Phytochemical Screening of Stephania Abyssinica and Brassica Oleracea Var. Capitata L. Leaves and Evaluation of Antibacterial Activity

Contents

Introduction

Materials and methods
Chemicals and Equipment
Plant Collection
Experimental procedure for in vitro-antibacterial activity test
Result and Discussion

Conclusion

Recommendation

References

Appendix
Appendex 1: In vitro antibacterial activities of s.abyssinica and B.oleracea var capitata L leaves crude extracts
Appendix 2: Phytochemical screenings of crude extracts of s.abyssinica and B.oleveracea var capitata L
Appendix 3: Photograph of Brassica oleracea var. capitata L and Stephania abyssinica

ABSTRACT:Medicinal plants have played a crucial role as a source of traditional medicine in Ethiopia from time immemorial to combat different ailments and human sufferings. Leaf of Stephania abyssinica are used in Ethiopia as traditional medicine for the treatment of stomach aches, headaches, and inflammation. And Brassica oleracea L. var. Capitata (cabbage) has been widely used as an herbal medicine to treat gastrointestinal disorders and can also be used as food. This study aimed to carry out a phytochemical analysis and evaluation of the antibacterial activities of Stephania abyssinica and Brassica oleracea L. var. Capitata leaf. The leaves were collected and dried, powdered, and subjected to extraction using maceration technique. The results showed that there are alkaloids, flavonoids, glycosides, terpenoids, phenols, and saponins in the petroleum ether, acetone, and 50% chloroform in methanol leaves extracts of both plants. The leaf extracts of both plants were tested against E. coli, S. typhus, P.aerugenosa, and S. aureus. The results showed that the S. abyssinica crude extracts had better zone of growth inhibition against S. aureus and the acetone crude extracts of B. oleraceae var. capitatal had good activity against the four strains. Further isolation and characterization of pure compounds are recommended.

From the leaf extracts of S. abyssinica 2.7, 12.8, and 1.3 (% w/w) crude extracts were obtained from petroleum ether, and 50% chloroform in methanol and acetone, respectively. Similarly, 6, 2, and 0.4 (%w/w) crude extracts were obtained using petroleum ether, and 50% chloroform in methanol and acetone respectively were obtained from leaves of B. Oleracea Var. Capitata L. Hence, the results of the phytochemical analysis indicated that as there are alkaloids, flavonoids, glycosides, terpenoids, phenols, and saponins in the petroleum ether, acetone and 50% chloroform in methanol leaves extracts of S.abyssinica and B. oleraceae var. capital.The S. abyssinica crude extracts have shown better zone of growth inhibition against S. aureus 13±3.53, 12±3.46 and 11±3.39 mm and the acetone crude extracts of B. oleraceae var. capitatal have good activity against the four strains 12±1.65 and 13.5+3.39 mm against S. typhus and S. aureus. Its antifungal activity test and further isolation, and characterization of pure compounds are recommended.

Keywords: crude extracts, phytochemical analysis, Traditional medicine, Jimma

Introduction

Traditional medicine is the major source of treatment for large portions of human populations in developing countries. It is estimated that 80% of developing countries' population rely on traditional medicine, mostly plant drugs, for their primary health care needs1. Particularly in resource-poor communities, local therapy using traditional medicine is the only means of treatment.2 Herbal remedies are becoming popular throughout the world because, though allopathic medicine can cure a wide range of diseases, its high prices and occasional side effects are causing many people to return to herbal medicines which tend to have fewer side effects.3

In Ethiopia, large populations treat different human and livestock ailments using traditional medicine using medicinal plants. Despite modern medicine becoming more widespread in the county, about 80 to 90 % of Ethiopia's population relies on traditional medicine to meet their primary healthcare needs.4‒6 The current healthcare system in Ethiopia is a primarily health care focused system that improves access to modern medicine more than ever.7 However, the majority of populations continue to use traditional medicine. This is because traditional medicine is the most affordable and easily accessible source of treatment to the poor community2 and cultural acceptance of traditional medicine.8

The use of medicinal plants as a source of traditional medicine has been inherited through generations in Ethiopia. It is an important component of the health care system in the county. The skills are however fragile and easily forgettable as most indigenous knowledge transfer in Ethiopia is based on oral transmission.9 With the current rate of modernization, it's logical to assume that traditional knowledge of medicinal plants is under threat of extinction.1 Caring comprehensive studies are therefore important to document traditional knowledge on medicinal plant uses. Based on the above insight, the present study is aimed to evaluate the antibacterial activities of the two selected plants and underlines the importance of traditional knowledge used for the treatment of fire burn wound bacteria diseases in Gida Ayana woreda, Koneji kebele, East Wollega.

