Universities Research Journal 2011, Vol. 4. No. 1

Demonstrator, Department of Botany, East Yangon University

Chemical Investigation and Antimicrobial Activities of Sesbania grandiflora L.

Aye Aye Aung

Abstract Sesbania grandiflora L. belongs to the family, Fabaceae, order Fabales. It is commonly known in Myanmar as Pauk-pan-phyu and Pauk-pan-ni. The plants were collected from Hpa-an Township, Kayin State and prepared for chemical studies in this work. According to the phytochemical investigation, alkaloid, α-amino acid, carbohydrate, starch, reducing sugar, glycoside, phenolic compound, saponin, tannin, terpenoid/steroid and flavonoid were found to be present and cyanogenic glycoside were absent in these plants. In physicochemical studies, the methanol and ethanol soluble matter content were found to be greater than soluble matter content of other organic solvents in leaves. These plants analysis are to determine the abundance amount of elements in Sesbania grandiflora L. (Pauk-pan-phyu and Pauk-pan-ni) which are a quantitative indication of the level of nutrients in plant and is obtained by using EDXRF (Energy Dispersive X-ray Fluorescence) spectrometer. In this experiments, chloroform extracts of leaves showed more significant activity against all test organisms especially on Pseudomonas aeruginosa.

Key words: Sesbania grandiflora L., Qualitative and Quantitative analysis, Antimicrobial Activities.

Introduction Sesbania grandiflora L. belonging to Fabaceae commonly known as

Sesbania which is often planted for its edible flowers and pods in tropical countries. It was known as Pauk-pan-phyu and Pauk-pan-ni in Myanmar. It is believed to have originated either in India or Southeast Asia and grown primarily in hot and humid areas of the world. From present generation, it is a green manure crop useful in paddy cultivation; it is one of the most valuable medicinal herbs. The tree is grown as an ornamental shade tree, and for reforestation. (Watt, 1972 and James, 1983). The crude methanolic extracts of the flowers, seeds, rachis and leaves of Sesbania grandiflora were strongly positive for saponin (by Fehling’s A and B test). The flowers, seeds and leaves were slightly positive for alkaloids. Sterols were markedly found in the rachis and leaves; strongly present in the flowers; and slightly present in the seeds. The rachis and leaves contained plenty of tannins

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(Rastogi, 1969).This paper deals with chemical investigations including phytochemical tests, physicochemical characterization, elemental analysis, nutritional values.

Materials and Methods

Collection, classification and identification of Sesbania grandiflora L. Sesbania grandiflora L. plant parts were collected from Hpa-an

Township, Kayin State, during the months of July to December in 2009. The dried samples were made into fine powder using a mortar and pestle and stored in air tight container to prevent moisture and other contamination.

Qualitative and quantitative analysis of Sesbania grandiflora L.

Qualitative analysis Qualitative analysis concerned with the presence of alkaloids, amino

acid, carbohydrate, starch, reducing sugar, cyanogenic glycoside, glycoside, phenolic compound, saponin, tannin, steroid and flavonoid were investigated by the methods of British Pharmacopoeia (1968), Trease and Evans (2002). Preliminary phytochemical examination was carried out in the phytochemical laboratory of Chemistry Department, University of Yangon.

Quantitative analysis Quatitative analysis including moisture content, total ash, acid

soluble, water soluble ash, acetone soluble, chloroform soluble, petroleum ether soluble, ethyl acetate soluble, ethanol soluble, methanol soluble and water soluble contents of leaves were determined by the methods of in B P, (1968).

Elemental analysis and nutritional values of Sesbania grandiflora L.

Elemental analysis of leaves The relative concentration of elements in Sesbania grandiflora L.

powdered leaves were analyzed by using Energy Dispersive X-ray Fluorescence (EDXRF) spectrometer, Shimadzu Co. Ltd., Japan, at the Universities’ Research Center, University of Yangon.

