Pharmacological Investigation of Methanolic Extract of Ficus glomerata Linn. Bark

Nowshin Nowaz Rumzhum*, Md. Mostafizur Rahman, Sadia A. Chowdhury, Most. Nazma Parvin, Mahabub Hasan

Laboratory of Pharmacognosy and Pharmacology, Department of Pharmacy, Stamford University Bangladesh, Dhaka-1217, Bangladesh

ABSTRACT:

The methanolic extract of the bark of Ficus glomerata Linn. (Family: Moraceae) was evaluated for in vitro antioxidant activity by determination of hydrogen peroxide scavenging activity, total antioxidant capacity, assay of nitric oxide scavenging activity and reducing power test and in vivo antinociceptive effect in acetic acid induced writhing model in Swiss albino mice. The plant extract was also subjected for brine shrimp lethality bio-assay to evaluate its cytotoxic property. The results revealed the presence of pronounced antioxidant property as compared with ascorbic acid used as standard and a dose-dependent (250 and 500 mg/kg) analgesic effect. Investigation also showed that it has strong lelthality (LC₅₀ 1.247 µg/ml) against brine shrimp nauplii, Vincristine Sulphate was used as positive control. The antioxidant, antinociceptive and cytotoxic properties observed seem to be in good accordance with the traditional uses of Ficus glomerata.

KEYWORDS: Ficus glomerata Linn., Moraceae, Antioxidant, Antinociceptive, Cytotoxicity.

INTRODUCTION:

The traditional medicine involves the use of different plant extracts or the bioactive constituents. Researchers are increasingly turning their attention to folk medicine and looking for new leads day by day to develop better drugs against different diseases. This type of study provides the health application at affordable cost. The study such as ethno medicine keenly represents one of the best avenues in searching new economic plants for medicine. Historically plants have provided a good source of medicine which is highly effective instruments in the fight against various diseases. Natural products, either as pure compounds or as standardized plant extracts, provide unlimited opportunities for new drug leads because of the unmatched availability of chemical diversity¹. Large scale evaluation of the local flora exploited in traditional medicine for various biological activities is a necessary first step in the isolation and characterization of the active principle and further leading to drug development². A large number of plants have been screened as a viable source of natural antioxidants including tocopherols, vitamin C, carotenoids and phenolic compounds which are responsible for maintenance of health, to help the human body reduce oxidative damage and protection from coronary heart diseases and cancer³. In view of these, methanolic extract of bark of Ficus glomerata was studied exhaustively for its potential antioxidant, analgesic and cytotoxic effects.

Ficus glomerata Linn. (Moraceae) is an evergreen, moderate to large sized spreading, lactiferous, deciduous tree, without much prominent aerial roots found throughout greater part of India in moist localities and is often cultivated in villages for its edible fruit⁴. It is commonly known as Gular fig or Cluster fig in English and Dumor in Bengali. Previous phytochemical studies revealed that the stem bark of Ficus glomerata contains tannin, wax, saponin gluanol acetate, β-sitosterol, leucocyanidin-3-O-β-D-glucopyrancoside, leucopelargonidin-3-O-β-D-glucopyranoside, leucopelargonidin-3-O-α-L-rhamnopyranoside, lupeol, ceryl behenate, lupeol acetate, α-amyrin acetate, leucoanthocyanidin, and leucoanthocyanin from trunk bark,lupeol, β-sitosterol and stigmasterol were isolated⁵. Fruit contains glauanol, hentriacontane, β sitosterol, glauanolacetate, glucose, tiglic acid, esters of taraxasterol, lupeolacetate, friedelin, higherhydrocarbons and other phytosterol⁶. A new tetra triterpene glauanol acetate which is characterized as 13α, 14β, 17βH, 20 α H-lanosta-8, 22-diene-3βacetate and racemosic acid were isolated from the leaves. An unusual thermostable aspartic protease was isolated from latex of the plant. The stem bark and fruit showed the presence of glauanol acetate⁷. Ficus glomerata has been used in traditional system of medicine for treating liver diseases, diarrhoea, piles, asthma, leprosy and other ailments⁸. The fruits of Ficus glomerata have been used since olden times in the ethnomedicine for many varied medicinal purposes including as a remedy of diabetes mellitus⁹. In addition, this plant is considered to possess tonic, expectorant, emollient, stomachic and carminative properties¹⁰. In this paper, the antioxidant, antinociceptive and the cytotoxic activities of the methanolic extract of Ficus glomerata are being reported to validate the traditional use of the crude drug through in vivo and in vitro evaluation.