Materials and methods

Chemicals and Equipment

Chemicals used for extractions were petroleum ether, chloroform: Methanol and Acetone, and those used for phytochemical and antimicrobial activity tests were concentrated HCl, lead acetate solutions, water, concentrated H2SO4, ferric chloride solution, chloroform, Muller Hinton Agar (MHA), 4% Dimethyl sulphoxide (DMSO). The equipment used for extraction is a round bottom flask and rotary vapors that are used to concentrate the extracted at reduced temperature and pressure. Those that are used for phytochemical and antibacterial tests are beakers, pipets, measuring cylinders, test tubes, and discs.

Plant Collection

The dried leaf ofS. abyssinicaandB. oleveraceavar.capitat L. were collected from East Wollega, Gida Ayana woreda, and Jimma zone, local markets respectively. The air-dried leaves were collected. Then, the leaves were grounded into a fine powder using an electric grinder and stored at room temperature.

Extraction

The 63.3 g and 224.2g of air-dried powdered leaves of S.abyssinica and B. oleracea var. capitata L. were taken respectively and the samples were soaked in 500 mL of each solvent for 48 hr. Then the soaked samples were filtered out using Whatman No.1 filter paper. The filtrated was evaporated in a rotary evaporator at reduced temperature and pressure. The crude extract was collected in vials which were washed with detergent followed with acetone and stored at room temperature for further phytochemical test and antimicrobial activity.

Bacterial strains

For the antibacterial tests, four different clinical microbial isolates Gram negative and Gram positive were obtained and identified by using conventional biochemical tests and cultivated in pure culture, obtained from at Biology Department at the Microbiology laboratory, Jimma University.

Preliminary phytochemical screening

The methanol; chloroform, petroleum ether, and acetone extract of S. abyssinica and B. leverage var capitata l. leaves were screened according to Trease and Evans (1996), cited by (Musa et al., 2009), for its phytochemical constituents according to standard procedures.

Test for Terpenoids

Salkowski test: 5 mL of various solvent extract was mixed in 2 mL of chloroform followed by the careful addition of 3 mL concentrated H2SO4. A layer of the reddish brown coloration was formed at the interface thus indicating a positive result for the presence of terpenoids.

Test for flavonoids

NaOH test: 2 mL of the extract was dissolved in 10% aqueous NaOH solution and filtered to give yellow color, a change in color from yellow to colorless with the addition of dilute HCl indicated the presence of flavonoids.

Detection of Phenols

FeCl3 Test: Extracts were treated with 3-4 drops of FeCl3 solution. The formation of bluish-black color indicates the presence of phenols.

Test for Quinones

One milliliter of each of the various extracts was treated separately with FeCl3 solution. Quinines give coloration ranging from red to blue.

Test for Steroid

Salkowski test: A little quantity of the extract was dissolved in 1 mL chloroform and 1 mL of concentrated H2SO4 was added down the test tube to form two phases. The formation of red or yellow coloration was taken as an indication of the presence of sterols.

Detection of Saponins

Foam Test:0.5 g of extract was shaken with 2 mL of water. If the foam produced persists for ten minutes it indicates the presence of saponins

Test for alkaloids

The extract (0.5 g) was stirred with 5 mL of 1% aqueous HCl in the water bath and filtered. Three ml of the filtrate was divided into three. To the first 1 mL, a few drops of Dragenroff's reagent were added and observed for the formation of orange to brownish precipitate.

Test for Glycosides

Formation of the brown ring at the interface by the addition of 2 mL of glacial CH3COOH followed by a few drops of FeCl3 solutions and 1mL of conc. H2SO4 to the extracts revealed the presence of glycosides.

