STUDIES ON ANTIBACTERIAL, CYTOTOXIC AND ANTIOXIDANT PROPERTIES OF THE SEEDS AND LEAVES OF FICUS RACEMOSA

STUDIES ON ANTIBACTERIAL, CYTOTOXIC AND ANTIOXIDANT PROPERTIES OF THE SEEDS AND LEAVES OF FICUS RACEMOSA

Salman Bin Hosain*¹, Shapna Sultana ² and Afroza Haque ²

Atish Dipankar University of Science and Technology ¹, Manik Nagar, Dhaka, Bangladesh

Department of Pharmacy, Southeast University ², Banani, Dhaka, Bangladesh

ABSTRACT

The seeds and leaves of Ficus racemosa have shown considerable antioxidant and cytotoxicity properties while their antibacterial activities were found low. The extracts were found to have different levels of antioxidant and cytotoxicity properties in different concentration. For antioxidant activity, here we used free radial scavenging activity of plant extracts against stable DPPH (1, 1-diphenyl 2-picrylhydrazyl) which was determined spectrophotometrically by the method described in Brand William et al., (1995). Methanolic extracts of these plants were used. As we know that ascorbic acid is a perfect antioxidant, that’s why it was used as a standard reference. The absorbance was taken at 517nm by UV spectrophotometer. The IC₅₀ (Inhibitory Concentration) value of standard was 8.287µg/ml. The IC₅₀ value of Ficus racemosa seeds and leaves were 3.893µg/ml and 14.5µg/ml respectively. For Cytotoxicity, brine shrimp lethality bioassay was used. We used Vincristine sulfate as a standard reference. The LC₅₀ value of standard was 0.057µg/ml. The LC₅₀ (Lethal Concentration) value of the seeds and leaves were 0.56µg/ml and 0.467µg/ml respectively. The plant has shown significant activities against Surcina lutea

 Ficus Racemosa

INTRODUCTION: The medicinal plants find application in pharmaceutical, cosmetic, agriculture and food industry. Recently the uses of natural drugs are under review and have been increasing day by day. Traditional use of medicines is recognized as a way to learn about potential future medicines. In 2001, researchers identified 122 compounds used in mainstream medicine which were derived from "Ethnomedical" plant sources; 80% of these compounds were used in the same or related manner as the traditional ethnomedical use ¹. It is becoming clearer that the medicinal herbs have a potential in today’s synthetic era as numbers of medicines are becoming ineffective against resistant microorganisms.

Many of the herbs and spices used by humans to season food yield useful medicinal compounds ^{2, 3}. Since antioxidant, cytotoxicity and antibacterial constituent are an important medicinal substance; recently research is being continued on various plants to derive antioxidant, cytotoxicity and antibacterial ingredients. But, the endeavor in this regard is not adequate enough. The present research is therefore, an effort to give a detailed survey of the literature on its pharmacological properties.

Plant review: The local name of the plant is Cluster and the taxonomy is as follows-

Kingdom- Plantae

Division – Magnoliophyta

Class – Magnoliopsida

Order – Rosales

Family – Moraceae

Genus – Ficus

Species – Ficus racemosa

Chemical constituents: Leaves contain Tetra triterpene, glauanol acetate, racemosic acid ⁴. Bark contains ceryl behenate, lupeol, lupeol acetate, α & β-amyrin, gluanol acetate, β -sitosterol, stigmasterol and ketone ⁵.

Medicinal Uses: Scientific studies indicate F. racemosa to posses various biological effects such as hepatoprotective ⁶, chemopreventive ⁷, antidiabetic ⁸, anti inflammatory ⁹, antipyretic ¹⁰, antitussive ¹¹ and antidiuretic ¹² properties. Bark is astringent, and is used in asthma, menorrhagia, bronchitis and bilious affections ¹³.

MATERIALS AND METHODS:

Collection and Identification of the Plant: The fresh seeds and leaves of the Ficus racemosa were collected during the month February 2009 from the area of Puran Dhaka. The National Herbarium taxonomically identified the plant.

Drying and Pulverization: The fresh seeds and leaves of the plant were first washed with water to remove adhering dirt and then cut into small pieces, sun dried for 4 days. After complete drying, the entire portions were pulverized into a coarse powder with the help of a grinding machine and were stored in an airtight container for further use.

