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JPRDP 3

                       Kola-Mustapha  et al., J. Pharm. Res. Dev. & Pract., December, 2016, Vol. 1 No. 1, P 25-33 ISSN:2579-0455

 

Antimicrobial Screening of the Combination of Vernonia amygdalina and Calotropis procera Methanol Leaf Extracts

 

                   A. T. KOLA-MUSTAPHA*, Y.O. GHAZALI Y, T. A. IRANLOYE

 

Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmaceutical Sciences, University of Ilorin, Nigeria

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           *Corresponding Author’s E-mail: atkmusty@yahoo.com.  GSM: +2348033475485

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 ABSTRACT

Herbal therapies and medicinal plants are fast growing in relevance in most societies around the world today due to their availability, relative safety and efficacy against numerous disease conditions. Vernonia amygdalina and Calotropis procera were investigated for their antimicrobial activities singly and in combination to determine their actions against skin infections. A sensitivity test was conducted for the extracts against Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Aspergillus niger and Trichophyton rubrum. The extracts were tested individually and in combination at 0: 100, 25: 75, 50: 50, 75: 25 and 100: 0 Vernonia : Calotropis (V: C) ratios against the five organisms. Antimicrobial activities were determined via the agar diffusion method. Griseofulvin was used as the positive control against the fungal organisms while Augmentin/Clavulanate was used as control for the bacterial organisms. Results of sensitivity test showed that all the organisms were sensitive to each of the extracts at a concentration of 200 mg/mL. The extracts gave zones of inhibition ranging from 5.00 ± 1.24 to 30.00 ± 0.00 mm across the test organisms with the 75: 25 V: C combination yielding the highest zones across all the organisms and the Vernonia amygdalina only extract ranking second. This implies that the extracts of Vernonia amygdalina and Calotropis procera used in combination results in a broad spectrum and increased intensity of activity as compared to when the extracts are used individually in what can be described as a ‘synergistic herb-herb’ interaction. This combination holds a great promise in the treatment of skin infections and exhibit potentials for topical pharmaceutical dosage formulations.

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KEYWORDS: Vernonia amygdalina, Calotropis procera, bacterial, fungal, skin infection

 

 

INTRODUCTION

The interest of people around the world today in natural product remedies is fast growing. Individuals are getting more conscious of the side effects of various synthetic drugs causing them to turn more towards nature1. Over the years, various efforts have been made to evaluate plants for

 

 

 

their antimicrobial activities. The availability and abundance of plants on the earth surface has accounted for the increased evaluation of these plants and how they are used traditionally as anti-infective agents. Medicinal plants are a potential source of new antimicrobial agents2. Phytochemicals are the components of plants responsible for their medicinal actions. They are biologically active compounds and have been of great interest to researchers. These biologically derived agents are eco-friendly and are being explored to substitute synthetic chemicals that can result in toxicity. There are well-documented problems regarding the harmful effects and a rise in the count of organisms that are resistant to chemical antibiotics3. This emphasizes the need for better strategies and agents with lower toxicities and higher specificity of action. Despite the remarkable potencies of synthetic drugs, the challenge of availability, affordability, side effects, even resistance have favored the use of herbal medicines recently. Several plants have been evaluated for the treatment of infections and skin diseases. In this study, Vernonia amygdalina and Calotropis procera are investigated for antimicrobial activities as individual extracts and when combined.

Vernonia amygdalina is of the family Asteraceae4. It is commonly called bitter leaf (due to its bitterness to the tongue) and is either consumed after extraction as tonic for the treatment of various illness or used as vegetable for cooking African soups5. The bitter taste is suspected to result from the presence of alkaloids, saponins, tannins and glycosides6.In fact chimpanzees are said to eat Vernonia leaves when suffering from parasitic infections7

The plant is a shrub of 2 to 5 m with petioate leaf of about 6 mm diameter and elliptic shape. The leaves are green with a characteristic odour and bitter taste4. It is known in Nigerian  local languages as etidot (Efik), uzi (Ebira), onugbu (Igbo), chusar duki (Hausa) and ewuro (Yoruba).Elsewhere in Africa, it is called muop or ndole (Cameroon), tuntwano (Tanzania), and  mululuza (Uganda)8. There are about 200 species of Vernonia4. According to Audu et al. (2012), the leaves are used traditionally as vegetable to stimulate the digestive system, and to reduce fever. The leaf extract of Vernonia amygdalina is used in medicine as an antimalarial, antimicrobial, laxative, antihelminthic, and antithrombotic. In addition, both hypoglycemic and  hypolipidaemic effects have been reported for Vernonia in diabetic hyperlipidemic and normoglycemic hyperlipidemic rats4. Phytochemical screening of the plant confirms the presence of saponins, glycosides and tannins as responsible for purgative effects, flavones for antioxidant activity and may be related to its actions against diabetes and artherosclerosis9.

