1. INTRODUCTION:

Medicinal plants have been used as traditional, folk and modern medicinal systems for treatment of various diseases. Characterization of phytochemicals present in the medicinal plants is desirable for the discovery of new therapeutic agents for newly arising diseases [1]. Information about bioactive components present in the medicinal plants may provide the new idea to identifying and developing new drugs [2]. Free radicals are unstable species induce cellular damage that are generated in our body during the metabolic activity [3]. Synthetic antioxidant agents have been reproached due to their low solubility with minimum activity and to possible toxic effects [4, 5].

For that reason, there come up a need to development of new prospective natural resources of antioxidants. Plants contain phytochemicals such as Phenolics, Flavonoids, Tannins and Alkaloids have potential to scavenging the free radicals [6].

In the present study, the ethanolic extract of Ceiba pentandra leaves was assessed for their phytochemical constituents using Gas chromatographic technique and antioxidant and antimicrobial activity. Ceiba pentandra (L) belongs to the family Malvaceae and mostly cultivated in tropical West Africa, Southeast Asia, India and Srilanka. It is commonly called as cotton tree or silk cotton tree. Ceiba pentandra is a deciduous tree grown into large up to 60 m [7]. The bark has been used as a diuretic, aphrodisiac, and to treat headache, as well as type II diabetes [8]. Young fruits are used to treatment for snake bite. Whole plant parts of this plant are used to treat fever, asthma, dysentery, diarrhea and kidney diseases [9].

2. MATERIALS AND METHODS:

2.1 Collection of plant material:

The medicinal plant Ceiba pentandra were collected from Kaligapura(v) , Tiruvanamalai district Tamilnadu, India.

2.2 Preparation of plant extract and GC-MS analysis:

The leaves of Ceiba pentandra collected were surface sterilized using ethanol and shade dried for 3-4 days. The dried plant leaves were pulverized into coarse powder by using dry grinder and passed through sieve. 250 g of dried, powdered leaf sample was defatted by treating with pet-ether and then extracted with ethanol solvent by using Soxhlet apparatus for 24 hrs at 30±1°C. As a result, the solid mass was obtained by removing the solvent under vacuum. The solid mass of residue was weighted and stored in air and water proof containers, kept in refrigerator at 4⁰C and used for further works by making dilutions from the stock. Phytochemical components of leaf extract was characterized by GC-MS analysis which carried out on a GC clarus 500 Perkin Elmer system and gas chromatograph interfaced to a mass spectrometer (GC-MS) instrument.

2.3 Antimicrobial activity:

Antibacterial activity of ethanolic extract Ceiba pentandra leaves was assessed by following Agar well diffusion method against pathogenic bacteria. The test cultures Streptococcus sp, Bacillus subtilis, Escherichia coli, and Klebsiella pneumonia were maintained in Luria Bertani broth. Typically, 24 hrs freshly prepared each cultures were swabbed on the freshly prepared petridish containing Muller Hinton Agar. About 5 wells were made with 6 mm diameter using gel puncture. Different concentrations like 20 – 100 µg/ml of ethanol extract of leaves were transferred into each well in aseptically. The commercial antibiotic disc Chloroamphenicol (100 µg) also placed in petridish as control. Then the plates were incubated at 30 °C for 24 hrs and noted the zone of inhibition around the well.

2.4 Free radical scavenging activity:

The free radicals like DPPH, nitric oxide and hydroxyl radical scavenging activity of ethanolic extract of Ceiba pentandra leaves were determined by following the method of Gyamfi [10], Madan et al [11] (Griess Illosvoy reaction), and Halliwel et al [12], respectively. The percentage of inhibition is calculated by following equation:

% Inhibition = [Control _abs – Test sample _Abs/ Control _abs] ×100

2.5 Statistical analysis:

The results of in vitro study were given as Mean ± Standard Error (SE) obtained from three independent experiments.

