INTRODUCTION:

Nature has been a source of medicinal agents for thousands of years and an impressive number of modern drugs have been isolated from natural sources, many based on their use in traditional medicine. Herbal medicine is the oldest from of healthcare known to mankind. India has rich medicinal plants flora of more than 7500 species¹.

Phytochemical compounds are found in plants that are not required for normal functioning of the body, but have a beneficial effect on health or play an active role in amelioration of diseases. This is due to increased awareness of the limited ability of synthetic pharmaceutical products to control major diseases and the need to discover new molecular structures as lead compounds from the plant kingdom. Plants are the basic source of knowledge of modern medicine. The basic molecular and active structures for synthetic fields are provided by rich natural sources².

In order to promote the use of medicinal plants as potential sources of antimicrobial compounds, it is pertinent to thoroughly investigate their composition and activity and thus validate their use. Some phytochemicals present in plants have antimicrobial activity allowing these plants to be studied and used for the development of new antimicrobial drugs³. The effectiveness of phytochemicals in the treatment of various diseases may lie in their antioxidant effects. Secondary plant metabolites are largely unexplored in ‘conventional’ animal production systems. In the past, plant metabolites were generally considered as sources of antinutritional factors. Recent bans and restrictions on the use of animal antibiotic growth promoters stimulated interest in bioactive secondary metabolites of plant source as alternative performance enhancers⁴.

Mimusops elengi L. (Sapotaceae) is a small to large evergreen tree found all over the different parts of India. It is cultivated in gardens as an ornamental tree. It has been used in the indigenous system of medicine for the treatment of various ailments⁵. Bark of Mimusops elengi possesses cardiotonic, alexipharmic, stomachic, anthelmentic and astringent activity. Phytochemical review reveals the presence of taraxerol, taraxerone, ursolic acid, betulinic acid, α-spinosterol, β-sitosterol glycoside, quercitol, lupeol, alkaloid, isoretronecyl tiglate and mixture of triterpenoid saponins in the bark of Mimusops elengi⁶.

The present study was undertaken to evaluate the phytochemical analysis, in-vitro antioxidant activity and antimicrobial activity of Mimusops elengi bark extracts.

MATERIALS AND METHODS:

PLANT MATERIAL:

The air dried powder of Mimusops elengi bark was provided by Amsar Pvt ltd.

TEST ORGANISMS:

Test organisms used for the study were obtained from National Chemical laboratory, Pune, Maharashtra.

REAGENTS AND CHEMICALS:

All reagents and chemicals used in the experiments were of analytical grade and obtained from Sigma Aldrich (USA). Solvents used for extraction of plant material were of analytical grade. Ascorbic acid, DPPH (1,1-diphenyl-2-picrylhydrazyl), sodium nitroprusside, sulphanilic acid and a-naphthyl-ethylene diamine were purchased from Sigma Aldrich (USA).

EXTRACTION OF PLANT MATERIAL:

The air dried powder of Mimusops elengi bark was subjected to continuous hot extraction with methanol, ethyl acetate and petroleum ether (60-80) for 18 hours. Aqueous extract was prepared by refluxing the plant material with water for 8 hours. The extracts were concentrated using rotary vacuum evaporator below 40ºC. The dried extracts were stored in vacuum desiccator for further use.

PHYTOCHEMICAL ANALYSIS: ^7,8

Preliminary qualitative phytochemical screening of the aqueous and methanol extracts were carried out to know the different phytoconstituents present in Mimusops elengi bark as per the standard procedure.

Total Phenolic content:⁹

The amount of total phenolic and tannins in the plant extract was determined calorimetrically with the Folin-Ciocalteu reagent (FCR). The reaction mixture contained 50µl of the extract (1mg/ml) in methanol, 250µl of FCR, 750µl of sodium carbonate solution. The volume was made upto 5 ml with distill water and was incubated in dark under ambient conditions for 2 hours to complete the reaction. In the control tube, the extract volume was replaced by methanol.

The absorbance of the resulting solution was measured at 760nm in a UV spectrophotometer. The concentration of total phenolics and tannins was expressed as mg of gallic acid equivalents (GAE) per g of dried extract, using a standard curve of gallic acid. All the measurements were carried out in triplicates.