Some nutrient elements such as Copper (Cu), Potassium (K), Calcium (Ca), Iron (Fe) and Phosphorus (P) in the leaves were also quantitatively analyzed by A A Analyst.800 Atomic Absorption

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Spectrophotometer England (A A S) at the Universities’ Research Center, University of Yangon.

Nutritional values of leaves Carbohydrate, protein, fibre, fat, vitamin B1 and vitamin C contents

have been determined in National Nutritional Centre, Department of Health, according to the procedures given in the methods of A.O.A.C. (Horwitz, 1980).

Antimicrobial activity of different solvent extracts of leaves

Extraction and test organisms The powdered leaves were extracted with chloroform, acetone, petroleum-ether, methanol, ethyl acetate, ethanol and distilled water by percolation method. The filtrate solvents were evaporated by using water bath .

Crude extracts of various solvents were tested on six pathogenic microorganisms such as Bacillus pumalis, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans. The test organisms used in this research were supplied from the Development Centre for Pharmaceutical Technology (DCPT) for determination of antimicrobial activities.

The study of antimicrobial activities was determined by agar-well diffusion method described by Cruickshank, et al., (1975). Nutrient agar was prepared and boiled and 20 - 25 ml of the medium was poured into a test tube and plugged with cotton wool and steriled at 121˚C for 15 minutes in an autoclave. Then the tubes were cooled down to 30 - 35˚C and poured into the sterile petridishes and 0.1 - 0.2 ml of the test organisms were also added into the dishes. They were allowed to set for 2 - 3 hours. Then 10 mm agar - well was made by the help of sterilized agar-well cutter. After then about 0.2 ml of sample was introduced into the agar-well and incubated at 37˚C for 24 hours. The inhibition zones which appeared around the agar - well indicated the presence of antimicrobial activities. The diameter of the inhibition zones were measured and recorded in mm, the controlled experiments using solvent only were prepared for the comparison with leaves plant extracts.

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Results

Morphological Characters of Sesbania grandiflora L. Small tree, stems cylindrical pubescent. Leaves alternate,

unipinnately compound, imparipinnate. Inflorescence axillary racemes. Flower creamy white and dark red. Stamens (2+3), synsepalous. Pistil 1- carpel, ovary linear-oblongoid (Fig. 1 – 5).

Qualitative and quantitative analysis of powdered leaves

Qualitative analysis Qualitative analyses were concerned with determination of presence or absence of phytochemical in qualitative value. The investigation of these tests confirmed the presence of alkaloids, α-amino acid, carbohydrate, starch, reducing sugar, glycoside, tannin, saponin, phenolic compound, steroid/ terpenoid and flavonoid in Pauk-pan-phyu and Pauk-pan-ni leaves. Cyanogenic glycoside were absent in the leaves of both plants. The results were tabulated in Table (1).

Fig.(1) Habit Fig.(2) Habit Fig.(3) Leaves Fig.(4) Flowers Fig.(5) Fruits

Pauk-pan- phyu Pauk-pan-ni

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Table (1) Qualitative analysis of Sesbania grandiflora L.

No. Test Solvent used for extraction Test reagent Observation

Results

Pauk-pan-phyu leaves

Pauk-pan-ni leaves

1 Alkaloids 1%HCl Dragendroff’s reagent Mayer’s regent Wagner’s reagentSodium picrate solution

Orange ppt White ppt Reddish Brown ppt Yellow ppt

+

+

+ +

+

+

+ +

2 α-amino-acid DW Ninhydrin reagent

Pink spot + +

3 Carbohydrate DW 10% α-naphtha and conc: H2SO4

Red ring + +

4 Starch EtOH Iodine solution Blue black ppt + +

5 Reducing sugar Dil H2SO4 Benedict’s solution

Brick red ppt + +

6 Cyanogenic glycoside

DW Sodium picrate solution

No colour - -

7 Glycoside EtOH 10% lead acetate solution

Pale yellow ppt + +

8 Phenolic compound EtOH 3% ferric chloride solution

blue ppt

+ +

9 Saponin DW Distilled water Frothing + +

10 Tannin EtOH 3% ferric chloride solution

Yellow ppt + +

11 Steroid/Terpenoid Pet-ether Acetic anhydride and conc: H2SO4

Green ppt + +

12 Flavonoid EtOH EtOH, Mg, conc: HCl

Pink colour + +

DW=Distilled Water

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Quantitative analysis of Sesbania grandiflora L. From the results of quantitative analysis, the moisture content was

