In vitro Validation and Phyto-constituent Analysis of Turmeric Extract: An Ethnological Alternative for Eye Treatment
The essential oil from the rhizomes of Roma cultivar of turmeric (Curcuma longa) from Orissa was examined for its antimicrobial activity against the pathogens causing eye infections. The purpose of this study was to authenticate the use of turmeric rhizome oil against eye infections so as to giving an approach to formulate turmeric rhizome oil as potential eye drop in place of traditional antibiotics after undertaking its in vivo pharmacological studies. Essential oil from rhizomes of Roma cultivar obtained by hydrodistillation extraction method using clenevger apparatus. The antimicrobial effects of oil towards Staphylococcus aureus, Pseudomonas aerugenosa, Candida albicans and Aspergillus niger were tested by Inhibition Zone Diameter (IZD) test to screen the antimicrobial activity, Minimum Inhibitory Concentration (MIC) test and Minimum Killing Time (MKT) test to determine the minimum concentration of oil and minimum time required to kill the pathogens. Oil showed very good activity against all four microbial strains used at concentration of 10 μL except Pseudomonas sp. Very low concentration of 1.95 μL mL-1 oil was needed to inhibit the growth of most highly infecting pathogen Staphylococcus aureus within 15 min of its exposure in comparison to other microbial strains. High turmerone content (49.76%) of elite turmeric cultivar Roma released from Orissa (India) might be assigned to be responsible for such excellent anti microbial activity against the tested pathogens.
Ailments related to eye continue to be a major health problem worldwide (Lowy,
). Severe eye infections like blepharo conjuctivities, corneal ulcers,
abscesses, styes, dacryocystitis, periorbital-celluties, orbital cellutitis and
blebs are mainly caused by Staphylococcus aureus
, a normal flora (Groden
et al., 1991
) and Pseudomonas aeroginosa
, an opportunistic human
pathogen (Stapleton et al., 1995
). Candida albicans
and Aspergillus niger
are other most common cause of endogenous endophthalmitis,
leading to scarring of the chorioretina and blindness (Anonymous,
). The present treatment of choice for these pathogens is antibiotics
which brings about severe side effects like hypersensitivity reactions, gastric
disturbances, ototoxicity and nephrotoxicity (Cosgrove et
) and incites resistance against these pathogens (Poole,
). On the other hand, there exist many advantages in using antimicrobials
obtained from medicinal plant
s such as fewer side effects, relatively less expensive,
better patient tolerance, acceptance due to long history of use and being renewable
in nature (Kim, 2005
; Prabu et al.,
). Ethno botanical search and review of ancient traditional medicinal
scripts on palm leaves form different districts of Orissa accentuates the use
of turmeric for treating eye infections by tribal community Kond, Kui and Dongria
of Kandhamal district and Bondas, Bhunia, Paroja and Dora of Koraput district
Other ethnological groups like common people of remote villages, traditional healers
and as well as qualified medicinal practitioners of Ayurved, Naturopathy, Unani
and Sidha have been found treating several eye ailments using turmeric nationwide
even till today. Drops of strained water extract, obtained from boiling turmeric
powder few minutes in water have been used for treatments of eye problems by these
tribal groups in few districts of Orissa. Turmeric has got enormous ethnological
use in treatment of several diseases in different states of India (Ammon
et al., 1992
) including Orissa (http://www.shroong.com
and also in many Asian and African countries like China (Araujo
and Leon, 2001
), Nepal (Eigner and Scholz, 1999
Thailand (Amphawan et al., 1995
et al., 2002
), Nigeria (Usman et al., 2009
and Brazil (Araujo and Leon, 2001
) for its range of medicinal
properties. It has been cultivated at a commercial scale in all these states for
its use in medicine, spice and flavoring agent. Orissa has been famed as the 2nd
largest producer of turmeric in India and is the home state for releasing of many
promising turmeric cultivars Surama, Roma, Ranga etc. possessing high rhizome
and/or drug yielding potential (Ravindran et al., 2007
and in vivo
experimental verification of ethnological usage
of turmeric has been done to establish its medicinal properties like anti inflammatory,
antioxidant (Prakash et al., 2003
), photo receptor,
etc. Jorge (2004)
including its anti infecting
potential (Srimal and Dhawan, 1985
) by various workers
by using essential oil or other chemical extracts and its derived compounds (Chattopadhyay
et al., 2004
). Some of the properties are well documented and validated
by pharmacological and clinical trials, while many remains still to be validated
). Review of literature does not illustrate any
single report where a concerted effort was taken to find the effect of turmeric
rhizome essential oil on inhibition of group of microorganisms responsible for
causing various eye infections e.g., Staphylococcus aureus, Pseudomonas aerogenosa,
and Aspergillus niger
. Phytoconstituents of essential
oil of medicinal plant
s are established to be responsible for the biological activity
against microbial pathogens (Kaur and Arora, 2009
an attempt is taken to authenticate the traditional use of turmeric by ethnological
groups against common diseases related to eye, in terms of evaluating the effectiveness
of rhizome essential oil collected from a high ar-turmeorne containing elite turmeric
cultivar Roma in inhibiting the growth of specific eye infection causing pathogens.
