Bioactivity Studies On Cerbera Manghas Leaves

Published Date: 02 Nov 2017

Several pharmacological investigations were carried out to ascertain analgesic, antioxidant, and antimicrobial activity of the ethanol extract of the leaves of Cerbera manghas Linn. (Family- Apocynaceae). The presence of reducing sugars, tannins, steroids, alkaloids, glycosides and flavonoids were indicated by phytochemical analysis of the extract. To evaluate analgesic activity acetic acid induced writhing method was utilized. The ethanolic extract exhibited statistically significant (p < 0.01) analgesic effect in acetic acid induced writhing of white albino mice (Swiss-webstar strain). The extract produced 17.75% and 30.64% writhing inhibition at the doses of 250 mg/ kg-body weight and 500 mg/kg-body weight respectively. To evaluate antioxidant activity free-radical-scavenging assay using 1,1,-diphenyl-2-picrylhydrazyl (DPPH) was used. IC50 value of 292 µg/mL was found in DPPH assay. Antibacterial activity was evaluated by using disc diffusion method. In antibacterial test, the extract, showed no activity against the bacterial strains namely S. aureus, E. coli, S. paratyphi, S. dysenteriae, S. typhi, and S. pyogens at the doses of 250 and 500 µg/disc. The investigations were carried out using crude extract of the plant. Separation and isolation of pure compounds of the extract might be proved responsible for these pharmacological effects.

Key words: Cerbera manghas, Apocynaceae, writhing test, 1, 1-Diphenyl-2-picrylhydrazyl (DPPH), Disk diffusion

Introduction

The local name of Cerbera manghas Linn. (Family- Apocynaceae) is dabor, dagor. It is a small evergreen coastal tree growing up 12 m tall and the shiny dark-green leaves are alternate, ovoid in shape. C. manghas is naturally distributed from the Seychelles Islands in the Indian Ocean eastward to French Polynesia. It occupies lowland and coastal habitats and is often associated with mangrove forests. In Bangladesh it is distributed mainly in the Sundarban and southern region.

The leaves and the fruits contain the potent cardiac glycoside cerberin, which is extremely poisonous if ingested. People in olden times used the sap of the tree as a poison for animal hunting. A potent drug cerberin has been extracted from the extremely poisonous seeds; it has some resemblance to digitalis in its effect on the heart, and has been used in medicine in very small amounts (1).

In the previous study, the bark of C, manghas showed cytotoxic activity against HepG2, MCF-7, and HeLa cell lines (2). 2'-epi-2'-O-Acetylthevetin B extracted from seeds of Cerbera manghas L. induces cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells (3). β-d-Glucosyl-(1-4)-α-l-thevetosides of 17β-digitoxigenin (GHSC-73) is a cardiac glycoside isolated from the seeds of Cerbera manghas L. showed that GHSC-73 inhibits the growth of HepG2 cells through caspase dependent and independent apoptosis pathways (4). Neriifolin, a cardiac glycoside isolated from Cerbera manghas L. reduced viability of HepG2 cells, induced S and G2/M phase arrests of the cell cycle, and stimulated apoptosis of HepG2 cells and induced activation of caspase-3, -8, and -9, and up-regulated expression of Fas and FasL proteins. So, neriifolin could be considered a candidate for the treatment of hepatocellular carcinoma (5).

To justify the traditional uses of C. manghas this project work has been performed. To ascertain its analgesic, antioxidant and antibacterial activities several pharmacological investigations were performed.

Materials and Methods

Plant material

The leaves of C. manghas was collecred from Dumuria upazilla in Khulna district, Bangladesh. The plant was collected on the 12th February, 2011. Any type of adulteration was strictly avoided during collection.

The plant was identified by the experts of Bangladesh National Herbarium, Mirpur, Dhaka (Accession no: DCAB-35569) and a voucher specimen was also deposited.

Preparation of plant extract

The leaves were subjected to shade drying to avoid any photochemical degradation. After proper drying, the leaves were grinded into coarse powder with a suitable mechanical grinder. To avoid any possible fungal attack the powder was stored in an air-tight container, and kept in a cool, dark, and dry place. The leaves were extracted by cold extraction method. 100gm grinded leaves powder was soaked in 500 ml of 98% of ethanol in a glass container for eight days accompanying regular shaking and stirring. The extract was separated from the plant debris by filtration by a piece of clean, white cotton material & it was done two times. At room temperature the solvent was evaporated with an electric fan to get the dried crude extract (yield value 7.34%). It rendered concentrate of deep purple type and the concentrate was designated as crude extract of ethanol. The crude extract was stored in refrigerator at 4Ëš C to avoid any possible fungal attack.