usually determined by drying to constant weight and taking the loss in weight as moisture. Total ash, acid insoluble ash, and water soluble ash content were also recorded. All these values were useful for the quality control system regarding ash and it was used for medicinal purposes. The solubilities of leaves powder plant in acetone, chloroform, petroleum ether, ethyl acetate, ethanol, methanol and distilled water were carried out to determine the amount of total solids soluble in an individual solvent. Sesbania grandiflora L. (Pauk-pan-phyu and Pauk-pan-ni) leaves were found to be significantly soluble in methanol and ethanol than those of other solvents. (Table 2)

Table (2) Quantitative analysis of Sesbania grandiflora L.

No Physicochemical characters Content in %

Pauk-pan-phyu leaves

Pauk-pan-ni leaves

1 Moisture content 9.75 9.09

2 Total Ash content 10.15 11.38

3 Petroleum ether soluble content 3.33 3.00

4 Chloroform soluble content 3.40 3.25

5 Ethyl Acetate soluble content 3.35 3.00

6 Acetone soluble content 6.00 6.67

7 Methanol soluble content 33.3 30.0

8 Ethanol soluble content 28.5 28.0

9 Distilled water soluble content 10.05 10.0

10 Acid soluble ash content 2.00 1.82

11 Acid insoluble ash content 3.08 3.20

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Elemental analysis and nutritional values of Sesbania grandiflora L. Elemental analysis was to determine the amount of elements in

Sesbania grandiflora L. (Pauk-pan-phyu and Pauk-pan-ni) leaves. It was a quantitative indication of the level of nutrients in plant obtained by using EDXRF (Energy Dispersive X-ray Fluorescence) spectrometer. It was found that Calcium (Ca), Phosphorus (P), Potassium (K), Sulphur(S), Chlorine (Cl) and Iron (Fe) are macronutrient elements, where as Strontium (Sr), Brominr (Br), Manganese (Mn) and Zinc (Zn) are found to be as micronutrient elements. Above the elements were relatively abundant in the leaves of both samples heavy toxic metals Lead (Pb), Arsenic (As) and Mercury (Hg) were absent. (Table 3)

Table (3) Comparative studies on relative elements of Sesbania grandiflora L. by EDXRF

No. Elements Relative abundance in %

Pauk-pan-phyu leaves

Pauk-pan-ni leaves

1 Calcium (Ca)** 1.327 1.072

2 Phosphorus(P)** 0.997 0.700

3 Potassium(K)** 0.447 0.548

4 Sulphur(S)** 0.288 0.203

5 Chlorine(Cl)** 0.225 0.106

6 Iron(Fe)** 0.021 0.022

7 Strontium(Sr)* 0.013 0.008

8 Bromine(Br)* 0.011 0.006

9 Manganese(Mn)* 0.011 0.011

10 Zninc(Zn) 0.003 -

**= macronutrient, *= micronutrient Elemental analysis by AAS

Due to the results of AAS analysis, Cu content in powdered leaves of Pauk-pan-phyu and Pauk-pan-ni were 3.65 and 4.71 ppm respectively which was found to be higher than the other elements. (Table 4)

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Table (4) Mineral values of Sesbania grandiflora L. by using AAS

No. Mineral elements Pauk-pan-phyu leaves (ppm) Pauk-pan-ni leaves (ppm)