Validation of ethnological use of turmeric (cv. roma) essential oil in vitro
is done through performing all the basic tests like; determination of IZD (Inhibition
Zone Diameter), MIC (minimum inhibitory concentration
), bactericidal and fungicidal
effect, MKT (Minimum killing time) so as to enable an opening to formulate turmeric
oil as potential eye drop in place of traditional antibiotics after undertaking
its in vivo
MATERIALS AND METHODS
Plant material: The rhizome of Curcuma longa (cv. roma) were
collected from the High Altitude Research Station Pottangi, Orissa and grown
in medicinal plant garden of Centre of Biotechnology, Bhubaneswar, Orissa (India).
Fresh leaves and rhizomes on harvest were collected, washed under running tap
water and were used immediately to extract the essential oils.
Microorganism and media: The test organisms used in this study were Staphylococcus aureus (MTCC-3160), Pseudomonas aerogenosa (MTCC-424), Candida albicans (MTCC-183) and Aspergillus niger (MTCC-281) obtained from the Microbial Type Culture Collection, Chandigarh, India. All the strains maintained in recommended media purchased from Hi-Media India private Ltd., Mumbai. This research project was conducted from 2nd December 2009 to 2nd June 2010.
EXTRACTION OF ESSENTIAL OILS
The rhizome of Curcuma longa (cv. roma) were collected from the High
Altitude Research Station, Pottangi, Orissa and grown in medicinal plant garden
of Centre of Biotechnology, Bhubaneswar, Orissa (India). Fresh rhizomes on harvest
were collected, washed under running tap water and were used immediately to
extract the essential oils. The fresh rhizomes of turmeric were washed to remove
soil, peeled and sliced. Sliced rhizomes of fresh turmeric (100 g) were mixed
with distilled water. The essential oil was extracted by hydro-distillation
using a Clevengers apparatus following the method of Guenther
(1948) out at room temperature. A flask containing the homogenate was heated
for 6-10 h and the condensed vapor was separated throughout an auto-oil/water
separator. The oil present at the upper most layers was collected in the ependroff
tube. Each essential oil extraction was run in duplicate to evaluate the oil
yield. The solubility of essential oil in water as well as other organic solvents
The total amount of oil in rhizome was calculated by following method. Yield percentage was recorded as dry weight basis.
ANTIMICROBIAL ACTIVITY ASSAY OF ESSENTIAL OIL
The test organisms used in this study were Staphylococcus aureus (MTCC-3160), Pseudomonas aerogenosa (MTCC-424), Candida albicans (MTCC-183) and Aspergillus niger (MTCC-281) obtained from the Microbial Type Culture Collection, Chandigarh, India. All the strains maintained in recommended media were purchased from Hi-Media India private Ltd., Mumbai.
Determination of Inhibition Zone Diameter (IZD): Initial screening through
inhibition zone diameter was determined by the disc diffusion method as described
previously (Pattnaik et al., 1995) with slight
modifications. Briefly, Nutrient Agar (NA) and Potato Dextrose Agar (PDA) plates
were swabbed with freshly grown cultures of the test pathogens by the help of
a pre-sterilized cotton swab. Sterile filter paper discs (5 mm diameter) were
kept on the above plates at equidistance. Varying volumes (2, 5 and 10 μL)
of rhizome oil was loaded over the sterile filter paper discs. The plates were
incubated at 37°C for 18-24 h for bacteria, 48 h for fungi and observed
for a zone of clearance around the discs which indicated positive microbicidal
activity of the oil. All the experiments were carried out in triplicate.