Experimental Animals

From animal resources department of the International Center for Diarrhoeal Disease Research, Bangladesh (ICDDR, B) Swiss-Albino mice of either sex Was collected having the age of 4-5 weeks, average weight of 20-25 gm. The animals were kept in animal house, Pharmacy Discipline, Khulna University under the standard laboratory condition (relative humidity 55-60%, room temperature 25±20 C, and 12 hours light: dark cycle) for period of one week prior to the pharmacological experiment. According to the animal ethics guidelines of Institutional Animal Ethics Committee the experiment were performed(6).

Test Pathogens

Antimicrobial assessment was performed by using the supplied (From ICCDR, B) both Gram-negative and Gram-positive bacteria namely Salmonella paratyphi, Escherichia coli, Salmonella typhi, Shigella dysenteriae, , Staphylococcus aureus and Streptococcus pyrogens.

Chemicals, Reagents and Standard Drugs

We purchased acetic acid and ascorbic acid from Merck, Germany. We obtained 1,1-Diphenyl-2-pycrylhydrazyl (DPPH) from Sigma-Aldrich, USA. Tween-8o was supplied from Loba Chemie Pvt Ltd, India. All other reagents and Solvents were of analytical grade. . Diclofenac sodium was obtained from Beximco Pharmaceuticals Ltd, Bangladesh.

Phytochemical Screening

The crude ethanol extract was subjected to different preliminary phytochemical screening to identify major phytochemical groups (7,8) . The presence of reducing sugars, tannins, steroids, alkaloids, glycosides and flavonoids were indicated by phytochemical analysis of the extract.

Evaluation of Analgesic Activity

Analgesic activity of the ethanol extract of C. manghas L. was performed using the model of acetic acid induced writhing in mice (9). The experimental mice were selected randomly and divided into four groups denoted as control, positive control, and test group I and II each group containing 5 mice. Test group I and II were treated with the test sample at the doses of 250 and 500 mg/kg body weight. Control group received 1% Tween-80 in distilled water at the dose of 10 mL/kg body weight, and positive control group received diclofenac sodium at the dose of 25 mg/kg body weight. Test samples, control and Diclofenac-Na were given orally by means of a feeding needle. A thirty minutes interval was given to ensure proper absorption of the administered treatments. . Then the writhing inducing chemical, acetic acid solution (0.6%, 15 ml/kg) was administered intraperitoneally to each of the animals of a group. After an interval of 5 minutes, which was given for absorption of acetic acid, number of squirms (writhing) was counted for 10 minutes. For the assessment of analgesic activity the percent inhibition of writhing for each group was calculated and compared with the control group.

In Vitro Antioxidant Activity Test

Using stable free radical DPPH (1,1-Diphenyl-2-pycrylhydrazyl) the antioxidant activity of the ethanol leaves extract was estimated both qualitatively and quantitatively (10).

Qualitative Analysis

For qualitative assessment Thin Layer Chromatographic (TLC) technique was applied (10). With non-polar, medium polar and polar solvent systems TLC plates were developed to resolve compounds of different polarities. With 0.02% DPPH in ethanol the TLC plates were sprayed. For the period of 30 min the bleaching of DPPH (yellow on purple background) radical was observed and noted.

Quantitative Analysis

Based on their scavenging activity of the stable DPPH radical the anti-oxidant potential of the ethanol extract was estimated (10). At first 10 test tubes were taken to make aliquots of 9 conc. (1.57, 3.13, 6.25, 12. 5, 25, 50, 100, 200 and 400) μg/mL for plant extract & 7 test tubes were taken to make aliquots of 7 conc.(1.57, 3.13, 6.25,12.5, 25, 50,100) μg/mL for ascorbic acid. Plant extract and ascorbic acid were weighed 3 times and dissolved in ethanol to make the required concentrations by dilution technique. Here ascorbic acid was taken as positive control. DPPH was weighed and dissolved in ethanol to make 0.004% (w/v) solution. To dissolve homogeneously magnetic stirrer was used. After making the desired concentrations, 6 mL of 0.004% DPPH solution was applied on each test tube by pipette and then 2 mL of different concentrations was mixed in each test tube. Test tubes were kept for 30 minutes in dark to complete the reactions. DPPH was also applied on the blank test tubes at the same time where only 2mL ethanol was taken as blank. After 30 minutes, absorbance of each test tube was determined by UV spectrophotometer at 517 nm. . Percent inhibition was calculated using the following formula:

% inhibition = [(Absorbance of control - Absorbance of sample or standard) / Absorbance of control] × 100

IC50 value was determined from % inhibition vs. concentration graph.