Total phenolic content was calculated using the following formula:

C=c×V/ m,

Where, C= total content of phenolic compounds in mg/g plant extract in GAE or mg GAE/g extract

c= the concentration of gallic acid established from the calibration curve in mg/ml

V= the volume of extract in ml; m= the weight of plant extract in g

Total Flavanoid content¹⁰

The flavonoids content in the plant extract was determined by aluminium chloride method using rutin as a reference compound. The 100µL of plant extract (10mg/ml) in methanol was mixed 100µL with 20% aluminium trichloride in methanol and a drop of acetic acid, and then diluted with methanol to 5ml. The absorption at 415nm was read after 40 minutes. Blank consists of 100μL of extract, a drop of acetic acid and adjusted to 5mL with methanol. The absorption of standard rutin solution (0.5mg/ml) in methanol was measured under the same conditions. All measurements were carried out in triplicates. The amount of flavonoids in the extract in rutin equivalents (RE) was calculated using following formula-.

X = (A. m_o)/(A_o.m)

Where, X is the flavonoid content of extract in mg/g extract in RE or mg RE/g extract, A is the absorption of plant extract solution, A₀ is the absorption of standard rutin solution, m is the weight of extract in mg and mis the weight of rutin in the solution in mg.

ANTIOXIDANT ACTIVITY

DPPH Free radical scavenging activity ^11,12

The free radical scavenging activity of the extracts were measured in terms of hydrogen donating or radical-scavenging ability using the stable radical DPPH (1,1- diphenyl- 2- picryl-hydrazyl). 0.1 mM solution of DPPH in methanol was prepared and 1.0 ml of this solution was added to 1.0 ml of dilutions of extracts at different concentrations (2-20 µg/ml). Thirty minutes later, the absorbance was measured at 517 nm. Ascorbic acid was used as standard. Lower absorbance of the reaction mixture indicates higher free radical scavenging activity. The capability to scavenge the DPPH radical was calculated using the following equation:

DPPH Scavenged (%) = (A _cont − A _test)/A _cont × 100

Where, A_cont is the absorbance of the control reaction and A_test is the absorbance in the presence of extract.

Nitric oxide (NO) radical scavenging activity¹³

NO radical inhibition was estimated by Griess assay. Sodium nitroprusside (10 mM, 1.5 ml) in phosphate –buffered saline (PBS) was mixed with 0.5 ml of different concentrations (10-100 µg/ml) of the extract dissolved in the suitable solvent systems and incubated at 25⁰C for 150 min. The sample mixtures were then reacted with 1 ml Griess reagent for 15 min (1% sulphanilamide, 2% H₃PO₄ and 0.1% napthylethylenediamine dihydrochloride). The absorbance of the chromophore formed during the diazotization of nitrite with sulphanilamide and subsequent coupling with napthylethylenediamine was read at 546 nm with reference to the absorbance of standard solutions of ascorbic acid, treated in the same way with Griess reagent.

The % of NO scavenging activity is calculated as follows:

Scavenging Effect (%) = (A _cont− A _test)/A _cont × 100

Where, A_contis the absorbance of the control reaction and A_testis the absorbance in the presence of the sample of the extracts.

ANTI- BACTERIAL ACTIVITY:

Two techniques were used to test the anti- microbial activity of Mimusops elengi extracts; ditch plate technique and agar well diffusion technique against the reference bacterial and fungal strains.

Strains tested:

Gram positive bacteria- Staphylococcus aureus (NCIM- 5022), Sterptococcus pyogens (NCIM- 2608), Clostridium perfringens (NCIM- 2677).

Gram negative bacteria- Pseudomonas aeroginosa (NCIM- 2200), Escherichia coli (NCIM- 2065), Klebsiella pneumonia (NCIM- 5082), Klebsiella aerogenes (NCIM- 2239)

Fungi- Candida albicans (NCIM- 3471), Aspergillus niger (NCIM- 1196)

Authentic pure cultures of bacteria were cultivated in nutrient broth (NB) or nutrient agar at 37 ± 0.2°C. Fungi and yeasts were cultured on sabouroud dextrose agar at 28 ± 0.2°C. The cultures of bacteria and fungi were maintained in their appropriate agar slants at 4°C throughout the study and used as stock cultures. Microbial cultures were suspended in a saline solution (0.85% NaCl) and adjusted to a turbidity of 0.5 MacFarland standards (10⁶ CFU/ml).

