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Phytochemical screening, cytotoxicity, antioxidant capacity and antibacterial potentiality of methanol extract of Antidesma ghaesembilla Gaertn.

Introduction

There is growing interest in exploiting plants for medicinal purposes especially in Asia. This stems from the fact that microorganisms are developing resistance to many drugs and as such created situation where some of the common & less expensive antimicrobial agents are loosing effectiveness (Mostefore D. et al 1989).Herbal medicine, which uses medicinal plants primarily present as an alternative to such situation (Sofowara A. 1993).These medicinal plants have immensely contributed to the development of human health & welfare. Concomitantly, there is an increase in data and huge patronage to herbal products round the world (Omoseyindemi B.X., 2003). Medicinal plants such as Antidesma ghaesembilla Gaertn. has been asserted to provide various culinary & medicinal properties. The acceptance of traditional medicine as an alternative form of health care and the development of microbial resistance to the available antibiotics has led researchers to investigate the antimicrobial herbal extracts (Bisignano G. et al, .2000, Hammer K.A. et al, .1999).There are many reports on antibacterial activity of various plants growing in different parts of this region.

Description and Medicinal Uses

Antidesma ghaesembilla also known as black currant tree (Family- Euphorbiaceae) grows extensively in deciduous or mixed dry forests in tropical parts of Central and East Asia starting from India to Papau New Guinea also available in Bangladesh and locally named as Khudijam. This tree can even also be seen in North Australia. It is also known as jhondri, umtea, and a small to medium tree up to 20 m tall; young twigs pubescent. Leaves oblong, more rarely ovate or obovate, (2-) 3-7(-16) x (2-)3-5(-9) cm, Fruit an ellipsoid drupe, laterally compressed, 3-4(-5) x 2.5-3(-3.5) mm, sparsely pubescent; style terminal to more rarely sub terminal; fruits borne on spike and ripen over a period of a few weeks and therefore harvested in more than one picking; fruits taste sour due to dominance by acids in spite of the presence of sugar. This plant widely used as a medicine for headaches; the stem is used as a medicine to stimulate the menstrual flow. The fruit is eaten locally or used as a purgative. Lactating women eat the leaves for breast milk production.

Materials and methods

Collection of Plant Material and Extraction:

After cutting and slicing, the collected plant sample was dried in the sun as well as in a mechanical dryer at 60 to 70[degrees]C. The dried sample was ground to coarse powder with a mechanical grinder and powdered sample were kept in clean closed glass container. The dried sample was then extracted by methanol with a volume of 800 ml for 3 days for allowing total extraction process. After that the Antidesma ghaesembilla plant extract was filtered with sterilized cotton filter and the filtrate was collected in a beaker. The plant extract then kept in a water bath at 60[degrees]C to evaporate the solvent from the solution. The container allowed to airtight for 72 hours and filtrate thus obtained was concentrated by using a rotary evaporator

Chemicals and Drugs:

DPPH (1, 1-diphenyl, 2-picrylhydrazyl), TCA (trichloroacetic acid) and ferric chloride were obtained from Sigma Chemical Co. USA; Ascorbic acid was from SD Fine Chem. Ltd. India, ammonium molybdate from Merck, Germany.

Phytochemical Screening:

To identify the chemical constituents of plant extract standard procedures are followed. Freshly prepared crude extract of A. ghaesembilla were qualitatively tested for the presence of chemical constituents using the following reagents and chemicals: flavonoids with the use of Mg and HCl; tannins with ferric chloride and potassium dichromate solutions and saponins with ability to produce stable foam and steroids with Libermann Burchard reagent, reducing sugars with Benedict's reagent and observed color change in respective (Ghani, A. 2003)..

Determination of Total Phenolic Content:

Folin-Ciocalteu method was used to determine the total phenolic content; Folin-Ciocalteu oxidized the extract whereas sodium carbonate neutralized it (Singelton V.R. et al 1999). Blue color formed and the absorbance was measured at 760 nm after 60 min by using gallic acid (GA) as standard. Total Phenolic content was expressed as mg GA equivalent/gm of extract.

Determination of Total Flavonoid Content:

Kumaran and Karunakaran method was followed to determine the flavonoid content (Kumaran A. et al., 2007) where quercetin is used as standard. 1 mg of plant extract in methanol was mixed with 1 ml of aluminium trichloride in ethanol (20 mg/ml) and a drop of acetic acid added. Then diluted up to 25 ml with ethanol and measured the absorbance at 415 nm after 40 min. The absorption of blank samples and standard quercetin solution (0.5 mg/ml) in methanol was measured under the same conditions.