MATERIAL AND METHODS:

Plant materials:

Ficus glomerata Linn. bark was collected from Gagipur in May 2008 and was identified by Bangladesh National Herbarium, Mirpur, Dhaka, where a voucher specimen (DCAB accession no: 32781) has been deposited. The bark of the plant was first sun dried and then ground into coarse powder.

Extraction of plant materials:

About 90 gm of powered material was taken in a clean, flat-bottomed glass container and soaked in 900 ml of methanol. The container with its contents was sealed and kept for a period of 24 days accompanying occasional shaking and stirring. The whole mixture then underwent a coarse filtration by a piece of clean, white cotton material. The filtrate (methanol extract) obtained was evaporated under ceiling fan and in a water- bath until dried. It rendered a gummy concentrate of blackish color. The gummy concentrate was designated as crude extract of methanol (2.0 gm).

To get preliminary idea about the active constituents present in the plant bark extract different chemical tests were performed and showed the presence of steroids, alkaloid, flavonoids and tannins¹¹.

Antioxidant property

i) Qualitative assay: A suitably diluted stock solutions ( sample solutions) were spotted on pre-coated Silica gel TLC (Thin layer chromatography) plates and the plates were developed in solvent systems of different polarities (polar, medium polar and non-polar) to resolve polar and non-polar components of the extract and to choose the solvent system in which stock solutions run well. The plates were dried at room temperature and were sprayed with 0.02% DPPH (2,2-diphenyl-1-picrylhydrazine) in ethanol. Bleaching of DPPH by the resolved bands was observed for 10 minutes and the color changes (yellow on purple background) were noted¹².

ii) Quantitative assay: Free radical scavenging activity of the methanol extract was evaluated by determination of hydrogen peroxide scavenging activity, total antioxidant capacity, assay of nitric oxide scavenging activity and reducing power test. In all methods ascorbic acid is used as standard.

a. Scavenging of hydrogen peroxide:

The ability of the methanolic extract of Ficus glomerata to scavenge hydrogen peroxide was determined according to the method of Ruch et al.¹³. A solution of hydrogen peroxide (2 mmol/l) was prepared in phosphate buffer (pH 7.4). Hydrogen peroxide concentration was determined spectrophotometrically from absorption at 230 nm with molar absorbtivity 81 (mol/l)^-1 cm^-1 in a spectrophotometer (Hach, DR-4000U). Extracts (50–250mg/ml) in distilled water were added to a hydrogen peroxide solution (0.6 ml, 40 mM). Absorbance of hydrogen peroxide at 230 nm was determined after ten minutes against a blank solution containing in phosphate buffer without hydrogen peroxide. For each concentration, a separate blank sample was used for background subtraction. The percentage of scavenging of hydrogen peroxide of methanolic extract of Ficus glomerata and standard compounds was calculated using the following equation:

Percent scavenged [H₂O₂] = (A₀ − A₁)/ A₀ × 100

Where, A₀ was the absorbance of the control, and A₁ was the absorbance in the presence of methanolic extract of Ficus glomerata and standards¹⁴.

b. Determination of total antioxidant capacity:

The antioxidant activity of the extract was evaluated by the phosphomolybdenum method according to the procedure of Prieto et al.¹⁵. The assay is based on the reduction of Mo (VI) – Mo (V) by the extract and subsequent formation of a green phosphate / Mo (V) complex at acid pH. 0.3 ml extract was combined with 3 ml of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes containing the reaction solution were incubated at 95^οC for 90 min. Then the absorbance of the solution was measured at 695 nm using a spectrophotometer (Hach, DR-4000U) against blank after cooling to room temperature. Methanol (0.3 ml) in the place of extract is used as the blank. The antioxidant activity is expressed as the number of equivalents of ascorbic acid.

c. Assay of Nitric oxide scavenging activity:

