Introduction:

The aim of this study was screen the antimicrobial activity of Ethanol extracts of Calotropis gigantea isolated from different plant parts such as: stem leaves, flower, fruit and seeds against clinical isolates of microbes. The aqueous extract of the C. gigantea was studied for its antagonistic activity against Candida albicans, Staphylococcus aureus, Escherichia coli, Salmonella typhimurium and Pseudomonas aeruginosa. In vitro antimicrobial activity was performed by well diffusion method in MH agar [1].

In current scenario it is the challenge for medical and pharmaceutical advancement, microbes to acquire resistant against the drugs used in the treatment of common infectious disease [2, 3]. These drug resistant candidates are more pathogenic with high mortality rate and become a great challenge in the pharmaceutical and healthcare industry. To overcome microbial drug resistant, scientists are looking forward for the development of alternative and novel drugs. Natural sources such as plants, algae and animals provide an array of natural medicinal compounds for the treatment of various infectious diseases [4].

Plants are exploited as medicinal source since ancient age. The traditional and folk medicinal system uses the plant products for the treatment of various infectious diseases. In recent times, plants are being extensively explored for harbouring medicinal properties. Studies by various researchers have proved that plants are one of the major sources for drug discovery and development [5, 6]. Plants are reported to have antimicrobial, anticancer, antiinflammatory, antidiabetic, hemolytic, antioxidant, larvicidal properties etc.

Calotropis gigantea is a wasteland weed better known as milkweed, habitat of Asian countries that includes, India, Indonesia, Malaysia, Philippines, Thailand, Sri Lanka and China. This plant is popularly known because it produces large quantity of latex [7]. The plant has potential pharmacological properties. Fractionation of the latex into its rubber and rubber-free fractions affords better insight into its potentials and limitations. A large quantity of latex can be easily collected from its green parts. The abundance of latex (containing alkaloids) in the green parts of the plant reinforces the idea that it produced and accumulated latex as a defence strategy against organisms such as bacteria, fungi and insects [8].

Current study was focused to investigate the antibacterial activity of ethanol extracts obtained from the crude leaves, stem, flower, seed and fruit of C. gigantea against clinical isolates of fungi and bacteria. The prevalence of invasive, opportunistic microbial and fungal infections has increased at an alarming rate especially in immune- compromised individuals. Although it appears to be a great array of antimicrobial and antifungal drugs, there is at present a quest for new generations of antimicrobial and antifungal compounds due to the low efficacy, side effects or resistance associated to the existing drugs [9]. This plant has potential antimicrobial properties against microbial infections [11, 12].

MATERIALS AND METHODS:

Collection of plant and preparation of extract:

Plants of Calotropis gigantea were collected from botanical garden City forest Noida, Uttar Pradesh, India. The leaves, stem, fruit, seed and floor were segregated and dried in the shade at the temperature of 30 ± 2ᵒC and were authenticated at Microbiology Quality Control Laboratory, Dabur Pvt. Ltd. Ghaziabad, India. Parts of plants were separately powdered in an electric grinder. 100 gram of plant powder was soaked in 100 ml of ethanol in conical flask and loaded on an orbit shaker at a speed of 120 rpm for 24 hours [13]. The mixture was filtered using Whatman filter paper number-1. The filtrate was concentrated using rotary evaporator and dried using lyophilizer [14]. The filtrates were concentrated under reduced pressure at 37°C and extract was collected and store in dark airtight plastic containers at 4–8°C, until use.

The extracted powder of different plant parts were dissolved in sterilized distilled water to make 1mg/ml solution. These solutions were used to perform antimicrobial activity assay [10].

Test microorganisms:

The following five clinical isolates of bacteria and fungi were used in this study: Candida albicans (ATCC 10231), Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC 8739), Salmonella typhimurium (NCTC 6017) and Pseudomonas aeruginosa (ATCC 9027). All stock cultures were obtained from Microbiology Quality Control Laboratory, Dabur Pvt. Ltd. Ghaziabad (UP), India. All these cultures were maintained on nutrient agar slants for bacteria and Potato Dextrose Agar slants for fungus at 4°C.

Phytochemical analysis of the extract:

Specific qualitative tests were performed to identify bioactive compounds of pharmacological importance through standard methods. In brief, the phytochemicals such as tannins, alkaloids, saponins, flavonoids, terpenoids, and phenols/polyphenols were qualitatively determined [13].