Ditch plate method:¹⁴

Ditch plate method is used for primary screening of extracts against various organisms. The antibacterial susceptibity of each organism to the test extracts is tested in this method.

In this technique 30ml of molten agar (nutrient agar/sabouroud dextrose agar) was poured into a sterile petri plate. The molten agar was allowed to set and harden at room temperature. Ditch (1cm×3 cm) was made in the agar plate using a sterile scalpal. Solution of each extract (1 ml solution containing 50 mg of extract) was placed in ditch. Loopful of inoculum of each organism (gram positive, gram negative and fungi) were streaked across the agar at right angle to the ditch. All the petri plates containing bacterial cultures were incubated at 37⁰C for 24 hours. Plates containing fungal cultures were incubated at 28⁰ C for 48 hours.

Agar well diffusion method:¹⁵

The extracts were then evaluated for antimicrobial activity using agar well diffusion method. Nutrient agar plates were seeded with 1 ml of bacterial suspension and Sabouroud dextrose agar plates with fungal suspension (equivalent to 10⁶cfu/ml). The seeded plates were allowed to set. A sterile cork borer of 11 mm diameter was then used to cut equidistant wells on the surface of the agar. The wells were filled with 0.4 ml solution of each extract at various concentrations (20, 40 and 80 mg/well). The plates were incubated at 37°C for 24 hours and 28 º C for 48 hours after which the diameter of zones of inhibition were measured. Chloramphenicol (100mg) and Fluconazole (15mg/well) were used as positive control.

STATISTICAL ANALYSIS

All experiments were carried out in triplicates. The values are expressed as mean ± SEM. Students paired t-test was performed to determine significance level. p ≤ 0.05 was considered significant.

RESULTS:

PHYTOCHEMICAL ANALYSIS

The qualitative phytochemical analysis of extracts of Mimusops elengi bark revealed the presence of carbohydrates, proteins, glycosides, alkaloids, tannins, phenols and saponins (Table 1).

Table No.1 Qualitative analysis of extracts of Mimusops elengi bark

Test	Aqueous extract	Methanol extract	Ethyl acetate extract	Pet ether extract
Carbohydrates	+	+	+	+
Proteins/amino acid	+	+	+	+
Glycosides	-	+	+	-
Saponins	+	+	-	-
Alkaloids	-	-	-	-
Tannins	+	+	+	+
Flavonoids	+	+	+	-
Steroids	-	+	+	+
Gums/mucilage	-	-	-	-
Fixed oil and fats	-	-	+	+

(+) = indicates presence, (-) = indicates absence

The total phenolic contents in Mimusops elengi plant extracts using the Folin-Ciocalteu’s reagent is expressed in terms of gallic acid equivalent. The values obtained for the concentration of total phenols are expressed as mg of GA/g of extract. The concentration of flavonoids in extracts of Mimusops elengi bark was determined using spectrophotometric method with aluminum chloride. The content of flavonoids was expressed in terms of rutin equivalent as mg of RU/g of extract.

The total phenolic contents in the extracts ranged from 29.767 to 102.874 mg GAE/g of extracts, whereas the total flavonoid were in the range of 13.876 to 36.682 mg of RU/g of extract. The highest concentration of phenols and flavonoids was measured in methanol extract followed by aqueous extract. Ethyl acetate and petroleum ether extract contains considerably smaller concentrations of phenols and flavonoids.

ANTIOXIDANT ACTIVITY:

DPPH Free radical scavenging activity:

DPPH is a stable free radical at room temperature and accepts an electron or hydrogen radical to become a stable diamagnetic molecule. The reduction capability of DPPH radicals was determined by the decrease in its absorbance at 517nm, which is induced by antioxidants. Mimusops elengi strongly scavenged the DPPH radical in a dose- dependent manner. It has been shown that the scavenging effects on the DPPH radical increases sharply with the increasing concentration of the extracts and standards to a certain extent and hence are said to be strongly dependent on the extract concentration. The IC₅₀ values of ascorbic acid, aqueous, methanol, ethyl acetate and pet ether extracts are shown in table 3.