1 Copper 3.65 4.71

2 Potassium 0.01 2.08

3 Calcium 0.37 0.56

4 Iron 0.03 0.03

5 Phosphorus 0.05 0.06

Nutritional values of Sesbania grandiflora L. The nutritional values of powdered plant were carried out at the

National Nutritiional Center, Department of Health and Ministry of Health. According to the experiments, the amount of carbohydrate and protein were greater than fiber and fat. Although vitamin B1 was 0.21 mg and 0.19 mg, and the amount vitamin C 105.74 mg and 120.63 mg were found to be respectively. (Table 5)

Table (5) Nutritional values of Sesbania grandiflora L.

No. Type of nutrients Pauk-pan-phyu leaves

Pauk-pan-ni leaves

1 Carbohydrate 32.81 g 21.31 g

2 Fat 6.59 g 9.53 g

3 Fibers 6.02 g 6.94 g

4 Protein 34.68 g 40.75 g

5 Vitamin B1 0.21 mg 0.19 mg

6 Vitamin C 105.74mg 120.63 mg

Antimicrobial activity of different solvent extracts of

Sesbania grandiflora L.

In the experiment, the antimicrobial activities of crude extracts were carried out by using various solvent such as petroleum ether, ethyl acetate, distilled water, ethanol, acetone, chloroform and methnol. In this results,

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among various extracts of leaves Sesbania grandiflora L. (pauk-pan-phyu and pauk-pan-ni), chloroform extract showed the highest activity on all six different types of microorganisms especially on Pseudomonas aeruginosa. The results were shown in Figure (6 -11).

Discussion and Conclusion In this research, chemical studies such as preliminary phytochemical

investigation, physicochemical characterization, elemental analysis, examination of nutritional values and vitamins contents of Sesbania grandiflora L. (Pauk-pan-phyu and Pauk-pan-ni) leaves had been studied.

For preliminary phytochemical investigation, Sesbania grandiflora L. (Pauk-pan-phyu and Pauk-pan-ni) leaves plant samples were collected from Hpa-an and prepared, alkaloids, α-amino acid, carbohydrate, starch, reducing sugar, glycoside, tannin, saponin, phenolic compound, steroid/terpenoid and flavonoid were detected in leaves. Cyanogenic glycoside was absent in the leaves of both plants. Therefore, the plants Sesbania grandiflora L. (Pauk-pan-phyu and Pauk-pan-ni) have phytoconstituents. Physicochemical characteristics such as moisture contents, ash content and different solubility of organic constituents in organic solvents were determined. The solubility in methanol and ethanol were found to be the greatest in these solubility tests. The more polar phytoconstituents can also be found.

According to the EDXRF result of elemental analysis, it was found that the macronutrient elements in calcium, phosphorus, potassium, sulphur, chlorine and iron. The micronutrient elements in strontium, bromine, manganese and zinc were found in Pauk-pan-phyu and Pauk-pan-ni leaves.

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Antimicrobial activity of different solvent extracts of Sesbania grandiflora L. (Pauk-pan-phyu and Pauk-pan-ni) leaves

Table (6) Organisms - Bacillus pumalis Fig .( 6 )

Agar-well = 7mm

Table (7) Organisms - Bacillus subtilis

Fig.(7)

No Solvent Pauk -pan -phyu Pauk -pan -ni

1 Petroleum -ether

11 mm 10 mm

2 Chloroform 15 mm 20 mm

3 Methanol 12 mm 13 mm

4 Acetone 10 mm 11 mm

5 Ethyl acetate 11 mm 10 mm

6 Ethanol 15 mm 15 mm

7 Distilled water 12 mm 8 mm

No Solvent Pauk -pan -phyu

Pauk -pan -ni

1 Petroleum -ether

12 mm 9 mm

2 Chloroform 16 mm 20 mm

3 Methanol 8 mm -

4 Acetone 8 mm -

5 Ethyl acetate 10 mm 9 mm

6 Ethanol 14 mm 10 mm

7 Distilled water 13 mm -

Pauk-pan-ni

Ba

Ba

Agar-well = 7mm

Pauk –pan-phyu

Pauk-pan-ni

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Table (8) Organisms - Escherichia coli

