INTRODUCTION:

Plants have been efficacious, fruitful and convenient source of natural products with miscellaneous therapeutic applications. The historic review by De Pasquale[1] reveals that the use of natural products with therapeutic properties is as ancient as human civilization and, for a long time, mineral, plant and animal products were the main sources of drugs[2]. From the ancient times, humans have relied on nature for their basic needs particularly plants which have formed the basis of sophisticated traditional medicine systems from 2600 BCE which documents around 1000 plant derivates[3]. Plant based medicines continues to play an essential role in healthcare as well as against menacing disease such as cancer. Cancer is one of the serious and major public health threats worldwide. WHO in 2012, has estimated that by 2030, the global burden is expected to grow to 21.4 million with new cancer cases and 13.2 million cancer deaths simply due to the growth and aging of the population, as well as reductions in childhood mortality and deaths from infectious diseases in developing countries.

Received on 22.12.2014 Modified on 10.01.2015

Research J. Pharm. and Tech. 8(2): Feb. 2015; Page 198-203

DOI: 10.5958/0974-360X.2015.00036.0

The increase in statistics of cancer patients and infectious diseases states that there is an urgent need to discover therapeutic agents. In ancient medical history, physicians used to recommend plant products to be used after surgery which represents the efficiency of natural products and can be correlated with current therapeutic pharmaceutical drugs[4]. Plants have a long history of use in the treatment of several types of cancer [5] and most of the plant derived anticancer drugs in clinical use, some of the best known are the so-called vinca alkaloids, vinblastine and vincristine isolated from the Madagascar periwinkle. Paclitaxel, plant-derived anticancer drug was new hope for novel drugs and it has proven to be effective anticancer drug along with several key precursors (the baccatins) in the leaves of various Taxus species[6]. There are more than 119 drugs which have been extracted from plant sources and have been globally used in allopathic medicines. The extracts of traditional medicinal plants have been tested to identify the source of the therapeutic effects and development of novel drugs[7].

Adenanthera pavonina which is commonly known as red wood and red-bread tree belongs to the family Leguminosae and subfamily Mimossoideae. It is an important medicinal plant which is known as Saga in Malaysia, Raktakambal in India, Lopa in Samoa and Tango and Red-Bead or Red Sandalwood in English[8]. Adenanthera pavonina is a deciduous tree which is 18-24 m tall, erect and 60 cm in diameter[9] which is widely distributed in the Asian and African countries whereas as an indigenous plant it is grown and cultivated mostly in the south-eastern region of Bangladesh. The seeds of Adenanthera pavonina are bright red, hard and heart-shaped, these seeds are formed in curved pods which are then excited from the pods when it is mature[10]. The seeds of Adenanthera pavonina has medical importance in treating various human ailments including inflammation, arthritis, rheumatism, cholera, treatment of boils, blood disorders, convulsion, spasm and indigestion [11,12]. In India, A. pavonina has been traditionally used in the treatment of diabetes mellitus and lipid disorders [13,14]. In Ayurveda, the extracts of plant A. pavonina are widely used in various ayurvedic herbal preparations for treating cancer [16]. The phytochemical constituents of A. pavonina possesses octacosanol, dulcitol, glucosides of β-sitosterol and stigmasterol in leaves, the bark constitutes the major component as stigmasterol glucoside [10] whereas seeds consists of glycosides, saponins and steroids [17,18].

As A. pavonina has been widely studied for various diseases such as anti-inflammatory, diabetes milletus and arthiritis focus was shifted to anticancer activity of seeds and leaves as well leaves of this plant. Hence the antimicrobial and anticancer properties of seeds and leaves from Adenanthera pavonina were evaluated.

MATERIALS AND METHODS

Plant collection: The seeds and leaves of Adenanthera pavonina was collected from Thanjavur, Tamil Nadu, India in the month of November 2013. Sample was completely dried for 48-72 h and was finely ground in mixer grinder to obtain powder.