Nitric oxide radical scavenging activity:

Nitric oxide radical generated from sodium nitroprusside (SNP) at physiological pH was found to be inhibited by extracts of Mimusops elengi. The scavenging of NO radical by the extracts was increased in dose dependent manner. Suppression of released NO may be partially attributed to direct NO scavenging, as the extracts of Mimusops elengi decreased the amount of nitrite generated from the decomposition of SNP in vitro. The IC₅₀ values of ascorbic acid, aqueous, methanol, ethyl acetate and pet ether extracts are shown in table 3.

Table No. 3 Antioxidant activity of Mimusops elengi bark extracts and ascorbic acid

Sample	IC50 (µg/ml)
Sample	DPPH Free radical scavenging activity	Nitric oxide radical scavenging activity
Aqueous extract	5.76	43.370
Methanol extract	2.95	34.847
Ethyl acetate extract	16.37	39.736
Pet ether extract	29.45	98.969
Ascorbic acid (standard)	11.181	27.117

ANTIMICROBIAL ACTIVITY:

The antimicrobial potential of extracts was compared according to their zone of inhibition against the several pathogenic organisms. Antimicrobial screening of different extracts of Mimusops elengi by ditch plate and cup plate method are given below.

Ditch plate method:

The extracts did not show susceptibility towards all strains of microorganisms. Aqueous and methanol extract was susceptible to S. aureas, E.coli, K. pneumonia, K. aerogens, S.pyogens and C. perfringes. Methanol extract was also susceptible to P.aeroginosa. Ethyl acetate extract showed low susceptibility towards very few bacterial strains. Petroleum ether extract did not posses any antibacterial activity. All the extracts were inactive towards the fungal strains.

All the extracts at different concentrations were analyzed further by agar cup late technique.

Cup plate method:

Table No. 4 Antimicrobial activity of extracts Mimusops elengi by Cup plate method

Organisms	Zone of Inhibition (mm)
	Std 100µg	Methanol extract			Std 100µg	Aqueous extract
	Std 100µg	50 mg	100 mg	200 mg	Std 100µg	50 mg	100 mg	200 mg
S. aureus	10.8 ± 0.02	4.1 ± 0.12	10.2 ± 0.01	12.1 ±0.04	10 ± 0.12	4.7 ± 0.13	6.7 ± 0.14	9.8 ± 0.13
E. coli	12.2 ± 0.10	3.4 ± 0.01	12.1 ±0.12	14.8 ±0.12	12.2 ± 0.16	8.3 ± 0.11	11.2 ± 0.12	16.2 ± 0.22
K. pneumoniae	14.7 ± 0.1	10.1 ± 0.1	13.1 ± 0.13	16.8 ± 0.12	13.7 ± 0.01	6.5 ± 0.12	10.2 ± 0.13	13.2 ± 0.12
K. aerogens	11 ± 0.12	6.5 ± 0.14	10.1 ±0.12	12.1 ± 0.02	11.2 ±0.12	-	-	-
P. aeruginosa	12.2 ± 0.12	8.1 ± 0.1	12.2 ± 0.21	14.7 ± 0.03	12.2 ± 0.13	3.4 ± 0.1	8.3 ± 0.12	12.1 ± 0.02
S. pyogens	10 ± 0.03	4.7 ± 0.15	9.8 ± 0.13	11.2 ± 0.12	10.7 ± 0.04	-	-	-
C. perfringens	13.8 ± 0.13	7.5 ± 0.12	13.2 ± 0.1	16.1 ± 0.14	16.8 ± 0.14	8.1 ± 0.12	14.8 ± 0.1	15.5 ± 0.15
C. albicans		-	-			-	-	-
A. niger		-	-	-		-	-	-

Table-4 continue..