Calotropis procera (Sodom apple) is a member of the plant family Asclepiadaceae, a shrub widely distributed in West Africa and other parts of the tropics10. The plant is erect, tall, large, much branched and perennial with milky latex throughout. The Shrub or small tree is up to 2.5 (– 6) m tall, stems are erect, simple or branched, woody at base, bark grey or pale brown, fissured, corky, and yellowish-white. The latex is copious; young branches are densely white, hairy, and almost glabrous. Leaves are opposite, decussate, simple and entire, almost sessile; stipules absent; blade oblong-obovate to broadly obovate, 5 – 30 cm × 2.5 – 15 cm, apex abruptly and shortly acuminate, base cordate, succulent, densely

 

 

 

white short-hairy below when young, pinnately veined with 6–10 pairs of lateral veins. Flowers are bisexual and regular. The fruit are a pair of follicles, each follicle ovoid, and fleshy, inflated, 6 – 10 cm × 3 – 7 cm, many-seeded. Seeds are ovoid, flattened, 6 mm long, with 3 – 4 cm long white coma at one end11. Various part of the plant contain cardenolide glycosides, calotropin, calotoxin, uscharin and uscharidin, and a base, choline. The plant also contains o-pyrocatechuic acid; cardenolides (calotropin, calotoxin, uscharin and uscharidin)12It has been used as a purgative, anthelmintic, digestive, stomachic, emetic, expectorant, sedative, blood purifier, and an antidote for snake poisoning and for the treatment of ulcers, tumors, leprosy, asthma, boils, dysentery, eczema, piles and diseases of liver, abdomen and spleen13.

The combination of active components of extracts derived from two plants may result in an enhanced or declined effect. For Vernonia amygdalina and Calotropis procera, the effects of combination on the antimicrobial spectrum and degree of activity of the extracts is a subject that warrants scientific investigation, as the need for improved potency and wider spectrum of action remains a priority in antimicrobial therapies.

 

 

 

 

 

 

 

MATERIALS AND METHODS

Materials

Leaves of Vernonia amygdalina and Calotropis procera collected from Ilorin, in March, 2016 and authenticated at the herbarium of the Department of Plant Biology, University of Ilorin, Nigeria where voucher numbers UILH/001/1023 and UILH/002/962 were deposited respectively. Stock cultures of Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Aspergillus niger and Trichophyton rubrum obtained from the Department of Pharmaceutical Microbiology, University of Ilorin. Reagents and chemicals used include Methanol (Guangdong Chemical Reagent, China), distilled water (Department of Pharmaceutical Chemistry, UNILORIN), sabouraud dextrose agar (SDA) (Biomalk, India), nutrient agar (Biomalk, India), peptone water (Biomalk, India), dimethyl sulphur oxide (DMSO) (Guangdong Chemical Reagent, China).

 

METHODS

Extraction of Plant

The method of extraction described by Adebayo and co-workers14 was utilized. A 250 g of the powdered leaves of Vernonia amygdalina was macerated in 70% methanol and allowed to stand for four hours. The mixture was decanted and more solvent added. This process was repeated three times and the final suspension was filtered through a large cloth sieve. The extract was then concentrated using a rotary evaporator and the resulting concentrate was freeze dried. The dried extract was then stored in a specimen bottle for later use. This same process was repeated for the same quantity of powdered Calotropis procera.

Characterization of Extract

The extracts of Vernonia amygdalina and Calotropis procera were examined for physical characteristics such as their colour, odour and texture. Also, the pH of each extract was determined by a pH meter (Hanna, England). All pH measurements were an average of three determinations and expressed as mean ± S.D.

 

Extract Preparation

Three concentrations: 50, 100 and 200 mg/mL of each of the extracts was prepared in 30% v/v DMSO. For the 200 mg/mL extract concentration, 2 g of each extract was dissolved in 10 mL of the prepared 30% v/v DMSO and agitated till complete dissolution was achieved. The preparation 200 mg/mL was further diluted to achieve the concentrations of the extracts (50 and 100 mg/mL). The same concentration and mode of dissolution was adopted for the preparation of both the Vernonia and Calotropis extracts.

Antimicrobial Screening of Extracts

The extracts were tested for their sensitivity against different microorganism selected based on their tendencies to cause skin infections using the prepared concentrations. Identified cultures of Staphylococcus aureus, Pseudomonas aeruginosa, Aspergillus niger, Candida albicans and Trichophyton rubrum were collected from the Department of Microbiology, University of Ilorin. Staphylococcus aureus and Pseudomonas aeruginosa which are bacteria organisms were sub cultured on nutrient agar plate and incubated at 37 °C for 24 hours. Similarly, Aspergillus niger, Candida albicans and Trichophyton rubrum were sub cultured on Sabouraud Dextrose Agar (SDA) plates and incubated at 25 C for 72 hours. The grown organisms were made into slants and stored for use at 4 C.

