INTRODUCTION:

The use of plant as medicine is as old as human civilization. Exploration of this traditional knowledge for curing common diseases is an attractive prospect ^(1). Plants have the ability to synthesize a wide variety of chemical compounds that are used to perform important biological functions, and to defend against the microorganisms. Many of these phytochemicals have beneficial effects on long term usage by humans and can be used to effectively treat human diseases. At least 12,000 such compounds have been isolated so far; a number estimated to be less than 10% of the total compounds ⁽²⁾. Most of the countries have a tradition of using medicinal plants for widely distributed ailments ^(3).

Traditional medicine using plant extracts continues to provide health coverage for over 80% of the World’s population, especially in the developing countries ⁽⁴⁾. Modern medicine now tends to use the active ingredients of plants rather than the whole plants. The phytochemicals may be synthesized, compounded or otherwise transformed to make pharmaceuticals. Few traditional remedies, however, have translated into modern drugs, although there is continuing research into the efficacy and possible adaptation of traditional herbal treatments ⁽⁵⁾. In recent years, plant derived secondary metabolites (phytochemicals), previously with unknown pharmacological activities have been extensively investigated as a source of medicinal agents ⁽⁶⁾. Thus, it is anticipated that phytochemicals with adequate antibacterial efficacy will be used for the treatment of bacterial infections ⁽⁷⁾. Since time immemorial, man has used different parts of plants for the treatment and prevention of various ailments ⁽⁸⁾. The main objective of this study to find out the presence of phytochemical constituents and evaluate the antibacterial activity of the various extracts of Glossocardia bosvallia against Gram-positive and Gram negative bacterial strains in in-vitro.

MATERIALS AND METHODS:

Fresh plants of Glossocardia bosvallia was collected from Madukarai Hills, Southern Western Ghats of Coimbatore District, Tamilnadu, South India. A voucher specimen was deposited in the Herbarium Botanical Survey of India Coimbatore for authentication of plant. After authentification plants were collected in bulk, washed, shade dried and extracted with petroleum ether, chloroform, methanol and aqueous for 48 h sequentially extracted in a soxhlet apparatus. The crude extracts were obtained and stored in room temperature for further usage.

Qualitative Method of Phytochemical Screening:

The petroleum ether, chloroform, methanol and aqueous extracts were analyzed for alkaloids flavonoids, phlobatannins, glycosides, phenols, saponins, lipids and fat, tannins, anthraquinones, quinines, cardiac glycosides, coumarines acids, steroids, phytosterols, proteins and carbohydrates ^{(9, 10, and 11)}.

Detection of Alkaloids:

About 50 mg of Solvent free extract was stirred with 3 ml of dilute hydrochloric acid and then filtered thoroughly. The filtrate was tested carefully with various alkaloids reagents as follows:

Mayer’s test-

To a 1 ml of filtrate, few drops of Mayer’s reagent are added by the side of the test tube. The white or creamy precipitate indicated test as positive.

Wagner’s test-

To a 1 ml of filtrate, few drops of Wagner’s reagent are added by the side of the test tube. The color change was observed. A reddish-brown precipitates confirms the test as positive.

Dragendorff’s test-

To a 1 ml of filtrate, 2 ml of Dragendorff’s reagent are added and the result was observed carefully. A prominent yellow precipitate confirms the test as positive

Detection of Carbohydrate:

Fehlings test-

One ml of extract was boiled on water bath with 1 ml each of Fehling solutions A and B. The color change was observed. A red precipitates indicated presence of sugar.

Barfoed’s test-

To 1 ml of extract, 1 ml of Barfoed’s reagent was added and heated on a boiling water bath for 2 minutes. The color change was noted and recorded. A red precipitates indicated presence of sugar.

Benedict’s test-

To 0.5 ml of extract, 0.5 ml of Benedict’s reagent was added. The mixture is heated on a boiling water bath for 2 minutes and the result was observed. A red precipitates indicated presence of sugar.

Detection of Glycosides:

Legals test-

Chloroform (3ml) and ammonia solution (10%) was added to 2ml plant extract. Formation of pink color indicated the presence of glycosides.

Detection of Proteins-

The extract was dissolved in 10 ml of distilled water and filtered through Whatmann No.1 filter paper and the filtrate is subjected to test for protein and amino acids.

Millon’s test-

To 2 ml of filtrate, few drops of Millon’s reagent are added. The result was observed. A white precipitates indicated presence of protein.

Biuret test-

An aliquot of 2 ml of filtrate was treated with drop of 2% copper sulphate solution. To this, 1 ml of ethanol (95%) was added, followed by excess of potassium hydroxide pellets. The pink color in ethanol layer indicated presence of protein.