Preparation of plant extracts:

Finely ground seeds of Adenanthera pavonina (32 g) was extracted using acetone and methanol separately in a Soxhlet extractor not exceeding the boiling point of the solvent whereas leaves of plant Adenanthera pavonina (28 g) was extracted with methanol. The obtained extracts were filtered through Whatman Filter Paper No.1 and then concentrated under reduced atmospheric pressure. The dry extracts were stored at -20°C for further experimental assays.

Phytochemical analysis

Fehling’s Test:

1 ml Fehling’s A solution and 1 ml of Fehling’s B solution were mixed and boiled for a minute. Equal volume of test solution was added to the above mixture. The solution was heated in boiling water bath for 5-10 min. The appearance of first yellow, then brick red precipitate indicates the presence of carbohydrates test [19].

Benedict’s test:

To perform Benedict’s test, equal volume of test solution and Benedict’s reagent was mixed in a test tube. The mixture was heated in boiling water bath for 5 min. The appearance of green color in solution indicates the presence of reducing sugar [20].

Molisch’s test:

Equal volume of Molisch’s reagent and test solution were mixed in a test tube. The mixture was heated in boiling water bath for 5 min. The formation of violet or purple color ring shows the presence of reducing sugar [19].

Biurret Test:

To the acetone and methanol extracts of Adenanthera pavonina, 1-2 drops of Biurret reagent was added. Formation of violet colour precipitate indicates presence of proteins in the extract [19].

Borntrager’s Test:

To the 3 ml of extract, dilute H₂SO₄ was added. The solution was then boiled and filtered. The filtrate was cooled and to it equal volume of benzene was added. The solution was shaken well and the organic layer was separated. Equal volume of dilute ammonia solution was added to the organic layer. The layer with ammonia turned pink which indicates the presence of glycosides in the extract [19].

Test for Cardiac glycosides (Keller- Killiani Test):

To the 5 ml of extract, 1 ml of conc. H₂SO₄, 2 ml of glacial acetic acid and 1 drop of FeCl₃ solution was added. The appearance of brown color ring indicates the presence of cardiac glycosides[21].

Test for Coumarins:

To the 2 ml of extract, 10% of NaOH was added and shaken well for 5 min. The yellow color appearance in the solution confirms the presence of coumarins [19].

Test for Quinone: To the 2 ml of extract conc. H₂SO₄ was added and shaken well for 5 min. The change of solution to red color confirms the presence of quinone in extracts [19].

Salkowski Test:

To 2 ml of extract, 2 ml of chloroform and 2 ml of conc. H₂SO₄ was added. The solution was shaken well. As a result chloroform layer turned red and acid layer shown greenish yellow fluorescence [19].

Hager’s Test:

To the 2-3 ml of filtrate, 1ml of dil. HCl and Hager’s reagent was added and shaken well. The formation of yellow precipitate indicates the presence of alkaloids.

Mayer’s Test:

1ml of dil. HCl and Mayer’s reagent was added to the filtrate and shaken well. Formation of yellow precipitate showed the presence of alkaloids [21].

Tests for flavonoids:

To the small quantity of extract lead acetate solution was added. The formation of yellow precipitate indicated the presence of flavonoids [19].

FeCl₃ Solution Test:

5% FeCl₃ solution was added to the extract, the appearance of deep blue black color indicated the presence of tannins and phenolic compounds.

Foam Test:

To 1 ml of the extract, 20 ml of distilled water was added and shaken well in measuring cylinder for 15 min resulting in 1 cm layer of foam which confirmed the presence of saponins [21].

Thin Layer Chromatography and characterization:

The thin layer chromatography of obtained extracts was carried out on silica coated thin sheets with various solvents systems such as Toulene:ethyl acetate (4:1), Acetic acid: chloroform (1:9), n Butanol:H₂O (1:1), chloroform: methanol: acetic acid (18:1:1), chloroform: isopropanol (9:1), Hexane :ethyl acetate (1:1). The separation of various components present in the extract was observed in UV-chamber and iodine chamber. The extracts of plant A. pavonina were characterized by UV-Vis spectrophotometer, FTIR analysis and Gas Chromatography, Mass Spectrophotometry (GCMS).