3. RESULTS AND DISCUSSION:

3.1 GC-MS analysis:

GC-MS characterization is an effective combination for chemical analysis of materials. Nine phytochemicals were identified using GC-MS method and the results of presence of various phytochemicals identified at retention time are represented in Table.1 and the chromatogram is shown in Figure 1. The presence of active phytochemicals were identify by the production of specific spectral peak at retention time which corresponding to specific component. The compound 1-9 were identified as 3,7,11,15-Tetramethyl-2-hexadecen-1-ol (C₂₀H₄₀O), Hexadecanoic acid (C₁₈H₃₆O₂), Phytol (C₂₀H₄₀O), 5,9,13-Pentadecatrien-2-one (C₁₈H₃₀O), Squalene (C₃₀H₅₀), Vitamin E (C₂₉H₅₀O₂), Olean-18-ene (C₃₀H₅₀), 2,4,4-trimethyl-3-hydroxymethyL-5A-(3-methyl-but-2-enyl)-Cyclohexe (C₁₅H₂₆O), and Cyclotrisiloxane (C₆H₁₈O₃Si₃). The compound 2 Hexadecanoic acid is well known as fatty acid. These bioactive components are known to exhibit medicinal property [13].

Figure 1: Ceiba pentandra leaves and GC-MS analysis of ethanolic extract of C. pentandra leaves

Table 1: Bio-active phytochemicals identified in Ceiba pentandra leaves using GC-MS study

Rt value	Compound	Molecular weight	Molecular formula
16.71	3,7,11,15-Tetramethyl-2-hexadecen-1-ol	296	C₂₀H₄₀O
18.29	Hexadecanoic acid, Ethyl Ester	284	C₁₈H₃₆O₂
19.89	Phytol	296	C₂₀H₄₀O
21.69	5,9,13-Pentadecatrien-2-one, 6,10,14-Trimethyl-, (E,E)-	262	C₁₈H₃₀O
24.98	Squalene	410	C₃₀H₅₀
27.54	Vitamin E	430	C₂₉H₅₀O₂
29.58	Olean-18-ene	410	C₃₀H₅₀
30.07	2,4,4-trimethyl-3-hydroxymethyl-5A-(3-methyl-but-2-enyl)-Cyclohexe	222	C₁₅H₂₆O
30.67	Cyclotrisiloxane, Hexamethyl-	222	C₆H₁₈O₃Si₃

3.2 Antimicrobial activity of Plant leaves:

The antibacterial activity of ethanolic Ceiba pentandra leaves extract was characterized by Agar well diffusion method it was shown in the Figures 2-5. There are five different plant extracts concentrations 20, 40, 60, 80 and 100 µg/ml were tested against the bacterium like Streptococcus sp, E. coli, Klebsiella pneumonia, and Staphylococcus aureus. It clearly indicates the antibacterial potential of the medicinal plant Ceiba pentandra leaves extract. These bacteria are known as pathogenic and opportune bacterial isolates. Antibacterial was determined by measuring the zone formation around the well and denoted in millimetre in diameter. In this analysis, antibacterial activity of leaf extract was increased as increasing the concentration of ethanolic extract of Ceiba pentandra leaves. The antibacterial activity is directly proportional to the extract concentrations. Zone of inhibition against Streptococcus sp, E. coli, Klebsiella pneumonia, and Staphylococcus aureus are 12, 15, 11, and 15 mm, respectively. Additionally, ethanolic extract of Ceiba pentandra leaves shows highest significant activity against multidrug resistant bacteria S. aureus.

Figure 2: Antibacterial activity of ethanolic extract of Ceiba pentandra leaves extract against Streptococcus sp.

Figure 3: Antibacterial activity of ethanolic extract of Ceiba pentandra leaves extract against E. coli

Figure 4: Antibacterial activity of ethanolic extract of Ceiba pentandra leaves extract against Klebsiella pneumoniae

Figure 5: Antibacterial activity of ethanolic extract of Ceiba pentandra leaves extract against Staphylococcus aureus.

3.3 Free radical scavenging activity:

The DPPH radical scavenging activity of ethanolic Ceiba pentandra leaves extract was increased in dose dependent manner ranges from 10-50 µg/ml. Figure 6 and Table 2 clearly shows the 50% radical scavenging activity was observed at 27.4 µg/ml concentration of ethanolic leaf extract. It confirms the high significant DPPH scavenging potential of leaf extract comparable than the standard BHT is used as positive control and shows the 50% inhibition at the concentration of 29.3 µg/ml.