Detection of Amino acid:

Ninhydrin test-

Two drops of ninhydrin solution (5 mg of ninhydrin in 200 ml of acetone) are added to two ml of aqueous filtrate. The color change was observed. A characteristic purple color indicated the presence of amino acids.

Detection of Phytosterols:

Liberdmann-Burchard’s test-

The extract (5 mg) was dissolved in 2 ml acetic anhydride and one or two drops of concentrated sulphuric acid was added slowly along the sides of the test tube. The formation of blue green color indicated the presence of triterpinoides and phytosteroids.

Detection of Tannin:

Ferric chloride test-

The extract (5 mg) was dissolved in 5 ml of distilled water and few drops of neutral 5% ferric chloride solution were added. The formation of blue green color indicated the presence of tannins.

Detection of Phenol:

Lead acetate test-

The extract (5 mg) was dissolved in distilled water and 3 ml of 10% lead acetate solution was added. A bulky white precipitates indicated the presence of phenols.

Detection of Flavonoids:

An aqueous solution of the extract was treated with ammonium hydroxide solution. The yellow fluorescence indicated the presence of flavonoids.

Detection of Coumarin:

10% NaOH (1ml) was added to 1 ml of the plant extracts to formation of yellow color indicated presence of coumarin.

Detection of Saponin:

Distilled water 2 ml was added of each plant extracts and shaken in a graduated cylinder for 15 minutes lengthwise. Formation of 1cm foam indicates the presence of saponin.

Detection of Quinone:

Concentrated sulphuric acid (1 ml) was added to 1 ml of each of the plant extract. Formation of red color indicated the presence of Quinone.

Detection of Cardiac glycosides:

Glacial acetic acid (2 ml) and few drops of 5% ferric chloride were added to 0.5% of the extract. This was under layered with 1 ml of concentrated sulphuric acid. Formation of brown ring at the interface indicated presence of cardiac glycosides.

Detection of Terpenoid:

Chloroform (2 ml) and concentrated sulphuric acid was added carefully to 0.5 ml of extract. Formation of red brown color at the interface indicated the presence of terpenoid.

Detection of Acids:

Plant extract 0.5 ml was treated with sodium bicarbonate solution. Formation of effervescence indicated presence of acids.

Detection of Phlobatannins:

Few drops of 10% ammonia solution were added to 0.5 ml of root extract. Appearance of pink color precipitates indicated the presence of phlobatannins.

Detection of Anthraquinones:

Few drops of 2% HCL were added to 0.5 ml of root extract. Appearance of red color precipitate indicated presence of anthraquinones.

Detection of Steroids and Phytosteroids:

To 0.5 ml of the plant extract equal volume of chloroform was added and subjected with few drops of concentrated sulphuric acid. Appearance of brown ring indicates the presence of steroids and appearance of bluish brown ring as phytosteroids.

Detection of Fixed oils:

A small quantity of extract was pressed between two filter papers. Oil stain on the paper indicated the presences of fixed oil.

Detection of Fat:

Saponification test-

A few drops of 0.5 N alcoholic potassium hydroxide solutions are added to small quantity of extract along with drop of phenolphthalein. The mixture was heated on water bath for 2 h. Formation of soap or partial neutralization of alkali indicated the presence of fixed oils and fats.

Detection of Gum and Mucilage:

The extract was dissolved in 5 ml of distilled water and to this 25 ml of absolute alcohol was added with constant stirring. White or cloudy precipitate indicated the presence of gums and mucilage.

In Vitro Antibacterial Activity:

Antibacterial screening-

The in vitro antibacterial activity of the G. bosvallia extracts were determined by using disc diffusion method. Two gram positive bacteria Streptococcus faecalis, Bacillus subtilis and four gram negative bacteria Staphylococcus aureus, Klebsiella pneumonia, Salmonella paratyphi, Pseudomonas aeruginosa, were used for this study. The bacteria were subcultured on nutrient broth, incubated at 37°C for 24 h and stored at 4°C in the refrigerator to maintain stock culture. Petri plates were prepared with 20 ml of sterile nutrient agar medium. The test cultures were swabbed on the top of the solidified agar media and allowed to dry for 10 minutes. The discs of 6 mm diameter were prepared from Whatman filter paper No. 1 and sterilized. The sterilized discs were then impregnated with respective petroleum ether, chloroform, methanol and aqueous extracts and placed on the surface of the medium and left for 30 minutes at room temperature for compound diffusion ⁽¹²⁾. Tetracycline (1 mg/ml) was used as positive control. The plates were incubated for 24 h at 37°C. The diameter of the zone of inhibitions was measured by measuring scale in millimeter ⁽¹³⁾. The sensitivity of the microorganisms to plant extract was determined by measuring the size of inhibitory zones on the agar surface around the discs ^{(14, 15)}.

Statistical analysis:

Triplicates were maintained in all experiments and means were segregated using Duncan’s Multiple Range Test (DMRT). Significant differences were recorded at 5% level (P=0.5).