Extraction of Plant Material: The powdered 60gm powders were extracted with 3 times methanol of their weight in a flat bottom glass container, through occasional shaking and stirring for 10 days. The extracts were then filtered through filter paper. The filtrates were concentrated at 50^oC under reduced pressure to afford a greenish mass of biological investigation.

In Vitro Antimicrobial screening: Disc diffusion method was used to determine the antimicrobial activity of the crude extracts ^{14, 15}, against the microbial strains listed in Table 1. These were collected as pure cultures from the microbiology lab of Atish Dipankar University of Science and Technology, Bangladesh. To get a concentration of 2000µg/ml, dry Methanol extract (4mg) was dissolved in 2ml methanol & carefully dried to evaporate the residual solvent. Here, Kanamycin (30μg/disc) was used as the standard.

Then, for 24 hours, these plates were kept at low temperature (4^oC) to allow maximum diffusion of the test materials and Kanamycin. To allow maximum growth of the organisms, these plates were then incubated for 12- 18 hours, at 37^oC. The test materials, which possess antimicrobial activity, suppressed the growth of the microorganisms and a clear, distinct zone of inhibition was seen surrounding the discs. The antimicrobial activity of the test agents was determined by measuring the diameter of zone of inhibition, expressed in mm.

Antioxidant Activity: The antioxidant activity, which refers to the free radical scavenging activity, of the extracts on the stable radical 1, 1-diphenyl-2-picrylhydrazyl (DPPH) was determined by the method of Brand-Williams et al (Use of a free radical method to evaluate antioxidant activity, 1995). 2mg of each of the extracts was dissolved in methanol. Solutions of varying concentration such as 500 μg/ml, 250 μg/ml, 125 μg/ml, 62.5 μg/ml, 31.25 μg/ml, 15.625 μg/ml, 7.8125 μg/ml, 3.90625 μg/ml, 1.953125 μg/ml and 0.9765625 μg/ml were obtained by serial dilution technique.

2 ml of a methanol solution of the extract of each concentration was mixed with 3 ml of a DPPH-methanol solution (20μg/ml) and was allowed to stand for 20 minutes for the reaction to occur. The absorbance was determined at 517nm and the corresponding percentages of inhibitions were calculated from these values by using the following equation:

% inhibition

= [1 - (ABS sample / ABS control)] x 100 [ABS = absorbed]

Then % inhibitions were plotted against respective concentrations used and from the graph IC50 was calculated by using tert-butyl-1-hydroxytoluene (BHT), ascorbic acid potential antioxidant, were used as positive control.

Brine shrimp lethality bioassay: Brine shrimp lethality bioassay ^{5, 15, 16,} technique was applied for the determination of general toxic property of the plant extracts. In this method, in vivo lethality in a simple zoological organism (Brine shrimp nauplii) is used as a favorable monitor for screening and fractional in the discovery of new biotic natural products. 2 mg of each sample was dissolved in specific volume of DMSO (Dimethyl Sulfoxide) to obtain the desired concentration of the prepared solution. For the experiment, 4 mg of each of the petroleum ether, carbon tetrachloride and chloroform soluble fractions were dissolved in DMSO and solutions of varying concentrations (500, 250, 125, 62.5, 31.25, 15.625, 7.8125, 3.90625, 1.953125, 0.9765625 μg/ml) were obtained by serial dilution technique using DMSO for each extract. Vincristine sulfate was used as positive control.

RESULTS & DISCUSSION: The outcome found for the antimicrobial, cytotoxic and antioxidant activities of seeds and leaves of Ficus racemosa are discussed in detail in this chapter. It was shown that the specimen has shown significant activities for cytotoxic and antioxidant activities while poor activities are found for antimicrobial properties.