DPPH Radical Scavenging Activity:

The free radical scavenging capacity of the plant extract was determined using DPPH (Hasan M.S. et al., 2006, Alam M.A. et al., 2008). The solution of methanol DPPH (0.004% w/v) was mixed with serial dilution (0 to 500 [micro]g) of A. ghaesembilla extract. The absorbance was taken at 517 nm by using the spectrophotometer after 10 min. Ascorbic acid used as standard then plotted the inhibition curve and calculated the [IC.sub.50] value.

Reducing Power:

The reducing power of A. ghaesembilla extract was determined according to the method of Oyaizu (Oyaizu M. 1986). Different concentration of A. ghaesembilla extract in 1 ml of distilled water was mixed with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and potassium ferricyanide [[K.sub.3]Fe [(CN).sub.6]] (2.5 ml, 1%) then the mixture was incubated at 50[degrees]C for 20 min. Trichloroacetic acid (10%) slightly added (2.5 ml) to the mixture and centrifuged at 3,000 rpm for 10 min. The upper layer of the solution (2.5 ml) was mixed with distilled water (2.5 ml) and Fe[Cl.sub.3] (0.5 ml, 0.1%) then taken the absorbance at 700 nm. The reference standard was Ascorbic acid and the blank solution contained phosphate buffer.

Cupric Reducing Antioxidant Capacity (CUPRAC):

Cupric ion reducing capacity was determined according to the method of Resat (Resat, A. et al., 2004). In this procedure, Cu[Cl.sub.2].2[H.sub.2]O solution, ammonium acetate buffer (pH 7.0), neocaproin solution and distilled water were added to A. ghaesembilla extract to adjust the final volume to 4.1 ml. The mixture was incubated for 1 hour at room temperature and the absorbance was measured at 450 nm using a spectrophotometer against blank solution. Ascorbic acid was used as a standard.

Brine Shrimp Lethality Bioassay:

Brine shrimp lethality bioassay was used for testing cytotoxic potential of the extract (Meyer et al., 1982, Persoone G. et al., 1988). The eggs of Brine shrimp (Artemia salina Leach) were collected and hatched in a tank at a temperature around 37[degrees]C with continuous oxygen supply. Two days were allowed to hatch and mature the nauplii. Stock solutions of the sample were prepared by dissolving required amount of extract in specific volume of pure dimethyl sulfoxide (DMSO). 4 ml of seawater was given to each of the vials. Then specific volume of sample was transferred from the stock solution to the vials to get final sample concentrations of 3.125, 6.25, 12.5, 25, 50, 100, 200 and 400 [micro]g/ml. In the control vials same volumes of DMSO (as in the sample vials) were taken. With the help of a Pasteur pipette 10 living nauplii were put to each of the vials. After 24h the vials were observed and the number of nauplii survived in each vial was counted. After that, the percentage of lethality of Brine Shrimp nauplii was calculated for each concentration of the extract.

Antibacterial Assay:

The antimicrobial assay was performed by using the disc diffusion method (Bauer A.W., et al., 1996, Barry A.L. et al., 1980). Seven pathogenic bacteria were used as test organisms for antibacterial activity of dried sample extract. The bacterial strains were collected from BCSIR Chittagong, Bangladesh. 0.5 mg/disc of the sample extract were used to observe the antimicrobial activity of the plant extract and compared with the standard kanamycin (0.1 [micro]g/disc). The test organisms were inoculated on 10 ml previously sterilized nutrient agar media, mixed thoroughly and transferred immediately to the sterile Petri dish in an aseptic condition using a sterile loop. Prepared sample and standard solutions were applied to the corresponding Petri dish. The plates were incubated for overnight at 37[degrees]C. After proper incubation, clear zone of inhibition around the point of application of sample solution were measured which is expressed in millimeter (mm).

Results and discussion

Phytochemical Screening:

Phytochemical analyses of the crude extract revealed the absence of carbohydrate steroid saponin, alkaloid and presence of glycoside, tannin and resin which has been shown in Table 01.

Total Phenol and Flavonoid Content:

The total phenol and total flavonoid contents of Antidesma ghaesembilla of methanol extract were expressed in gallic acid and quercetin equivalents respectively that are shown in Table 02. The content of phenolics of the extract under this investigation showed moderate result 120.818mg/g GAE and the amount of flavonoid was 95.72-mg/g quercetin equivalent.

Different studies suggest that different types of polyphenolic compounds such as flavonoids, phenolic acids which are found in plants have multiple biological effects, including antioxidant activity (Vinson et al.,1995).

DPPH Radical Scavenging Activity:

In case DPPH radical scavenging activity of methanol extract of Antidesma ghaesembilla shown in Figure 01. Where increasing the concentration of the extract, activity was found to increase remarkably. And the inhibitory capacity of the plant extract was comparatively lower than the ascorbic acid.