Culture media and inoculum preparation:

Nutrient agar media (Himedia, India) was used for the culturing of bacterial strains. Loops full of all the bacterial cultures were inoculated on the nutrient agar and incubated at 37˚C for 48 hrs and potato dextrose agar media (Himedia, India) were used for the culturing of fungal strain. Loops full of fungus were inoculated on the Potato dextrose agar (PDA) and incubated at 22-25˚C for 72 hrs.

Antibacterial activity:

The different parts of plant extracts obtained as above were screened for their antibacterial activity in comparison with standard antibiotic ciprofloxacin (100mg/ml) in vitro by well diffusion method [15, 16]. Lawn culture was used using the test organism on Nutrient Agar (NA). The inoculated plates were kept aside for few minutes using well cutter, four wells were made in those plates at required distance. In each step of well cutting the well cutter was thoroughly wiped with alcohol. A fixed volume (0.1ml) of the C. gigantea extracts were then introduced into the wells in the increasing concentration. The plates with bacteria were incubated at 37˚C for 24 hours. The activities of the extracts were determined by measuring the diameters of zone of inhibition. The standard antibiotic disc was placed on the agar surface as positive control. Plates were incubated at 37°C for 48 hours. Triplicate plates were maintained for each organism.

Antifungal activity:

The different parts of plant extracts obtained as above were also screened for their antifungal activity in comparison with standard antibiotic Ketoconazole (10mg/ml) in vitro by well diffusion method [15, 16]. Lawn culture was prepared using the test organism on Sabouraud’s Dextrose Agar (SDA). The inoculated plates were kept aside for few minutes using well cutter, four wells were made in those plates at required distance. A fixed volume (0.1ml) of the C. gigantea extracts were then introduced into the wells in the increasing concentration. The plates with fungi were incubated at 22-25°C for 48 hours. The standard antibiotic disc was also placed on the agar surface as positive control. Plates were incubated at 22-25°C for 48 hours. The activities of the extracts were determined by measuring the diameters of zone of inhibition. Triplicate plates were maintained for each organism.

Positive and negative control:

Amphotericin B (10 µg/disc) was used as positive control for Candida albicans and Tetracycline (10µg/disc) for Staphylococcus aureus, Escherichia coli, Salmonella typhimurium and Pseudomonas aeruginosa. Sterilized distilled water was used as negative control.

Determination of relative zone inhibition percentage:

The relative percentage inhibition of the test extract with respect to positive control was calculated by using the following formula [17, 18].

Relative percentage inhibition of the test extract

=100 x (x-y)

z-y

Where,

x: total area of inhibition of the test extract

y: total area of inhibition of the solvent

z: total area of inhibition of the standard drug

Statistical analysis:

The values of antimicrobial activity of the aqueousextracts of different parts of C. gigantea plant were expressed as mean ± standard deviation (n= 3) for each sample.

RESULTS AND DISCUSSION:

Medicinal plants are being probed as an alternate source to get therapeutic compounds based on their medicinal properties. C. gigantea is easily available in most of the agricultural and non-agricultural fields and the usage of this plant for medicinal purpose was reported by several researchers. The aqueous extract of leaves exhibited more efficient antibacterial activity in comparison to other plant extracts such as fruit, flower, stem and seeds against four clinical isolates of bacteria and one clinical isolates of fungal (Figure-1).

Figure-1: (A) Slants of the different strains, (B) Extracts of Calotropis gigantea.

Determination of minimum inhibitory concentration (MIC):

MIC of the leaves, fruit, flower, stem and seeds extract were performed by modified agar well diffusion method. Two fold serial dilution of the stock solution was prepared in sterilized distilled water to make a concentration range from 0.1-100 mg/ml [19].

The concentrations of test cultures were adjusted to 0.5 McFarland standards. The bacterial suspensions were seeded on MHA plates using a sterilized cotton swab. In each of these plates three wells were cut out using a standard cork borer (7 mm). Using a micropipette, 100 µl of each dilution was added in to wells. All the plates were incubated at 37ºC for 24 hours. Similarly done for fungal suspension and incubated at 22-25ºC for 24 hours.