For the bacteria culture, nutrient agar media was prepared by dissolving 14 g of the dry agar in 500 mL of distilled water, slightly heating while mixing for complete dissolution on a burner. For the fungal organisms, SDA media was prepared by dissolving 27.5 g of the dry SDA in 500 mL of distilled water and heating slightly to ensure complete dissolution. Following this, the prepared agar solutions were sterilized in an autoclave. The sterile media on cooling was then poured into sterile petri dishes and allowed to solidify in the plates.

The stored organisms were allowed to stabilize under room temperature. Using an inoculating loop, each of the organisms was transferred into a tube containing 5 mL peptone water and adequately mixed afterwards to form a broth. The organisms were incubated for 24 hours at 37 C after which they were standardized to MacFarland turbidity by visual mean to obtain an organism density of 106 cfu/ mL. The prepared Staphylococcus aureus and Pseudomonas aeruginosa were streaked on petri dishes containing nutrient agar, while the standardized Aspergillus niger, Candida albicans and Trichophyton rubrum were streaked on previously prepared SDA plates using sterile cotton swabs15.

 

Antimicrobial Activity of Extract

Three concentrations: 200, 100 and 50 mg/mL of each of the methanol extracts of Vernonia amygdalina and Calotropis procera was first used to conduct a sensitivity test on each of the five tested organisms. The method used was the Agar Well Diffusion method16with a slight modification. To test for the sensitivity of Vernonia amygdalina extract against the bacterial and fungal organisms, three holes were bored into the prepared inoculated plates using a sterile cork borer size 6 mm. These wells were sealed with molten nutrient agar for the bacteria plates and molten SDA for the fungal plates. Then into each well, the prepared concentration was applied until each well was filled with the extract. This same procedure was repeated on extract concentrations of Calotropis procera. The plates now with the extract were allowed to stand for 1 hour after which the bacterial plates were incubated for 24 hours at 37 C and the fungal plates for 72 hours at 25 C.

From the sensitivity test, an extract concentration of 200 mg/mL of Vernonia amygdalina and Calotropis procera was selected for the antimicrobial activity test. For this test, 200 mg/mL of Vernonia amygdalina, 200 mg/mL of Calotropis procera, and combinations of these extracts in the ratio 75:25, 25:75 and 50:50 at a concentration of 200 mg/mL were tested against Staphylococcus aureus, Pseudomonas aeruginosa, Aspergillus niger, Candida albicans and Trichophyton rubrum using the agar well method earlier described. Zones of inhibition were measured after 24 hours for the bacterial organisms and after 72 hours for the fungal organisms using a transparent vernier caliper. Each of these tests was conducted in triplicates. Griseofulvin was used as the positive control for the fungal tests; Amoxicillin/Clavulanate was used as positive control for the bacterial tests and 30% DMSO solution as the negative control.

 

Statistical Analysis

Results are presented as mean ± standard deviation. Statistical comparison was done by one way analysis of variance (ANOVA). The difference between the means of zones of inhibitions of the different formulation and control was also evaluated. The statistical analysis was carried out with Graph pad prism version 7.0 software. Significant differences were set at p values lower than 0.05.

Null Hypothesis (H0): There is no significant difference between the means of the antimicrobial activities of the extracts in their various combinations and the control drugs used. Alternate Hypothesis (HA): There is a significant difference between the means of the antimicrobial activities of the extracts in their various combinations and the control drugs used.

 

RESULTS

 The methanol extract of Vernonia amygdalina was dark green in colour, sticky to touch and had an astringent odour.  The pH of the extract was 4.6 ± 0.14.  The methanolic extract of Calotropis procera was light green in colour, rough to touch, and had a leaf-like smell. Its pH was 5.1 ± 0.22. The extract combined was greenish and had an overwhelming bitter leaf odour. The concentrations of extracts that had activities by inhibition zones around the extract wells of the three concentrations of 50, 100 and 200 mg/mL tested are presented in Table 1. Based on the sensitivity of the various organisms to the extracts mostly at 200 mg/mL, the extracts were combined in five different combination ratios at 200 mg/mL and the results obtained expressed as inhibition zones in millimeters are presented in Table 2. At 200 mg/mL of the extracts, the extract combination ratio of 75: 25 Vernonia: Calotropis (V: C) gave the broadest zone of inhibition across all organisms, followed by the Vernonia only extract of V: C ratio 100: 0.