[FIGURE 1 OMITTED]

Furthermore the methanol extract of Antidesma ghaesembilla showed moderate to good DPPH scavenging activity in which [IC.sub.50] value for the plant extracts was 632.528[micro]g m[L.sup.-1]. In this test, Ascorbic acid was used as the reference antioxidant and the [IC.sub.50] value for ascorbic acid was 13.37-[micro]g m[L.sup.-1].

Cupric Reducing Antioxidant Capacity (CUPRAC):

[FIGURE 2 OMITTED]

The cupric reducing power of the plant extract shows good increase at the beginning while increasing the concentration and the increasing trend sustain with concentration. The reducing properties are generally associated with the presence of reductones, which have been shown to exert antioxidant action by breaking the free radical chain by donating a hydrogen atom (Duh et al., 1999).

[FIGURE 3 OMITTED]

Figure 03 the plant extracts was identified by using the potassium ferricyanide reduction method. The reductive capabilities of this extract were compared with ascorbic acid, which demonstrated at figure 03. The reducing power of the extract was moderately strong while increasing dose it shows steady state condition.

Brine Shrimp Lethality Bioassay:

The Methanol extract of Antidesma ghaesembilla was tested for Brine shrimp lethality bioassay by using brine shrimp nauplii and DMSO as a solvent. Control was used to see whether DMSO had any effect on brine shrimp lethality or not. The control group of brine shrimp nauplii with and without DMSO exhibited no mortality. For the extract, the number of nauplii died and percent mortality was counted. The result is shown in the following table:

Antibacterial Assay:

Antibacterial activities of the extract were tested against seven pathogenic bacteria and were compared with the standard antibiotic kanamycin by measuring the zone of inhibition diameter and expressed in millimeter (mm) showed in table IV.

In the antimicrobial screening, the extract showed average zone of inhibition 0-16 mm (Table IV) at concentration 400 [micro]g/disc to 1200 [micro]g/disc. No zone of inhibition was noticed against the growth of Shigella sonnei, Vibrio cholera, Bacillus megaterium at concentration 400 [micro]g/disc. The concentrations at 1200 [micro]g/disc, showed markable effect against number of bacteria.

Conclusion:

In conclusion, the present study, using in vitro experiments established that MEAG distinctly inhibit the bacterial growth, In case of anti cancer drug preparation this plant extract may treated as a good candidate as it has notable cytotoxic effect. On the other hand to minimize the free radical production MEAG may perform considerable role because of its moderate to good anti oxidant capacity. This is only a preliminary study but 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.

Refferences

Alam, M.A., M.A.B. Nyeem, M.A. Awal, M. Mostofa, M.S. Alam, N. Subhan and M.M. Rahman, 2008. Antioxidant and hepatoprotective action of the crude methanolic extract of the flowering top of Rosa damascena. Oriental Pharmacy and Experimental Medicine, 8: 164-170.

Barry, A.L., 1980. Procedures for testing antimicrobial agents in agar media. Antibiotics in laboratory medicine; (V.Lorian Ed.); Willams and Wilkins Company; Baltimore; USA, pp: 1-23.

Bauer, A.W., W.M.M. Kirbey, J.C., 1996. Sherries and M. Truck: Antibiotic susceptibility testing by standardized single disc method. Am. J. Clin. Pathol., 45: 493-496.

Bisignano G., R. Sanogo, A. Marino, R. Aquino, V. D'Angelo, M.P. Germand, R. De Pasquale and C. Pizza, 2000. Antimicrobial activity of Mitracarpus scaber extract and isolated constituents. Lett. Appl. Microbiol., 30: 105-108.

Duh, P.D., Y.Y. Tu and G.C. Yen, 1999. Antioxidant activity of the aqueous extract of harng Jyur (Chrysanthemum morifolium Ramat). Lebensmittel-Wissenschaft und Technol., 32: 269-277.

Ghani, A., 2003. Medicinal Plants of Bangladesh, The Asiatic Society of Bangladesh, 2nd Revised Edn., Dhaka, Bangladesh, ISBN: 9845123481, pp: 603.

Hammer, K.A., C.F. Carson and T.V. Riley, 1999. Antimicrobial activity of essential oils and other plant extracts J. Appl. Microbiol., 86: 985-990.

Hasan, M.S., M.I. Ahmed, S. Mondal, S.J. Uddin, M.M. Masud, S.K. Sadhu, M. Ishibashi, 2006 Antioxidant, antinociceptive activity and general toxicity study of Dendrophthoe falcata and isolation of quercetin as the major component. Orient. Pharm. Exp. Med., 6: 355-360.

Kumaran, A., A.J. Karunakaran, 2007. In vitro antioxidant activities of methanol extracts of five Phyllanthus species from India. LWT, 40: 344-352.