The procedure is based on the method, where sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide, which interacts with oxygen to produce nitrite ions that can be estimated using Greiss reagent. Scavengers of nitric oxide compete with oxygen leading to reduced production of nitrite ions. For the experiment, sodium nitroprusside (10 mM) in phosphate buffered saline was mixed with different concentrations of methanolic extract of Ficus glomerata dissolved in methanol and incubated at room temperature for 150 min. The same reaction mixture without the ethanol extract but the equivalent amount of methanol served as the control. After the incubation period, 0.5 ml of Griess reagent (1% sulfanilamide, 2% H₃PO₄ and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride was added. The absorbance of the chromophore formed was read at 546 nm¹⁶.

d. Reducing power test:

The reducing power of methanolic extract was determined according to the method of Oyaizu¹⁷. Different amounts of methanolic extracts (100 - 1000 µg) in 1 ml of methanol were mixed with phosphate buffer (2.5 ml, 0.2 mol/l, pH 6.6) and potassium ferricyanide [K₃Fe(CN)₆] (2.5 ml, 1%). The mixture was incubated at 50^οC for 20 min. A portion (2.5 ml) of trichloroacetic acid (10%) was added to the mixture, which was then centrifuged (650 x g at room temperature) for 10 min. The upper layer of solution (2.5 ml) was mixed with distilled water (2.5 ml) and FeCl₃ (0.5 ml, 0.1%), and the absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicated increased reducing power.

Antinociceptive property

Evaluation of antinociceptive property was performed by acetic acid induced writhing model in mice¹⁸. The acetic acid induced writhing method is an analgesic behavioral observation assessment method that demonstrates a noxious stimulation in mice. The test consists of injecting the 0.7% acetic acid solution intraperitoneally and then observing the animal for specific contraction of body referred as ‘writhing’. A comparison of writhing was made between positive control (Diclofenac-Na). Control and test sample are given orally 30 minutes prior to acetic acid injection. If the sample possesses analgesic activity, the animal that received the sample will give lower number of writhing than the control, i.e. the sample having analgesic activity will inhibit writhing.

Cytotoxicity study

Brine shrimp lethality bioassay^{19, 20} technique was applied for the determination of cytotoxic property of the crude extract.

Preparation of positive control group

Vincristine sulphate was used as the positive control. Measured amount of the vincristine sulphate was dissolved in DMSO to get an initial concentration of 20 μg/ml from which serial dilutions were made using DMSO to get 10 μg/ml, 5 μg/ml, 2.5 μg/ml, 1.25 μg/ml, 0.625 μg/ml, 0.3125 μg/ml, 0.15625 μg/ml, 0.078125 μg/ml, 0.0390 μg/ml. Then then positive control solutions were added to the premarked vials containing ten living brine shrimp nauplii in 5 ml simulated sea water to get the positive control groups.

Preparation of negative control group

100 μl of DMSO was added to each of three premarked glass vials containing 5 ml of simulated sea water and 10 shrimp nauplii to use as control groups. If the brine shrimps in these vials show a rapid mortality rate, then the test is considered as invalid as the nauplii died due to some reason other than the cytotoxicity of the compounds.

Counting of nauplii

After 24 h, the vials were inspected using a magnifying glass and the number of survived nauplii in each vial was counted. From this data, the percent (%) of lethality of the brine shrimp nauplii was calculated for each concentration.

RESULTS AND DISCUSSION:

Antioxidant property

i) Qualitative assay: The color changes (yellow on purple background) on the TLC plates were observed due to the bleaching of DPPH by the resolved bands.

ii) Quantitative assay

a. Scavenging of hydrogen peroxide: Scavenging of H₂O₂ by extracts may be attributed to their phenolic constituents, which can donate electrons to H₂O₂, thus neutralizing it to water. The methanolic extracts of Ficus glomerata were capable of scavenging hydrogen peroxide in a concentration-dependent manner (Figure 1).

Figure 1: H₂O₂ scavenging activity of methanolic extract of Ficus glomerata Vs Ascorbic acid

b. Total antioxidant capacity: Total antioxidant capacity exerted by the extract was concentration dependent. It was observed that the extract was likely to have the capacity of reduction of Mo (VI) to Mo (V) by the antioxidant principle and the formation of a green phosphate / Mo (V) complex with a maximal absorption at 695 nm. The antioxidant activity is expressed as the number of equivalents of ascorbic acid (Table 1).