Experimental procedure for in vitro-antibacterial activity test

Preparation of cultural media

Pure Muller Hinton Agar (MHA) was prepared by dissolving about 38 g of MHA in 1000 mL of distilled water and adjusted to pH 7.4 ± 0.2, sterilized by autoclaving at 121 0C for 15 min at 15 psi pressure, and used for sensitivity tests in the microbiology laboratory, Jimma University.

Preparation of Discs

From the plant extracts, 250 mg of each crude extract was dissolved in 1 mL of 4 % DMSO and 1 mL of the prepared extracts were taken and the filter Paper discs (Sterilized Whatman No. 1 filter paper discs of 6 mm diameter) were soaked into extracts to get 25 mg/disc Concentration and allowed to dry at room temperature.

Antibacterial Activity Test

Previously prepared paper discs containing different extracts were placed individually on the surface of the Petri plates, containing 25 mL of respective media seeded with 0.1 mL of previously prepared microbial suspensions individually (10 CFU/ mL). Standard antibiotic gentamycin (20 μg/ disc) obtained from Hi-media was used as the positive control. The discs containing n-hexane, dichloromethane, chloroform, and methanol solvents served as negative controls. The assessment of antimicrobial activity will be based on the measurement of inhibition zones formed around the discs. The plates were incubated for 24 hr. at 37°C and the diameter of the inhibition zones was recorded.

Result and Discussion

Percent Recovery of the Crude Extracts

From air-dried powdered leaves of S.abyssinica (63.3g) leaves, 2.7, 12.8, and 1.3% crude extracts were obtained from petroleum ether, and 50% chloroform in methanol and acetone solvents respectively. And from air-dried powdered B. oleracea Var. Capitata L (224.2 g) leaves1.6, 1.95, and 0.4% crude extract were obtained from petroleum ether, and 50% chloroform in methanol and acetone solvents respectively. The results indicated that S. abyssinica has a high amount of oil content than that B. Oleracea Var. Capitata L.

Phytochemical screening of the crude extracts

Table 1.Phytochemical screenings of crude extracts ofS.abyssinicaandB. oleracea

Abbildung in dieser Leseprobe nicht enthalten

Key: ++ = high concentration + = moderate concentration, - = absence

As it has been tried to be indicated in Table 1 above, the phytochemical analysis of leaves extracts of B. Oleracea Var. Capitata L and S.abyyssinica have confirmed the presence and absence of different classes of secondary metabolites. From this, it shows the presence of alkaloids, and glycosides in all crude extracts of S. abyyssinica leaves, alkaloids also found in all extracts of B. Oleracea, and absence of quinones in both plant's crude extracts of petroleum ether, and 50% MeOH in Chloroform. The more intense color implies a higher concentration (++) and medium or normal coloration implies the presence of a sensible concentration (+). Therefore, phytochemical analysis helps to screen the phytochemical screening in a given medicinal plant. It has also taxonomical significance.

Abbildung in dieser Leseprobe nicht enthalten

Table 2. Antibacterial activities Test results of Leaves of S.abyssinica and B. Oleracea

Key: NI- No inhibition

The antibacterial activity of different solvent leaves extracts of Stephania abyssinica was summarized against four bacteria in Table 2. Petroleum ether extract of the plant showed a maximum zone of inhibition (13±3.53 mm) against S. aureus while a minimum zone of inhibition (6.5±2.55 mm) against E.coli. Chloroform: methanol extract of the plant also exhibited a maximum zone of inhibition (13±3.4 mm) against Pseudomonas aeruginosa while a minimum zone of inhibition (6.5±1.34 mm) against Salmonella typhus and acetone extracts shows a minimum zone of inhibition (6±2.5mm) against E.coli.

Conclusion

This research carried out phytochemical analysis and antibacterial activity evolution of S. abyssinica and B. Oleracea. The phytochemical analysis showed the presence of alkaloids, glycosides, saponins, and quinones in all crude extracts of both plants. The antibacterial activity test showed a maximum zone of inhibition against S. aureus and P. aeruginosa by 50%MeOH in Chloroform crude extract. The crude extract of both plants showed a high potential for antibacterial activities.

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