Fig.(8)

Agar-well = 7mm

Table (9) Organisms – Pseudomonas aeruginosa

Fig.(9)

Agar-well = 7mm

No Solvent Pauk -pan -phyu

Pauk -pan -ni

1 Petroleum -ether 11 mm 11 mm

2 Chloroform 15 mm 20 mm

3 Methanol 12 mm 14 mm

4 Acetone 11 mm 15 mm

5 Ethyl acetate 10 mm 10 mm

6 Ethanol 14 mm 16 mm

7 Distilled water 10 mm 8 mm

No. Solvent Pauk -pan -phyu

Pauk -pan -ni

1 Petroleum -ether 10 mm 12 mm

2 Chloroform 17 mm 21 mm

3 Methanol 12 mm 14 mm

4 Acetone 10 mm 15 mm

5 Ethyl acetate 9 mm 10 mm

6 Ethanol 12 mm 15 mm

7 Distilled water 10 mm 12 mm

Pauk –pan-phyu

Pauk-pan-ni

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Table (10) Organisms- Staphylococcus aureus

Fig.(10)

Agar-well = 7mm

Table (11) Organisms - Candida albicans

Agar-well = 7mm

No. Solvent Pauk -pan -phyu

Pauk -pan -ni

1 Petroleum -ether 10 mm 9 mm

2 Chloroform 15 mm 19 mm

3 Methanol 12 mm 12mm

4 Acetone 10 mm 12 mm

5 Ethyl acetate 10 mm 10 mm

6 Ethanol 14 mm 17 mm

7 Distilled water 10 mm -

No. Solvent Pauk -pan -phyu

Pauk -pan -ni

1 Petroleum -ether 8 mm 10 mm

2 Chloroform 19 mm 20 mm

3 Methanol 14 mm 13 mm

4 Acetone 13 mm 13 mm

5 Ethyl acetate 8 mm 10 mm

6 Ethanol 13 mm 17 mm

7 Distilled water 8 mm 9 mm

Pauk –pan-phyu

Pauk-pan-ni

Pauk –pan-phyu

Pauk-pan-ni

Fig.(11)

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There are six main classes of nutrients that the body needs: carbohydrates, proteins, fats, vitamins, minerals, and water. It is important to consume these six nutrients on a daily basis to build and maintain healthy bodily function. The important nutritional values found in this plant might also be major contributors to the medicinal value of Pauk-pan-phyu and Pauk-pan-ni.

The antimicrobial activities with six different microorganisms were also tested by using seven crude extracts such as chloroform, acetone, petroleum ether, methanol, ethanol, aqueous extract of Pauk-pan-phyu and Pauk-pan-ni leaves. In this experiment, chloroform extracts of Pauk-pan-phyu and Pauk-pan-ni leaves showed the highest activity on test organisms. Methanol, acetone and aqueous extracts were activity of Bacillus subtilis and aqueous extracts was no activity of Staphylococcus aureus Pauk-pan-ni leaves.

From the overall assessment of chemical studies of Pauk-pan-phyu and Pauk-pan-ni plants which can be used as nutritious food due to the results of the vitamins and nutrient elements. Furthermore, the presence of phytoconstituents, steroids, and flavones in these plants indicate the medicinal properties.

Acknowledgements

I would like to express my deepest gratitude and special thanks to Dr. Thet Thet May, Professor and Head and Professor Dr. Aye Pe, from the Department of Botany, University of Yangon for their invaluable advice and kind suggestions. I would like to express my deepest gratitude to my supervisor Dr. Moe Moe Lwin, Lecturer, Department of Botany, Pathein University for her close supervision, valuable guidance, constant encouragement for the success of my research work..

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