Table 1: Total antioxidant capacity of methanolic extract of Ficus glomerata

Materials	Concentration (μg/mL)	Equivalent to ascorbic acid
Methanol extract of Ficus glomerata	10 25 50 125 250 500	0.073±0.07 0.132±0.09 0.271±0.03 0.613±0.11 1.301±0.10 2.032±0.04

c. Nitric oxide scavenging activity: From Figure 2, it is observed that the extract is likely to have concentration dependent nitric oxide scavenging activity. The bark may have the property to counteract the effect of NO formation and in turn may be of considerable interest in preventing the ill effects of excessive NO generation in the human body. Further, the scavenging activity may also help to arrest the chain of reactions initiated by excess generation of NO that are detrimental to the human health. Nitric oxide is also implicated for inflammation, cancer and other pathological conditions²¹.

Figure 2: Nitric oxide scavenging activity of methanolic extract of Ficus glomerata

d. Reducing power activity: Reduction ability of the extract has been investigated from the Fe⁺⁺⁺ - Fe⁺⁺ transformation using the method followed by Oyaizu¹⁷. Earlier authors^{22, 23} have observed a direct correlation between antioxidant activity and reducing power of certain plant extracts. The reducing properties are generally associated with the presence of reductones²² which have been shown to exert antioxidant action by breaking the free radical chain by donating a hydrogen atom²⁴. Reductones are also reported to react with certain precursors of peroxide, thus preventing peroxide formation. Figure 3 shows the reduction ability of Ficus glomerata.

Figure 3: The reducing power of methanolic extract of Ficus glomerata

Antinociceptive property: The methanolic extract of plant bark produced 77.59% and 53.45% writhing inhibition at the doses of 500 mg/kg and 250 mg/kg body weight respectively, in acetic acid induced writhing model in mice which are comparable to Diclofenac sodium (51.72% at the dose of 25 mg/kg). Table 2 shows the antinociceptive activity of Ficus glomerata.

Table 2: Effect of methanolic extract of Ficus glomerata bark on acetic acid induced writhing in mice

Treatment	Dose* (mg/kg)	Route of administration	Number of Writhing** (% writhing)	% of writhing inhibition
Control (1% aq. tween 80 )	10 ml/kg	p.o.	29±0.17^a (100)	--
Diclofenac-Na	25	p.o.	14±0.08^a (48.28)	51.72
Methanolic extract of Ficus glomerata	250 500	p.o. p.o.	13.5±0.13^b(46.55) 6.5±0.04^a(22.41)	53.45 77.59

*Administered 30 min before 0.7% acetic acid administration (10 ml/kg, i.p.). **Counted for 15 min, starting 5 min after acetic acid administration; a, p <0.001 b, p <0.01 c , p <0.05 vs. control, Student’s t-test; values are mean ±S.E (n=5).

Cytotoxic effect: Following the procedure of Mayer²⁵ the lethality of the crude extract to brine shrimp was determined on Artemia salina. Table 3 shows the results of the brine shrimp lethality testing after 24 hours of exposure to the methanolic extract and the positive control, vincristine sulphate. The LC₅₀ obtained from the best-fit line slope was found to be 1.247±0.08 µg/ml in comparison with Vincristine Sulphate (LC₅₀ 0.322±0.11 µg/ml).

Table 3: LC₅₀ data of the test sample of Ficus glomerata in brine shrimp lethality bioassay

Sample	LC50 ( µg/ml)
Vincristin Sulphate	0.322±0.11
Methanolic extract of Ficus glomerata	1.247±0.08

CONCLUSION:

Our present study reveals the antioxidant, antinociceptive and cytotoxic properties of Ficus glomerata Linn. bark, which can be a scientific avenue to proceed further exhaustively to explore its active constituents responsible for its pharmacological effects. We believe, further detailed advanced studies may explore novel leads for new drugs development.

ACKNOWLEDGMENTS:

Authors wish to thank the authority of Stamford University Bangladesh and the Chairman, Department of Pharmacy of the same for extending their cordial supports to perform these investigations.

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Received on 13.12.2011

Modified on 23.01.2012

Accepted on 12.02.2012

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Research J. Pharmacology and Pharmacodynamics. 4(2): March - April, 2012, 111-115