Determination of MIC (minimum inhibitory concentration): Minimum inhibitory
concentration of oil was determined by the tube dilution method (Pattnaik
et al., 1997). The oil was diluted with NBT and PDBT (Nutrient broth
and potato dextrose broth supplemented with 0.75% of Tween-20) to give oil concentration
of 5 to 100 μg mL-1. Fifty microliter of (fresh culture) over
night growth of the test organisms in NB and PB was inoculated into 1 mL of
NBT and PDT containing various concentrations of the oil. The tubes were incubated
at 37°C, for 18-24 h (48 h for fungi) and the lowest concentration inhibiting
bacterial and fungal growth (no turbidity) was noted as MIC.
Determination of bactericidal and fungicidal effect: In order to evaluate the effect (microbicidal /microbiostatic) of the oils, one loop from the MIC tube was sub cultured on to the NA and PDA plates which were then incubated at 37°C over night to check whether the oil merely had bactericidal or fungicidal activity i.e., no growth on subculturing.
Determination of MKT (Minimum killing time): This experiment was designed to determine the time required to kill the bacteria in vitro by the oil. One milliliter of NB, supplemented with 0.75% of DMSO and 1 mL of PDB supplemented with 0.75% of DMSO at MIC level of the oil was prepared and inoculated with 0.1 mL of freshly grown test organisms and incubated at 37°C. One loop of the sample from the above test tubes were sub cultured onto NA plates at 0, 5, 10, 15, 30, 45, 60, 90, 120, 180 min intervals and incubated overnight. Two sets of tubes were incubated for each test organisms from which subculturing were carried out alternatively (to avoid time lapse during subculture).
Statistical analysis: Each experiment was carried out in triplicate. The data were statistically analyzed using software SPSS 10.0. A least significant difference (LSD 0.05) was used to test the effects of essential oil through a general linear model. The test was statistically significant at p<0.05.
RESULTS AND DISCUSSION
In vitro antimicrobial property test of essential oil: The yield
of essential oil from the rhizome of Roma cultivar was found to be 4%. From
the preliminary screening studies by disc diffusion method, it was observed
that the test pathogens were susceptible to the oil. However, differences in
the zone sizes were observed with different pathogens (Table 1).
Oil showed highest activity against S. aureus (IZD 49.83±0.76
mm), followed by, C. albicans (IZD 30±1), A. niger (IZD
25.33±0.57) and least activity against P. aerogenosa (IZD 14±1).
The oil was significantly effective (p<0.05) against the test pathogens at
all volumes. As evident from the Table 1, the IZD value for
standard antibiotic used was invariably less than the corresponding IZD value
obtained for essential oil against all pathogens except P. aeruginosa.
Bacterial and fungal susceptibility towards the oil was observed at 2.5 μL
per disc but higher concentrations 10 μL showed larger zones of inhibition
(Table 1), when tested by agar plate technique against all
four microbial stains.
|| Antimicrobial activity of roma rhizome oil by Disc Diffusion
|*Mean±standard deviation (SD) where n = 3 and data
is significant at p<0.05
|| MIC values for oil against tested organisms
The MIC of the oil against different test organisms was found to vary from
1.95 μL mL-1 against S. aureus to 7.81 μL mL-1
against P. aerogenosa amongst the pathogens analysed (Table
2). Though a variation was observed in the diameter of zones of inhibition
at different concentration of oil and the MIC values for all the test pathogens
but MKT result shows that S. aureus was killed soon after 15 min which
indicates its microbicidal effect whereas rest other species killed after 12
Experimental verification for validation of ethnological use of medicinal plants
in vitro and in vivo has been one of the principal criteria of
drug discovery worldwide since centuries. Validation reports like the attempt
taken in work in support of ethological use of turmeric against pathogens responsible
for eye infections across the globe including India, having a rich history of
ethno-medicinal use of turmeric, are few. Ethno medicinal use of turmeric rhizome
(popularly known as Ajo, Laali pupa or Obedo) by Yorubas of North Central Nigeria
for human ailments is reported but not is supported with any information corroborated
with experimental verification. (Maurice, 1993; Gul
et al., 2004) tried to experimentally validate the use of turmeric
in Unani treatment in Pakistan against UTI infecting pathogens using resins
and other extracts but found no activity to support essential oil against pathogens
responsible for eye infections. Bacterial and fungal susceptibility towards
the oil was observed at 2.5 μL per disc but higher concentrations at 10
μL per disc showed larger zones of inhibition when tested by agar plate
technique which was found to be larger than activity shown against the antibiotic
gentamycin against all the pathogens except P. aeruginosa (Table
1) which is found to be little more susceptible as compared to other three
microbial strains. Only report on antibacterial activity of turmeric rhizome
oil exhibits lower activity against S. aureus and P. aeruginosa but
lacks the chemical constituent analysis of oil (Singh et
al., 2002). Such lower in antimicrobial activity might be due to rhizome
oil used from a cultivar not having good quality in oil constituent in comparison
to that of roma cultivar with turmerone as major constituents, as it has been
established that the constituent of essential oil has got major role in attributing
to its biological activity (Singh et al., 2005).