Antimicrobial assay:

The antimicrobial activity of the extracts was performed by Kirby-Bauer well diffusion method against Gram positive and Gram negative pathogenic microorganisms. Escherichia coli, Staphylococcus aureus, Proteus mirabilis, Salmonella sp., Shigella sp., Enterococci faecalis, Pseudomonas aeruginosa, Klebsiella pneumoniae are the bacterial test organisms used in this study. All the test organisms were acquired from Microbial Biotechnology Laboratory, SBST, VIT University, Vellore, Tamil Nadu, India. 100 µl of the test pathogens were swabbed onto Muller- Hinton agar plates and 6 mm of four wells were punctured into it. The plant extract obtained after extraction were poured into wells with 25, 50, 75 and 100 µl of concentration. Petriplates were incubated at 37°C for 24-48 h to determine zone of inhibition.

In-vitro cytotoxicity assay:

The bone cancer cell line (MG 63) was obtained from National Centre for Cell Science (NCCS), Pune and was grown in Eagles Minimum Essential Medium (EMEM) containing 10% fetal bovine serum (FBS). All cells were maintained at 37°C, 5% CO₂, 95% air and 100% relative humidity.

The monolayer cells were detached with trypsin-ethylene diaminetetraacetic acid (EDTA) to make single cell suspensions and viable cells were counted using a hemocytometer and diluted with medium containing 5% FBS to give final density of 1×105 cells/ml. 100 µl per well of cell suspension were seeded into 96-well plates at plating density of 10,000 cells/well and incubated to allow for cell attachment at 37°C, 5% CO₂, 95% air and 100% relative humidity. Aliquots of 100 µl of seed and leaves extract of Adenanthera pavonina, dilutions were added to the appropriate wells. The microtitre plates were incubated for an additional 48 h at 37°C, 5% CO₂, 95% air and 100% relative humidity. The medium without test sample served as control and all the test was performed in triplicates [22].

After 48 h of incubation, 15 µl of MTT (5 mg/ml) in phosphate buffered saline (PBS) was added to each well and incubated at 37°C for 4 h. The medium with MTT was then drained off and the formed formazan crystals were solubilized in 100 µl of DMSO and then measured the absorbance at 570 nm using micro plate reader. The percentage of cell inhibition was determined using the following formula: percentage (%) cell Inhibition = 100- Abs (sample)/Abs (control) x100. Nonlinear regression graph was plotted between percentage (%) cell inhibition and log concentration and IC50 was determined using GraphPad Prism software [23].

RESULTS:

The soxhlet extraction of Adenanthera pavonina seeds yielded 1.94 g of acetone extract and 2.32 g of methanol extract whereas leaves of plant yielded 3.16 g of methanol extract. The extracts were completely dried by reduced atmospheric pressure and phytochemical studies were carried out which reveals the presence of saponins, alkaloids, phenols and tannins, cardiac glycosides and steroids in acetone and methanol extract of Adenanthera pavonina seeds. Flavonoids, reducing sugar, proteins, anthraquinone glycosides, coumarins and quinones were absent in seed as leaves extract of plant. The methanol extract of A. pavonina leaves did not show any phytochemical constituents. The phytochemical profile of acetone and methanol extract of seeds and methanol extract of leaves of A. pavonina has been illustrated in Table 1. The phytochemical components, saponins which have been reported for effectual anti-inflammatory and hemotoxic property has been detected in acetone as well as methanol extract of A. pavonina seeds. Alkaloids, known for anti-inflammatory activity has been reported in extracts of seeds which were confirmed by Hager’s and Mayer’s test. The presence of cardiac glycosides in both the seed extracts exhibits the extract has potential to be used in the treatment associated to heart, activity as an anti-inflammatory, anti-coagulant and in diarrhea and dysentery. Another phytochemical constituent, tannins which has been detected in both the seed extracts was confirmed by lead acetate correlates with astringent and detergent activity.