Antimicrobial activity of the leaves, stem, flower, fruit and seeds extracts were evaluated by measuring the zone of inhibition (Figure-2). Experiment was carried out in triplicates for each test organism.

Negative control and positive control also putt-up with specimen samples.

Values are expressed as mean ± standard deviation of the three replicates.

Table-1: Antimicrobial activity of Calotropis gigantea. The total area of the inhibition was calculated by using area = πr2; where, r = radius of zone of inhibition

Test Microorganisms	Inhibition zone diameter (in mm)
Test Microorganisms	Leave extract	Stem extract	Fruit extract	Seed extract	Flower extract	Positive control	Negative control
Staphylococcus aureus (ATCC 6538)	22.500	14.667	7.000	1.183	2.433	26.083	0
Pseudomonas aeruginosa (ATCC 9027)	19.700	13.200	9.117	1.967	3.967	20.683	0
Salmonella typhimurium (NCTC 6017)	21.100	13.933	8.058	1.575	3.200	23.383	0
Escherichia coli (ATCC 8739)	30.083	20.967	14.117	10.717	6.800	33.400	0
Candida albicans (ATCC 10231)	23.346	15.692	9.573	3.860	4.100	25.888	0

Values are expressed as mean ± standard deviation of the three replicates.

Previous studies report the presence of phytochemicals like cardenolides, flavonoids, terpenes, pregnanes, nonprotein amino acid and cardiac glycoside as major constituents in C. gigantea may acknowledge the medicinal property of this plant [8, 17]. Antimicrobial activity of different parts of plant extracts of C. gigantea showed varying degrees of antibacterial and antifungal activity against all microorganisms tested in this study. There are many reports of plants in the family Asclepiadaceae possessinganti-microbial activity [12, 13]. From this study it can be said that, Ethanol shade dried leaves extract of Calotropis gigantea showed wide range of Antibacterial and Antifungal activity can be used and administered in the medical practice. The present study has shown a spectrum of antimicrobial activities which provides a support to some tradition uses of these few medicinal plants [16]. But the effective biomolecules which act as antimicrobial have to be identified isolated and subjected to extensive scientific and pharmacological screening that can be used as sources for new drugs.

Commercially available antimicrobial agents (antibiotics) are now used to treat diseases arising from microbial infections. A major problem encountered with antibiotics in clinical use is drug resistance, which mostly leads to treatment failure. Other problems with antibiotics include toxicity, high cost, low cost efficacy, etc. This necessitates a continuous search for new antimicrobial agents. Medicinal plants have no doubt remained the major sources of traditional medicine worldwide. This study attempts to determine the phytochemical analysis and antimicrobial effect of Calotropis gigantea. In this report, we provide new information on the antimicrobial activities of C. gigantea using known microbial pathogens as tested organisms.

CONCLUSION:

We conclude that C. gigantea represents a rich source of valuable medicinal compounds and leaves of C. Gigantea contain high antibacterial and antifungal property and can be further be explored for the isolation of its bioactive compound. The bioactive compounds which are responsible for the In-vitro antimicrobial of C. gigantea over all fungi and bacteria strains in all extracts could be alkaloids, cardiac glycoside, tannins, saponins, flavonoids, steroids, terpenoids, reducing sugar and resins. The result of this work suggests that the whole plant extract of C. gigantea has number of medicinal properties. From this work it can be said that the shade dried Calotropis gigantea leaf extract of Ethanol and distilled water has more effective against these pathogenic organisms and can be used for the future references for the treatment other diseases.

FUTURE PROSPECTS:

Candida albicans (sometimes referred to as monilia) is a fungus that is normally present on the skin and cause skin disease. Bearers of this disease spent so much money for the treatment of same disease but results were not satisfactory many times. Therefore in this study we find out that ethanol leaves extract of C. gigantea may be utilised for the treatment of skin disease caused by C. albicans. These types of drugs are fall in over the counter drugs category; it will be an additional benefit to patients.

ACKNOWLEDGEMENTS:

We are thankful to Microbiology Quality Control Laboratory, Dabur India Ltd., Ghaziabad for their technical and infrastructure support.

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Received on 26.05.2016 Modified on 28.06.2016

Research J. Science and Tech. 2016; 8(3):129-134.

DOI: 10.5958/2349-2988.2016.00017.6