Chattopadhyay et al. (2004) have reported antimicrobial
activity against S. aureus which is in agreement with our work but restricted
to only one bacteria and the report also excludes MKT and microbicidal effects
of essential oil against microbes causing eye infections. Curcuma oil showed
positive activity against bacteria S. aureus including two more species
which are not eye infecting pathogen (Chopra et al.,
1941). The results of this study suggest that the antimicrobial activity
of the rhizome essential oil of roma cultivar is bactericidal against S.
aureus. The oil was significantly effective (p<0.05) against the test
pathogens (Table 1). Antimicrobial activity of essential oil
of turmeric with high tumerone content was reported by Singh
et al. (2005) but the strains used were other then infecting eye
except one A. niger. Babu et al. (2007)
and Amphawan et al. (1995) have published separately
their work denoting the antifungal activity of turmeric rhizome oil against
species responsible for causing infections other than eye.
The results were highly significant for all the treatments, determining MICs,
MKTs and even when the activities compared with standard antibiotics. In general,
there seemed to be overall agreement between the size of inhibition zones obtained
by the Disc Diffusion Method (DDM) and the Minimum Inhibitory Concentration
(MIC) values, i.e., larger zones of inhibition correlated with lower MIC values.
|| Mass spectrum and structure of Ar-turmerone
This relationship between inhibition zones and MIC values has been reported
in literature while studying the antibacterial activity of essential oils (Rath
et al., 2005). Roma rhizome essential oil was found contain 49.76%
of turmerone (Fig. 1) which is supported by the report showing
presence of ar-turmerone as most abundant constituent in rhizome essential oil
of South-West Nigerian grown turmeric by Ajaiyeoba et
al. (2008), Usman et al. (2009). Ar-turmerone
has been reported as the major constituent of rhizome oil of turmeric of different
origin (Singh et al., 2005) but all these reports
are not supported with any antimicrobial activity study against eye infecting
Better activity of roma rhizome oil could be due to presence of high percentage
of ar-turmerone (49.76%). Some reports support our inference that turmerone
is responsible for showing antimicrobial activity against specific pathogens
(Amphawan et al., 1995). Previous studies have
already shown the growth inhibition activity of Curcuma longa rhizome
essential oil on different microorganisms.
However, this is the first time that the bactericidal activities of Curcuma
longa rhizome essential oil have been demonstrated against eye disease causing
pathogens. The results appear promising, for possible use of this rhizome oil
of roma cultivar as bactericidal agents (Baratta et al.,
1998), more particularly eye infections which are very sensitive organs.
The use of antibiotics causes severe side effects like hypersensitivity reactions,
gastric disturbances, ototoxicity and nephrotoxicity (Cosgrove
et al., 2009). Different antibiotics such as chlorotetracyclin, oxytetracycline
and chloramphenicol at low concentrations have been used to prevent these infections.
These antibiotics have been proved to have severe side effects, now-a-days consumers
are concerned about the ill effects due to use of synthetic antibiotics. When
safety of synthetic products is questioned, natural compounds of plant origin
may appeal to the public.
Probably in our investigation, for the first time we have documented the antibacterial activity of rhizome oil of roma cultivar against eye infection causing pathogens, experimentally supported with low concentration of oil needed to affect the growth of a range of microorganisms responsible for dreaded eye diseases within a short period of killing time supplemented with information on its major chemical constituent, ar-turmerone. Furthermore, essential oil of rhizome of roma cultivar proved to have bactericidal properties at low concentrations and most of the essential oil components possess antioxidant properties holds a promise as an alternate to expensive, harmful antibiotics against these pathogens. Of course, other studies are highly necessary to study the toxicity of these oils in order to set an appropriate formulation like eye drop for this purpose.
The authors are grateful to Prof. Dr. S.C. Si, Dean, Centre of Biotechnology and Prof. Dr. M.R. Nayak, President, Siksha O Anusandhan University for providing financial support and encouraging throughout.
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