RESULTS:

Phytochemical analysis:

Qualitative phytochemical analysis of various chemical petroleum ether, chloroform, methanol and aqueous extracts of the ayurvedic herbal plant Glossocardia bosvallia showed the presence of most important phytoconstituents. The medicinal value of this plant can be correlated due to the presence of various bioactive chemical constituents (Table 1). Petroleum ether extract confirms the presence of phytosterols, saponins, terpenoids, steroids/phytosteroids, fat and gum/mucilage. Alkaloids, carbohydrates, glycosides, tannin, phenol, flavonoid, coumarin, saponin, quinine, cardiac glycosides and anthraquinones were absent where as proteins, amino acids, acids and phlobatannins were not noticeable. Chloroform extract gave positive result to phytosterols, tannins, steroids/phytosteroids and negative results to carbohydrates, glycosides, cardiac glycosides and anthraquinones. Phytoconstituents like alkaloids, proteins, amino acids, saponins, acids, phlobatannins, fats, gum and mucilage were not evident. Methanol extract showed significant result to more phyotochemical constituents like carbohydrates, tannin, flavonoid, coumarin, saponin, quinine, cardiac glycosides, terpenoid, phlobatannins, steroid/phytosteroids and fat. Alkaloids, glycosides, proteins, amino acids, phytosterols, phenol, acids were not perceptible and anthraquinone showed insignificant to the analysis. Aqueous extract was substantiate to carbohydrate, tannin, phenol, coumarin, saponin, cardiac glycosides, phlobatannins and gum/mucilage. Alkaloids, protein, amino acids, phytosterols, terpenoid, acids, steroid/phytosteroids and fats were not traceable. Glycosides, flavonoids, quinine and anthraquinone were lacking of this extract. The fixed oils showed strong positive indication to all the four extracts.

In vitro antibacterial activity

The in vitro antibacterial activity result of petroleum ether, chloroform, methanol and aqueous extracts of the medicinal herb Glossocardia bosvallia showed varied activity against the different pathogens used (Table 2). It was found that the zones of inhibition mm were ranged between 6.7 to 33.6 mm. Bacterial strain Streptococcus faecalis showed highest inhibition (29.3±1.2) in methanol extract and lowest inhibition zone (7.0±0.2) in petroleum ether extract followed by chloroform (9.3±0.5) and aqueous extracts (11.0±1.2). The gram negative bacteria Staphylococcus aureus showed maximum inhibition (32.0±1.6) in methanol extract compared to all other strains and extracts. On the other hand same bacterial strain showed less inhibitory concentration (6.6±0.5) in petroleum ether extract and chloroform (15.6±0.2), aqueous (7.3±1.2) respectively. Methanol extract showed highest (28.7±1.2) zone on Klebsiella pneumoniae and moderate (13.6±1.2) in aqueous extract where as petroleum ether (7.3±1.2) and chloroform (8.0±0.8) extract found to be very less. Salmonella paratyphi and Pseudomonas aeruginosa showed rational zone formation (13.3±1.2) and (12.3±1.7) respectively in methanol extract and the highest (18.3±1.2) inhibition attained in aqueous extract. Both petroleum ether (8.0±0.8 and 10.3±0.9) and chloroform (9.6±1.2 and 9.7±1.7) found to be very less in these strains. One of the gram positive bacteria Bacillus subtilis showed less zone (8.7±1.2) formation in petroleum ether extract utmost (13.3±1.2) in methanol extract. Aqueous extract and chloroform extract developed (10.3±1.2), (9.0±1.6) correspondingly by the same pathogen.

Table 1. Qualitative phytochemical analysis of various solvent extract of Glossocardia bosvallia whole plant.

S.no	Bioactive constituents	Petroleum	Chloroform	Methanol	Aqueous
1	Alkaloids	---	--	--	--
2	Carbohydrate	---	---	++	++
3	Glycosides	---	---	--	---
4	Proteins	--	--	--	--
5	Amino acid	--	--	--	--
6	Phytosterols	++	++	--	--
7	Tannins	---	++	+++	++
8	Phenol	---	++	--	+++
9	Flavanoid	---	---	++	---
10	Coumarin	---	++	++	++
11	Saponin	++	--	++	++
12	Quinone	---	++	++	---
13	Cardiac glycosides	---	---	++	++
14	Terpenoid	++	++	+++	--
15	Acids	--	--	--	--
16	Phlobatannin	--	--	++	++
17	Anthraquinone	---	---	---	---
18	Steroids/phytosteroids	++	++	++	--
19	Fixed oils	+++	++	+++	++
20	Fat	++	--	++	--
21	Gum and mucilage	++	--	++	++

+++ = strongly present ++ = moderately present --- = strongly absent -- = not detectable

Table 2. Evaluation of in vitro antibacterial activity of various solvent extract of Glossocardia bosvallia whole plant.