Antibacterial Activity

For the assessment of antibacterial activity of the ethanol leaves extract of C. manghas disc diffusion assay method was used against some Gram-positive and Gram-negative strains (11). At the doses of 250 and 500 µg/disc of the test extract sterile blank discs (BBL, Cocksville, USA) were saturated using micropipette. Test extract was prepared using ethanol at the desired concentrations and control discs (blank) were also prepared using ethanol. Then both sample and control discs were dried. Using sterile forceps sample containing discs, standard antibiotic discs (Kanamycin 30 μg/disc, Oxoid Ltd, UK) and control discs were placed in Petri dishes containing nutrient agar medium seeded with the test pathogens. Then Petri dishes were transferred into incubator and incubated at 37 °C for 16 h. After incubation period, digital slide calipers were used to measure the zone of inhibition (11, 12).

Statistical Analysis

Student’s t-test was used to determine significant differences between the control and test group. Results were considered as statistically significant when P<0.01.

Results

Phytochemical Tests

The phytochemical screening of ethanol extract was performed for the detection of different biologically active chemical groups and the results are summarized in the Table1.

Table 1: Phytochemical screening of C. manghas

Phytochemical groups

Results

Reducing sugars

+

Alkaloids

+

Glycosides

+

Steroids

+

Gums

-

Saponins

-

Flavonoids

+

Tannins

+

+ = Presence - = Absence

Acetic Acid-Induced Writhing Test

The ethanolic extract exhibited statistically significant (p < 0.01) analgesic effect in acetic acid induced writhing of white albino mice (Swiss-webstar strain). The extract produced 17.75% and 30.64% writhing inhibition at the doses of 250 mg/ kg-body weight and 500 mg/kg-body weight respectively which was highly comparable to diclofenac sodium 79.03% (P<0.001) at the dose of 25 mg/kg body weight.

see Table 2.

DPPH Scavenging Activity

The extract showed DPPH radical scavenging activity on concentration dependent manner. The ethanol extract showed IC50 value of 292 µg/mL, which was highly comparable to the ascorbic acid, IC50 value of 15 µg/mL.

see Figure 1.

Antibacterial Activity in Disc Diffusion Assay

In antibacterial test, the extract, showed no activity against the bacterial strains namely S. aureus, E. coli, S. paratyphi, S. dysenteriae, S. typhi, and S. pyogens at the doses of 250 and 500 µg/disc.

see Table 3.

Discussion

The tests were conducted to assess some phytochemical and pharmacological properties of C. manghas. To get preliminary idea about the phytochemicals present in the extract phytochemical screening was performed which revealed the presence of reducing sugars, tannins, steroids, alkaloids, glycosides and flavonoids.

Acetic acid induced writhing test is a simple for evaluating in vivo analgesic activity in mice, more specifically for evaluating peripheral analgesic activity. In this test, sensitization of pain receptors by prostaglandins release is used to evaluate the analgesic activity of the sample acting peripherally (13, 14). The active principle responsible for the analgesic activity of extract may be terpenoids, reducing sugar, gums, xanthoprotein, flavonoids and tannins (15, 16, 17).

In vitro antioxidant activity study of the extract was done by most widely used DPPH scavenging assay model. In this test, the conversion of free radical, DPPH to stable DPPH-H by accepting electron, or hydrogen radical. As a result the deep violet colour of DPPH is converted to light yellow colour. Among the phytochemicals investigated by phytochemical screening, flavonoids and tannins are responsible for antioxidant properties (18, 19). ). The DPPH radical containing an odd electron is detected by UV spectrophotometer at 517 nm against blank, and due to the development of its non‐radical form, DPPH–H, upon reduction with an antioxidant this absorption decreases (20). The extract showed concentration dependent DPPH radical scavenging activity that was comparable to the standard antioxidant ascorbic acid.

In antibacterial test, the extract, showed no activity against the bacterial strains namely S. aureus, E. coli, S. paratyphi, S. dysenteriae, S. typhi, and S. pyogens at the doses of 250 and 500 µg/disc.

The investigations were carried out using crude extract of the plant. Further investigations might be conducted using the pure compounds of extract.

Conclusion

The ethanol leaves extract of C. manghas showed potential antioxidant and analgesic activity showed no antimicrobial activity which resembles its traditional uses. The results suggest further advanced investigations to find out the active compounds and Beximco Pharmaceuticals Ltd. for providing standard drugs..