Table 2 DPPH Scavenging activity using ethanolic extract of Ceiba pentandra leaves

Concentration (µg/ml)	% inhibition
Concentration (µg/ml)	Plant extract	Standard
10	21.33±0.69	20.35±1.25
20	47.25±0.64	45.45±0.85
30	63.85±0.96	62.12±0.98
40	73.35±0.63	72.13±1.65
50	91.05±0.84	90.25±1.24

± Standard deviation

Figure 6: Effect of ethanolic extract of Ceiba pentandra leaves extract and Standard BHT on scavenging of DPPH radical Results

The results of Nitric oxide radical inhibition by ethanolic Ceiba pentandra leaves extract are shown in Figure 7 and table 3. The IC50 concentration of Nitric oxide radical scavenging activity of leaf extract was 24.45 µg/ml. It was more significant as compared to a positive control i.e the standard BHT (30.05µg/ml). These results confirmed the antioxidant potential of ethanolic extract of C. pentandra leaves.

Table 3 Nitric oxide Scavenging activity using ethanolic extract of Ceiba pentandra leaves

Concentration (µg/ml)	% inhibition
	Plant extract	Standard
10	19.93±0.84	15.48±0.63
20	36.68±1.21	35.95±0.78
30	55.36±0.96	50.02±0.69
40	78.02±0.22	76.87±2.25
50	92.45±0.64	91.65±1.34

± Standard deviation

Table 4 Hydroxyl radical scavenging activity of ethanolic extract of Ceiba pentandra leaves

Concentration (µg/ml)	% inhibition
Concentration (µg/ml)	Plant extract	Standard
20	18.87±1.34	16.32±0.22
40	36.21±0.63	32.98±0.84
60	58.02±2.25	55.28±0.96
80	74.77±0.69	70.63±0.64
100	86.25±0.78	85.45±1.21

± Standard deviation

Figure 7: Effect of ethanolic extract of Ceiba pentandra leaves on Nitric oxide radical inhibition assay

Figure 8 showed the ethanolic Ceiba pentandra leaves extract have high inhibition activity against hydroxyl radical. The scavenging activity of plant extract against hydroxyl radical was observed by deoxyribose assay in a concentration dependent manner. It showed hydroxyl radical scavenging activity with about 50 % at concentration of 51.65 µg/ml. The results are shown in Figure 8, the concentrations of 50% inhibition were found to be 30.0 µg/ml and 48.05 µg/ml for the extract and standard of vitamin E, respectively. The extract inhibition value was found to be lesser than the standard.

Figure 8: Hydroxyl radical scavenging assay of ethanolic extract of Ceiba pentandra leaves

The antioxidant activities of leaf extract shows the significantly scavenging the free radical due to the presence of alkaloids, phenols, vitamin and fatty acid. Odukoya et al. [14] has reported a strong relationship between phenolic content and antioxidant activity in selected fruits and vegetables. The fatty acid and vitamin E are strongly scavenging the DPPH, nitric oxide and hydroxyl free radicals [15]. These suggestions was confirmed in this present study where by ethanolic extract of Ceiba pentandra leaves with more phenolic, vitamin, alkaloids, and fatty acid content exhibited greater antioxidant activity.

4. CONCLUSION:

The present investigation showed antimicrobial and antioxidant activity of ethanol derived extract of Ceiba pentandra leaves and bioactive components was determined by using GC-MS analysis. GC-MS analysis of the extract revealed presence of alkaloids, vitamin and fatty acid. The extract of C. pentandra was subjected to determine the antimicrobial and antioxidant activity. The extract shows maximum zone of inhibition was noticed against E. coli and S. aureus. Free radical scavenging activity of extract shows remarkable activity as compared to the standard BHT. Consequently, the results concluded that the ethanolic extract of Cebia leaves could be considered as futuristic alternative medicine for treatment of human diseases.

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Received on 21.07.2016 Modified on 02.08.2016

Research J. Pharm. and Tech 2016; 9(11):1922-1926.

DOI: 10.5958/0974-360X.2016.00393.0