Table 1: Phytochemical analysis of extracts of plant Adenanthera pavonina

Phytochemical Test	Seed extract in acetone	Seed extract in methanol	Leaves extract in methanol
Fehling’s test	-	-	-
Benedict’s test	-	-	-
Biurret test	-	-	-
Test for saponins	+	+	-
Mayer’s test	+	+	-
Hager’s test	+	+	-
Quinone test	-	-	-
Lead acetate test for Flavonoids	-	-	-
Lead acetate test for Tannins and phenolic compounds	+	+	-
Molisch’s	-	-	-
Salkowski’s test	+	-	-
Keller Killiani	+	+	-
Test for coumarins	-	-	-
Borntrager’s test	-	-	-
Fecl₃ solution	-	-	-

The thin layer chromatography (TLC) of seed and leaves extract of A. pavonina exhibited various colored compound with different refractive index. The various bands obtained at different R_fvalues has been explained,

TLC for Phenolic acids: Seed extract in methanol and acetone extract exhibited one compound each with R_fvalues 0.675 and 0.540 respectively with acetic acid: chloroform (1:9) as solvent phase whereas leaves extract in methanol of Adenanthera pavonina has revealed the presence of three compounds with the R_fvalues 0.729, 0.837, and 0.945 when a solvent phase of acetic acid: chloroform (1:9) was used.

TLC for Phytosterols: the seed extract of Adenanthera pavonina revealed the presence of one compound each with R_f values 0.466, 0.636 respectively with hexane: ethyl acetate (1:1) as the solvent phase and leaves extract in methanol impart the presence of two compound with R_f values 0.575, 0.9909.

TLC for Alkaloids: TLC of methanol extract of leaves of Adenanthera pavonina has revealed the presence of three compounds with the R_fvalues 0.787, 0.829, 0.808 when a solvent phase of chloroform: methanol: acetic acid (8:1:1) was used.

TLC for Terpenoids: The leaves extract in methanol of Adenanthera pavonina revealed the presence of three compounds with R_f values of 0.268, 0.829 and 1 when a solvent phase of Toluene: ethyl acetate (4:1) was used. TLC of seed extracts in methanol and acetone revealed the presence of one compound each with R_f values of 0.512 and 0.390 respectively with toluene: ethyl acetate (4:1) as solvent system.

TLC for Saponins: The thin layer chromatography of methanol extract of leaves revealed the presence of one compound with R_f value of 0.606 when Butanol: Water (1:1) was used as a solvent phase.

TLC for Cardiac glycosides: TLC of leaves extract in methanol revealed the presence of three compounds with R_f values 0.833, 0.888 and 0.555 respectively when a solvent phase of chloroform: isopropanol (9:1) was used. The Rf value for various extracts of plant A. pavonina has been presented in Table 2.

The acetone extract of A. pavonina seeds (50 mg/ml and 75 mg/ml) exhibited no zone of inhibition against pathogens whereas 50 mg/ml of methanol extract of A. pavonina manifested antimicrobial activity against Salmonella sp. (17 mm), Serretia sp. (15 mm), Pseudomonas aeruginosa (20 mm), Klebsiella pneumoniae (12 mm) and 75 mg/ml of exhibited good antimicrobial activity against Klebsiella pneumoniae (21 mm), Shigella sp. (16 mm), Salmonella (12 mm), Serretia sp. (15 mm) and Staphylococcus aureus (8 mm). The leaves extract in methanol exhibited proficient antimicrobial activity in comparison to seed extracts against Salmonella sp. (31 mm), Shigella sp. (20 mm) and Proteus mirabilis (15 mm).