Meyer, B.N., N.R. Ferrigni, J.E. Putnam, L.B. Jacobsen, D.E. Nichols and J.L. Mclaughlin, 1982. Brine shrimp: A convenient bioassay for active plant constituents. Planta Med., 45: 31-34.

Mostefore, D., Y.O. Rotimi, F.A. Adeyemi-Doro, 1989. The problem of Antibacterial resistance to antibiotics among strains from hospital patients in Lagos and Ibadan. Niger. J. Antimicro. chemother, 23: 604.

Omoseyindemi, B.X., 2003. Plants as natural medicine. Paper presented at the annual conference of botanical society of Nigeria (boson) university of lagos.

Oyaizu, M., 1986. Studies on product of browning reaction prepared from glucose amine. Jpn. J. Nutr., 44: 307-315.

Persoone, G., 1988. Proceeding of the international symposium on brine shrimp; Artemia salina; University press, Belgium, pp: 1-3.

Resat, A., G. Kubilay, O. Mustafa and E.K. Saliha, 2004. Novel Total Antioxidant Capacity Index for Dietary Polyphenols and Vitamins C and E, Using Their Cupric Ion Reducing Capability in the Presence of Neocuproine: CUPRAC Method. J. Agric. Food Chem., 52: 7970-7981.

Singelton, V.R., R. Orthifer, R.M. Lamuela-Raventos, 1999. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Methods in Enzymol., 299: 152-178.

Sofowara, A., 1993. Medicinal Plants and traditional medicine.Spectrum books Ltd. Ibadan, pp: 224-227.

Vinson, J.A., Y.A. Dabbagh, M.M. Serry and J. Jang, 1995. Plant flavonoids, especially tea flavonols are powerful antioxidants using an in vitro oxidation model for heart disease. J. Agric. Food Chem., 43: 2800-2802.

Corresponding Author: Md. Razibul Habib, Lecturer, Department of Pharmacy, International Islamic University Chittagong, Bangladesh. Email: mrhjewel@gmail.com; cell: +8801618423453

(1) Md. Razibul Habib, (1) Md. Mominur Rahman, (2) Kaiser Hamid, (1) Md. Obayed Raihan, (1) Mohammed Aktar Sayeed

(1) Department of Pharmacy, International Islamic University Chittagong, Bangladesh.

(2) Department of Pharmacy, East West University, Bangladesh.
Table 01: Result of phytochemical screening of methanol extract of
A. ghaesembilla.

Extract   Carbohydrate   Glycoside   Steroid   Alkaloid   Tanin
                         Saponin

MEAG      -              +           -         -          +

Extract   Resin

MEAG      +

MEAG denote for Methanol Extract of Antidesma ghaesembilla (+):
Present, (-): Absent.

Table 02: Total amount of plant phenolics and flavonoid content of
methanol extract of A. ghaesembilla.

Extract   Total phenol        Total flavonoid
          (in mg/g, Gallic    (in mg/g, quercetin
          acid equivalents)   Equivalents)

MEAG      120.818             95.72

Table 03: Cytotoxic potential of methanol extract of Antidesma
ghaesembilla

Test       Conc.           Log Conc.   % Mortality   LC50
solution   ([micro]g/ml)                             ([micro]g/ml)

MEAG       3.125           0.507181    30            25
           6.25            0.79588     40
           12.5            1.09691     40
           25              1.39794     50
           50              1.69897     60
           100             2.0000      70
           200             2.30103     100
           400             2.60206     100

Table 04: Antibacterial activity of methanol extract of Antidesma
ghaesembilla

Sample   Name of the Bacteria     Sample Extract
code                              Diameter of zone of inhibition (mm)
                                  of MEAG

                                  400             800
                                  [micro]g/disc   [micro]g/ disc

B 01     Shigella dysenteriae     7               12
B 02     Salmonella typhi         7               12
B 03     Pseudomonas aeruginosa   9               12
B 04     Vibrio Cholerae          0                9
B 05     Bacillus cereus          8               12
B 06     Shigella sonnei          0               10
B 07     Bacillus megaterium      0               10

Sample   Sample Extract
code     Diameter of zone of inhibition (mm)

         1200            Standard
         [micro]g/disc   (K-30) *

B 01     13              32
B 02     16              33
B 03     15              34
B 04     12              36
B 05     17              36
B 06     12              40
B 07     13              40

* Standard: Kanamycin, MEAG=Methanolic Extract Antidesma ghaesembilla
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Title Annotation:Original Article
Author:Habib, Razibul; Rahman, Mominur; Hamid, Kaiser; Raihan, Obayed; Sayeed, Mohammed Aktar
Publication:Advances in Natural and Applied Sciences
Article Type:Report
Geographic Code:9BANG
Date:Apr 1, 2011
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