Antimicrobial activity: The Methanolic extract of Ficus racemosa was tested for antibacterial activities against a number of both gram positive and gram negative bacteria. Standard kanamycin discs (30 mg/disc) were used for the comparison purpose. The result of antimicrobial activity was measured in term of zone of inhibition (mm), have been shown in Table 1. The methanolic extracts were concentration used in 500 mgm/disc. From the above data, it is evident that the methanolic extract of Ficus racemosa (seed) has the activity against gram positive bacteria of surcina lutea and its zone of inhibition was 21 mm. The zone of inhibition for the Ficus racemosa (leaves) against both gram positive & gram negative bacteria were 6-10 mm, 6-11 mm, 6-14 mm & 8-14 mm respectively. The Methanolic extract of Ficus racemosa (leaves) has poor activity against gram (+) ve & gram (-) ve bacteria.

TABLE 1: DIAMETER OF THE ZONE OF INHIBITION OF DIFFERENT PLANT EXTRACTS

Cytotoxic activity through Brine Shrimp lethality bioassay: In this method, in vivo lethality in a simple zoological organism (Brine shrimp nauplii) is used as a favorable monitor for screening and fractional in the discovery of new biotic natural products. 2 mg of each sample was dissolved in specific volume of DMSO (Dimethyl Sulfoxide) to obtain the desired concentration of the prepared solution. For the experiment, 4 mg of each of the petroleum ether, carbon tetrachloride and chloroform soluble fractions were dissolved in DMSO and solutions of varying concentrations (500, 250, 125, 62.5, 31.25, 15.625, 7.8125, 3.90625, 1.953125, 0.9765625 μg/ml) were obtained by serial dilution technique using DMSO for each extract. Vincristine sulfate was used as positive control.

The Methanolic extracts of samples were subjected to brine shrimp lethality bioassay and Vincristine sulfate was used as standard in this investigation.  The extracts were found to have different cytotoxic activity. The standard Vincristine sulfate has the LC₅₀ value of 0.057 μg/ml. According to Table 2, methanol extracts of Ficus racemosa (seeds) the LC₅₀ value is 0.56 μg/ml which indicating good cytotoxicity properties. Table 3 indicates that the LC₅₀ Value of methanol extract of Ficus racemosa (leaves) is 0.467 μg/ml which indicates good cytotoxicity properties.

TABLE 2: LC50VALUE OF METHANOLIC EXTRACT OF FICUS RACEMOSA SEEDS

TABLE 3: LC50 VALUE OF METHANOLIC EXTRACT OF FICUS RACEMOSA LEAVES

Antioxidant activity: The methanolic extracts of the plant was subjected to free radical scavenging activity by the method of Brand Williams et al., (1995) ¹⁷ and ascorbic acid was used as standard in this investigation. The antioxidant activity of the plant was measured by free radical scavenging assay with DPPH. IC₅₀ value (concentration of sample require to scavenge 50% free radical or to prevent lipid peroxide by 50%) of all the extracts were calculated. IC₅₀ value of standard ascorbic acid was 8.287 μg/ml. The methanolic extract of Ficus racemosa (seeds) and Ficus racemosa (leaves), the IC₅₀ value is 3.8938 μg/ml and 14.55 μg/ml in table 4 and 5. This indicates significant antioxidant properties.

TABLE 4- IC₅₀ VALUES OF FICUS RACEMOSA (SEED)

TABLE 5- IC₅₀ VALUES OF FICUS RACEMOSA (LEAVES)

CONCLUSION: The plant has been being used for a long time in herbal medicine without knowing the exact phytopharmacological properties that works against certain disease. Ficus racemosa showed good cytotoxicity properties. The methanolic extract of Ficus racemosa (seeds) showed antibacterial activity against the gram positive bacteria of Surcina lutea. Ficus racemosa (seeds) and Ficus racemosa (leaves) shows strong antioxidant properties. The plant can be further screened against various diseases in order to find out its unexplored efficacy and can be a potential source of biologically important drug candidates.

ACKNOWLEDGEMENT: We would like to thank Professor Dr. Ekramul Haque, the Chairman of Pharmacy Department of the “Atish Dipankar University of Science & Technology” to permit us to work in the laboratories of Pharmacy department on the project “Antimicrobial, Cytotoxic and Antioxidant Activity of the seeds and leaves of “Ficus racemosa”.

During the project, we were helped a lot by Dr. Manirul Islam with his expertise in Phytochemistry. We are also thankful to our friend Md. Farhad Hossain Masum, to help us out in proof reading the manuscript.

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