Bacterial pathogens	Zone of inhibition (mm)
Bacterial pathogens	Control (Tetrcycline)	Petroleum ether	Chloroform	Methanol	Aqueous
Streptococcus faecalis	24.7±1.7^b	7.0±0.8^de	9.3±0.5^bc	29.3±1.^2ab	11.0±1.2^c
Staphylococcus aureus	26.6±1.2^a	6.6±0.5^f	15.6±0.2^a	32.0±1.6^a	7.3±1.2^e
Klebsiella pneumoniae	23.3±1.2^bc	7.3±1.2^d	8.0±0.8^d	28.7±1.2^b	13.6±1.2^b
Salmonella paratyphi	15.6±0.9^e	8.0±0.8^bc	9.6±1.^2bc	13.3±1.2^c	18.3±1.2^a
Pseudomonas aeruginosa	21.0±1.6^d	10.3±0.9^a	9.7±1.7^b	12.3±1.7^cd	18.3±1.2^a
Bacillus subtilis	12.0±0.8^f	8.7±1.2^b	9.0±1.6^c	13.3±1.2^c	10.3±1.2^cd

Values are mean±SD (n=3); Mean values followed by different superscripts in a column are significantly different (P<0.05) according to Duncan’s multiple range tests (DMRT).

DISCUSSION:

The Diospyros species biological and pharmacological activities may be attributed in the presence of quinone, terpenoid, steroid and phenolic compounds ^{(9, 16)}. Plants containing tannin, alkaloid, saponin, flavonoid and glycoside showed broad spectrum of antimicrobial activity ⁽¹⁷⁾. Same way coumarins play a vital role in the field of medicinal chemistry and pharmacotherapy ⁽¹⁸⁾. Flavonoid in chloroform and aqueous extract seems to have lot of medicinal properties compared to other phytoconstituents ⁽¹⁹⁾. Chandrasekharnath et al. ⁽²⁰⁾ studied that the tannin rich ellagitannins and phenolic acids of Punica granatum have antibacterial, antifungal and antiprotozoal activity. Yamuna et al.⁽²¹⁾observed the steroid and triterpenoids presence in the stem, leaves, root, flower and seeds of Aerva lanata and confirmed it in high performance thin layer chromatography. Govndasamy and Srinivasan ⁽²²⁾ reported the phytochemical analyses of Catharanthus roesus were showed the presence of soluble sugar, reducing sugar, protein, amino acids, lipids, total chlorophyll, phenol and ortho-dihydroxyphenols in the ethanolic extract.

Subbaiyan et al. ⁽²³⁾ reported that the antibacterial activity was identified in the methanol extract of Catharanthus pusillus showed potential activity against tested pathogens, due do their more phytochemical constituents. Wasim et al. ⁽¹²⁾ studied that the methanol extract of Lawsonia inermis leaves were active against six different bacterial strains and showed significant anti bacterial activity as compared to standard antibiotic. The antimicrobial activity of leaves of five South Indian medicinal plants such as Adhatoda vasika, Bacopa monnieri, Carica papaya, Cissampelos pareira and Cynodon dactylon revealed that the extract of five plants acts as a good source of antibiotics against various bacterial pathogens tested and exhibited a broad spectrum of antimicrobial activity ⁽²⁴⁾. Jaiganesh and Arunachalam ⁽²⁵⁾ concluded that Pterospermum canescens leaf extracts possess a broad spectrum of antimicrobial activity against pathogens responsible for various infectious diseases. The ayurvedic herb Glossocardia bosvallia showed some of the phytochemical components such as fixed oils, flavonoids, saponins, tannins, steroids/phytosterods, gum/mucilsge and terpenoids. There is no indecision that this plant is a collection of potentially useful bioactive compounds which serve as drugs, provide new leads and indications for modern drug design. Due to its many medicinal and ayurvedic properties there is enormous scope of future research. Clinical and pharmacological study should be conducted to investigate the unexploited potential of this plant. The methanol extract showed greater antibacterial activity on all selected human pathogenic bacterial strains which are comparable with the standard control, Tetracycline.

ACKNOWLEDGEMENT:

Authors are greatly thankful to Dr. M. Aruchami, Secretary, Kongunadu Arts and Science College (Autonomous), Coimbatore, Tamil Nadu, India carry out this research work successfully.

ABBREVIATIONS:

WHO-World Health Organization; mg-milligram; ml-millilitre; mm-millimetre; NaOH-Sodium hydroxide; HCl-Hydro chloric acid; N-Normality; °C-Celsius; h-Hour.

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Received on 09.06.2013 Modified on 30.06.2013

Research J. Pharm. and Tech. 6(12): Dec. 2013; Page 1391-1396