Organisms	Zone of Inhibition (mm)
	Std 100µg	Ethyl acetate extract			Std 100µg	Pet ether extract
	Std 100µg	50 mg	100 mg	200 mg	Std 100µg	50 mg	100 mg	200 mg
S. aureus	10 ± 0.21	4.8 ± 0.21	6.8 ± 0.15	9.7 ± 0.1	10 ± 0.2	-	-	-
E. coli	12.2 ± 0.16	3.4 ± 0.12	8.3 ± 0.13	12.1 ± 0.21	12.2 ± 0.1	-	-	-
K. pneumoniae	13.7 ± 0.2	6.5 ±0.13	10.2 ± 0.2	13.2 ± 0.12	13.7 ±0.12	-	-	-
K. aerogens	11 ± 0.13	-	-	-	11.2 ± 0.1	-	-	-
P. aeruginosa	12.2 ± 0.13	8.3 ± 0.21	10.2 ± 0.13	12.2 ± 0.11	12.2 ± 0.13	-	-	-
S. pyogens	10.8 ± 0.14	2.3 ± 0.12	6.6 ± 0.14	10.2 ± 0.2	10.7 ± 0.2	-	-	-
C. perfringens	13.8 ± 0.16	7.5 ± 0.2	11.2 ± 0.21	13.2 ± 0.13	16.8 ± 0.13	-	-	-
C. albicans	-	-	-	-		-	-	-
A. niger	-	-	-	-		-	-	-

N=3, values are expressed as mean ± SEM.

As seen in table 4, aqueous, methanol and ethyl acetate extracts obtained from Mimusops elengi bark have been shown to possess mild to moderately antibacterial activity against most of the tested bacteria. All the extracts were ineffective towards fungal strains. No inhibitory effect of petroleum ether extract was observed against all strains of bacteria and fungi indicating absence of antimicrobial activity. The results were compared with those of Chloramphenicol as a standard antibacterial. Methanol extract showed highest activity against K. pneumoniae, aqueous extract against E-coli and ethyl acetate extract against C. perfringes. On overall consideration, the antibacterial activity of extracts was in order of methanol > aqueous > ethyl acetate. However preliminary phytochemical analysis of the methanol extract revealed the presence of phenol, saponins, flavonoids and carbohydrates. The antimicrobial potency of the extract may be attributed to the single or combined effect of the above mentioned chemical groups.

DISCUSSION:

In this study aqueous, methanol, ethyl acetate and petroleum ether extracts of Mimusops elengi were taken for analysis, the extraction of antioxidant substances of different chemical structure was achieved using solvents of different polarity. Numerous investigations of qualitative composition of plant extracts revealed the presence of high concentrations of phenols and flavonoids in the extracts obtained using polar solvents. Phenols and flavonoids are very important plant constituent because of their scavenging ability on free radicals due to their hydroxyl group. Therefore the phenolic and flavonoid content of plant may contribute directly to their antioxidant action. Methanol extract exhibited higher antioxidant activity in both the antioxidant activity performed (DPPH and NO).

The extracts were also quantified for their total phenolic and total flavonoid content. The total phenolic and flavonoid content in the extract of Mimusops elengi depends upon the type of extract, i.e the polarity of solvent used for extraction. High solubility in polar solvents provides high concentration of these compounds (phenols and flavonoids) in the extracts obtained using polar solvents for extraction. The quantity of flavonoid and phenolics were higher in methanol extract. This makes us to interpret that Mimusops elengi bark posses promising health beneficial and phytochemicals. It is widely accepted that phenolic and flavonoid compounds may significantly contribute to overall antioxidant and antimicrobial properties. The antimicrobial activity for methanol extract was also high against the all tested strains of bacteria compared to other extracts. Results also indicated that the polarity of the solvent plays an important role in the extraction of the active ingredient and consequently on its antimicrobial and antioxidant activity.

The results obtained in the present investigation represent a worthwhile expressive contribution to the antimicrobial and antioxidant activity of extracts of Mimusops elengi bark. This study may be a lead for further ethnopharmacognostic investigation to identify and isolate new compounds with therapeutic promise.

ACKNOWLEDGEMENT:

We are thankful to Amsar Goa Pvt. Ltd., Goa for providing financial support.

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Received on 21.08.2014 Modified on 05.09.2014

Research J. Pharm. and Tech. 7(11): Nov. 2014 Page 1226-1230

Sr. no	*Mimusops elengi* extract	Total phenolic content (mg GAE/g) of extract	Total flavonoid content (mg RE/g) of extract/
1.	Aqueous extract	102.874 ± 1.231	36.682 ± 0.519
2.	Methanol extract	104.47 ± 1.653	39.077 ± 1.08
3.	Ethyl acetate extract	78.165 ± 1.402	29.476 ± 0.654
4.	Petroleum ether extract	29.767 ± 0.658	13.876 ± 1.179