 

 

 

 

 

Table 1: Organisms’ Sensitivity to Extract Concentrations

Organisms

Extract Concentrations of Calotropis procera

Extract Concentrations of Vernonia amygdalina

Staphylococcus aureus

200 mg/mL

100 mg/Ml

Pseudomonas aeruginosa

200 mg/mL

200 mg/mL

Candida albicans

200 mg/mL

200 mg/mL

Aspergillus niger

100 mg/mL

200 mg/mL

Trichophyton rubrum

200 mg/mL

200 mg/mL

 

 

Table 2: Means of Zones of Inhibition of Extract Combinations against Organisms

Extracts

                             Zones of Inhibition in mm

V: C

1

2

3

4

5

100: 0

18.33 ± 0.53

16.67 ± 1.11

14.33 ± 0.74

30.00 ± 0.00

16.00 ± 0.53

75: 25

18.00 ± 1.70

20.67 ± 1.06

18.33 ± 0.42

28.00 ± 1.14

22.43 ± 0.47

50: 50

12.00 ± 1.12

12.00 ± 0.82

15.00 ± 1.06

25.50 ± 0.25

20.33 ± 0.82

25: 75

5.00 ± 1.24

15.67 ± 0.68

10.00 ± 1.00

14.00 ± 0.81

8.00 ± 0.26

0: 100

8.50 ± 0.01

24.00 ± 0.43

16.67 ± 0.72

16.00 ± 0.25

12.00 ± 0.00

A

0.00

0.00

0.00

0.00

0.00

B

24.00

20.00

-

-

-

C

-

-

20.00

25.00

22.00

 

Legend:

V: C

Ratio of Vernonia amygdalina to Calotropis procera

1

Activities against Staphylococcus aureus

2

Activities against Pseudomonas aeruginosa

3

Activities against Aspergillus niger

4

Activities against Candida albicans

5

Activities against Trichophyton rubrum

 

Statistical Analysis

The mean of activities of each extract obtained as the average of their inhibition zones was compared against the five test organisms for the extracts and controls. Result of analysis of variance gave a P value of 0.0114. This implies that there is a significant difference between the average antimicrobial activities of the extracts in their various combinations and the control drugs. The alternate hypothesis is therefore taken and the null rejected. In addition, the results obtained are significant at p < 0.05.

DISCUSSION

The medicinal applications of plants and their derivatives for the management of various ailments; topical and systemic is gaining more grounds17. Vernonia amygdalina is a popular medicinal plant, especially in West Africa. Its extract has proven antimicrobial activities18.

 

Similar is Calotropis procera, known especially for its anti fungal activities19. These attributes informed the selection of these plant species for the possibilities of combining them to achieve greater antimicrobial results. In proving their individual activities, an antimicrobial activity screening was first conducted on the methanolic extract of these plants. The choice of methanol as the extraction solvent was on account of relatively high extraction yield as compared to other solvents from literature search. The plant extracts were tested at three concentrations of 50, 100, 200 mg/mL to determine which concentration was best suitable for the combined formulation on accounts of their sensitivities against five test organisms reputable for their involvement in various skin infections. As shown in Table 1, of these concentrations, the 200 mg/mL showed activity all across the organisms in the agar well sensitivity test, with very little or no inhibition at all occurring at lower concentrations. On this basis, the 200 mg/mL concentration was selected and at this concentration, Vernonia amygdalina and Calotropis procera were combined and tested at different ratios and individually against Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Aspergillus niger and Trichophyton rubrum. The essence of this combination tests was to evaluate and observe the activities of the extracts when combined as compared to when used individually. It was also imperative to determine what combination ratio was most adequate and most productive. Results of this test as presented in Table 2 showed that the combination of the extracts in the ratio 75: 25 Vernonia to Calotropis (V: C) broadened the spectrum of activity of these extracts against both bacterial and fungal organisms at zones of inhibition ranging from 18 mm to 28 mm. This activity ranked first on comparism with other combinations and even the extracts in isolation. This was seconded by the individual activity of 100% Vernonia amygdalina with lower activities against the fungal organisms with the exception of Candida albicans. As compared with the control antibiotics used, the extracts especially at V: C ratio 75: 25 and 100: 0 had activities similar to and for some organisms greater than the controls. This demonstrates the potency of the extract combination.

 

CONCLUSION

The methanol leaf extracts of both Vernonia amygdalina and Calotropis procera have antimicrobial activities. The combination of these extracts in the ratio 75: 25 Vernonia to Calotropis results in a greater degree of activity and a broader spectrum of action against microorganisms. This outcome can be a basis for combining the two extracts in a topical formulation for the management of skin infections.

 

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