Table 2: Rf value of different extracts of plant Adenanthera pavonina

S. No.	Solvent system	Leaves extract in methanol
S. No.	Solvent system	Pigment colour	Measured R_f value
1	Toluene: ethyl acetate	Yellow green	0.2682
		Green	0.8292
		Yellow	1
2	Acetic acid: chloroform	Light green	0.7297
		Green	0.8378
		Yellow	0.9459
3	Butanol: water	Light green	0.6060
4	Chloroform: methanol: acetic acid	Yellowish green	0.7872
		Green	0.8297
		Yellow	0.8080
5	Chloroform: isopropanol	Yellow	0.8333
		Green	0.8888
		Yellowish green	0.5555
6	Hexane: ethyl acetate	Yellowish green	0.5757
6	Hexane: ethyl acetate	Green	0.9090

The UV-Vis spectrophotometer of acetone extract of seeds showed the absorbance at 258 nm and 330 nm whereas methanol extract of seeds revealed the peak at 218 nm which represents that absorbance has taken place due to the excitation of π-π electron transitions. The methanol extract of leaves revealed the absorbance at 222 nm, 273 nm and 364 nm which demonstrates the presence of bioactive metabolites which helps in absorption. The UV-vis spectrum of various extracts of plant A. pavonina has been presented in Figure 1. The FTIR analysis of acetone extract of seeds represented various functional groups such as alcohols, phenols, aromatic, alkanes, nitro compound, primary and secondary amines. The functional groups present in acetone extract, methanol extracts of seeds and functional groups present in methanol extracts of leaves has been listed in Table 3. In order to confirm the presence of these functional groups, gas chromatography mass specrophotometry (GCMS) was carried out which revealed the presence of fatty acids, alkyl esters and oleic acid. GCMS analysis of seeds in methanol extract exhibited ethers at retention time of 13.3, 1,3-propylene glycol, o,o-di(pivaloyl) was obtained which is also known as 2.2-dimethylpropionic acid 3-pivaloxypropyl ester (C₁₃H₂₄O₄) and carbohydrate at retention time of 15.7, Myo-inositol, 4-methyl Myo-inositol, 2-c-methyl-3-o-methyl-d-glucose which is commonly known as inositol (C₆H₁₂O₆) was obtained. The chromatogram showing various peaks after GCMS analysis of seed extracted has been shown in Figure 2.

Table 3: FT-IR analysis of seed and leaves extract of plant A. pavonina

S. No.	Seeds in methanol extract		Leaves in methanol extract
S. No.	Frequency	Functional groups	Frequency	Functional groups
1	3417.86	Alcohols, phenols	3417.86	Alcohols, phenols
2	3032.10	Aromatics, carboxylic acid	3005.10	Aromatics, alkenes
3	2949.16	Alkanes	2951.09	Alkanes
4	2922.16	Aromatics, carboxylic acid	2922.16	Alkanes
5	2864.29	Alkanes	2864.29	Carboxylic acids, alkanes
6	2843.07	Carboxylic acid	2843.07	Carboxylic acids
7	2108.20	Alkynes	1641.42	Alkenes, primary amines
8	1641.42	Alkenes, primary amines	1631.78	Primary amines
9	1629.85	Primary amines	1450.47	Aromatics, alkanes
10	1402.25	Aromatics	1402.25	Aromatics
11	1328.95	Aromatic amines	1330.88	Aromatic amines, nitro compounds

Fig. 1: (a) UV-Visible spectrum of various extracts of plant A. pavonina (a) UV-spectrum of acetone extract A. pavonina seeds. (b) UV-spectrum of methanol extracts A. pavonina seeds. (c) UV-spectrum of methanol extract A. pavonina leaves.

Fig. 2: (a) GCMS chromatogram of methanol extracts of A. pavonina seeds. (b) GCMS chromatogram of acetone extracts of A. pavonina seeds.

CONCLUSIONS:

In the present investigation, important phytochemical constituents such as saponins, flavanoids and alkaloids have been reported in seed extracts of A. pavonina when compare to extract of leaves. Methanol extract of A. pavonina leaves exhibited effective antimicrobial activity against clinical pathogens in comparison to acetone and methanol extract of seeds of A. pavonina. Seed extract in methanol showed good anticancer activity against bone cancer cell line whereas acetone extract exhibited low cancer cell inhibition and leaves extract in methanol of A. pavonina had no anticancer effect against hepatocellular carcinoma. Therefore, it can be concluded that seeds of A. pavonina possesses good antimicrobial and anticancer activity whereas, leaves exhibited only antimicrobial activity.

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