberaceae family revealed to be having
antioxidant and antimicrobial activities by present investigation. K. galanga is a precious
medicinal plant, which is rarely available in few countries and becoming extinct due to
change of climatic conditions and human factors. These plant rhizomes shown good
secondary metabolite products and medicinal activities have not been widely recognized.
Currently the entire plant K. galanga are used for antifungal and antibacterial properties for
extracting plant metabolites against fungi and bacteria using agar diffusion and the zones of
inhibition methods. The antioxidant activity can be calculated using DPPH method.
The K. galanga rhizome extracts shown antioxidant activity with IC50 value for ethanol is
490µg/ml, 590µg/ml for methanol, ethyl acetate is 640µg/ml and aqueous extract shown is
720µg/ml. The IC50 value for standard (ascorbic acid) obtained in the experimentation is
230µg/ml (Figure 1, Table 1). Though the antioxidant activity of the tested plant extract is
less compared to the standard, the plant has shown good antioxidant activity
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Table 1: Antioxidant activity (IC50) for crude rhizome extracts of K. galanga
Table 1: Percentage of Inhibition of DPPH radical for antioxidant activities of K.
galanga
Log conc.
(µg/ml) Ethanol Methanol Ethyl
acetate Aqueous Standard
(Ascorbic
acid)
0 0 0 0 0 0
31.25 8.39 6.45 4.44 4.02 23.13
62.5 16.33 12.23 11.43 8.02 34.33
125 27.45 23.44 22.75 17.34 41.67
250 38.66 35.24 30.33 27.34 52.73
500 50.45 44.44 42.33 39.43 66.23
1000 89.54 76.33 72.33 65.43 92.78
The plant rhizome extracts were analyzed for antimicrobial activity against various test
microorganisms. All the prepared extracts were shown fine antimicrobial activity (Table 2).
Ethyl acetate, Ethanol, Methanol and aqueous rhizome extracts of K. galanga was revealed
good antimicrobial activity against the tested bacteria (9.5 to 15 mm). The extracts have
shown good activity against fungi (11 to 22) while compared with gram negative bacteria
(9.5 to 13) and gram positive bacteria (10 to 15mm). The zone of inhibition for the bacterial
standard, tetracycline has shown inhibition zone from 10 to 44 mm at 50µg/ml. The zone of
inhibition for fungal standard, Fluconazole has shown inhibition zone for 13 and 16 mm for
Aspergillus niger and Candida albicans respectively at 50µg/ml. Antifungal medicinal plants
belong to the Zingiberaceae family has also been previously reported in plants like Curcuma
longa L., Curcuma zedoaria Rosc., Curcuma malabarica Vel., and A. galanga supports the
use of their rhizomes in traditional medicine for the treatment of fungal and bacterial
infections [26].
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Table 2: Antimicrobial activity for crude rhizome extracts of K. galanga
Note: Standard antibiotic for bacteria is Tetracyclin and for fungi is Fluconazole
K. galanga belongs to Zingiberaceae family used in the treatment of several diseases. Most of
the medicinal plants are widely used for the treatment of several diseases in India and China
[27]. The practice of plants like turmeric may decrease the risk of different kinds of cancers
and provide other protective biological effects in humans. These biological effects are due to
constituent curcumin that widely studied for its anti-oxidant, wound healing, anti-cancer and
anti-inflammatory effects [28].
The possible compounds for developing antimicrobials from plants appear valuable, as it will
lead to the enhancement of a phytomedicine to act against microbes. The Plant based
antimicrobials have immense therapeutic potential as they can provide the purpose with
slighter side effects that are frequently associated with synthetic antimicrobials [29].
CONCLUSION
The crude rhizome extracts of K. galanga has shown antioxidant activity. The K.galanga has
shown extensive antimicrobial activity and hence the naturally available crude rhizome
extracts of K. galanga could be potential alternative against microbes. Hence the crude
rhizome extracts of K. galanga shows antioxidant and antimicrobial components.
ACKNOWLEDGEMENT
The author would like to thank GITAM University for providing lab facility and access to e-
journals to carry out the entire research work.
Microorganism Zone of inhibition in mm (including well size of 8mm) at 50 µg/ml
Methanol
extract Ethanol
extract Ethyl
acetate
extract
Aqueous
extract Antibiotic
Control
(DMSO)
Bacteria
Bacillus subtilis 11 12.5 15 11 26 Nil
Klebsiella pneumoniae 12 11.5 15 10 31 Nil
Serratia marcescens 13 13 12 10.5 11 Nil
Pseudomonas aeruginosa
9.5 11 9.5 10 38 Nil
Enterobacter aerogenes 11 12 11 9.5 44 Nil
Escherichia coli 10.5 12 11 10 10 Nil
Fungi
Aspergillus niger 18 20 22 13 13 Nil
Candida albicans 20 21 21 11 16 Nil
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