Journal of Microbial & Biochemical Technology

Evaluation of the Antimicrobial Activity of Erythrina abyssinica Leaf Extract

Wadzanai Chitopoa, Idaiishe Muchachaa and Rumbidzai Mangoyi^* Department of Biochemistry, University of Zimbabwe, Mt. Pleasant, Harare, Zimbabwe
^*Correspondence: Rumbidzai Mangoyi, Department of Biochemistry, University of Zimbabwe, Mt. Pleasant, Harare, Zimbabwe, Tel: 00263773650560, Email:

Received: 13-Mar-2019 Published: 17-Apr-2019

Introduction

About 80% of the African population uses herbal medicine for the treatment of known and unknown illnesses, and this has increased the world market for herbal medicine to approximately US 60 billion [1]. Different plant parts can be used as source of medicine and can be consumed raw, cooked, boiled or administered as decoction, syrup, poultice, infusion, or pounded paste which is applied topically, used in herbal baths or taken orally. Erythrina abyssinica is a plant commonly found in Zimbabwe, Sudan and Ethiopia south to Angola and Mozambique and is well known as an herbal plant used by traditional healers [2]. The bark is commonly used in traditional medicine to treat snakebites, malaria, sexually transmitted diseases like syphilis and gonorrhoea, amoebiasis, cough, liver inflammation, stomach ache, colic and measles.

Roasted or powdered bark is applied to burns, ulcers and swellings. The liquid from crushed bark of green bark is used to treat conjunctivitis caused by chlamydia trachomatis (trachoma). The sap is drunk as an anthelmintic and against vomiting. The pounded flowers are used to treat dysentery and a maceration of flowers is drunk as an abortifacient, also applied externally to treat earache [3]. The roots are taken to treat peptic ulcers, epilepsy, malaria, blennorrhagia and schistosomiasis. The leaves are also used to treat peptic ulcers diarrhoea and applied to wounds, painful joints and skin diseases. The fruit of the Erythrina abyssinica are also taken to treat asthma and meningitis. Thus, this study focused on validating the use of Erythrina abyssinica as a medicinal plant by investigating its antimicrobial activity against C. albicans and S. aureus.

Candida albicans is an example of dimorphic fungi and is the most cause of systemic mycoses. Candida albicans is a member of the human gut flora found at about 40% to 60% in healthy humans. However under favourable conditions, C. albicans can become pathogenic and can infect the skin, genitals, mouth and blood [4]. Staphylococcus aureus is another microorganism which frequently causes infections such as skin, wound and deep tissue infections and is responsible for some life threatening conditions like pneumonia, endocarditis and septicemia. The bacterium is one of the most common causes of nosocomial infections worldwide and has been reported to be multidrug resistant [5].

Materials and Methods

Chemicals and reagents

Chemicals and reagents used in this study were purchased from Sigma-Aldrich, USA. Reagents used include, 3-(4,5-dimethylthiаzol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT), nutrient agar, nutrient broth, absolute methanol, hexane, ethyl acetate, tetrazolium, ethanol, glycerol and sodium chloride. Distilled water was obtained from the department of biochemistry.

Plant collection and identification

Erythrina abyssinica bark was collected from Glen Norah suburb in Harare Province (Coordinates 17.9113oS, 30.9790oE) and was identified by a botanist at Harare Gardens. The bark was allowed to sun dry, then was pulverised by use of а mortar pestle, weighed and then stored at room temperature.

Extract preparation

The dried powdered bark of E. abyssinica was soaked overnight in absolute methanol in a ratio of 1:4, for the extraction of its constituents. The extract was filtered and allowed to evaporate at room temperature. About 75% of the dried methanol extract was dissolved in 30% methanol and solvent-solvent extraction followed. Different solvents were added to the 30% methanol extract in their increase in polarity and constituents were extracted according to their polarity. The solvents used for the extraction were hexane, ethyl acetate and dichloromethane. The extracts were dried, weighed and stored at room temperature in air tight bottles.

Test organisms

Glycerol stocks of the test organisms, S. аureus and C. albicans were provided by Prof. S. Mukanganyama from the Department of Biochemistry, University of Zimbabwe. The microorganisms were resuscitated by culturing them overnight in nutrient broth at 37°C and 150 rpm. S. аureus and C. albicans were subculture on nutrient agar in а 37°C non shaking incubator overnight. Some glycerol stocks were prepared and stored in a refrigerator at 4°C.

Agar disc diffusion assay

S. aureus and C. albicans cells from glycerol stocks were grown overnight at 37oC, 150 rpms in 20 ml of nutrient broth. Cells were then inoculated in nutrient agar to a final concentration of 1x106 CFU/ml. Each dried plant extract was dissolved in absolute ethanol and a certain volume (equivalent to 500 μg) was impregnated on whatman paper discs (6 mm in diameter). The extracts on paper discs were allowed to dry and then placed on inoculated agar in petri dishes. The petri dishes were refrigerated at 4°C for 2 hours to allow the diffusion of the extracts into the agar. The plates were then incubated overnight at 37°C. Growth or inhibition of growth was observed the following day.

Broth dilution assay

A volume of 100 μl of fresh broth cultures of 1x106 CFU/ml of each microorganism were placed in 96 well plates. Plant extracts (100 μl) prepared by two fold dilution from 500 μg/ml to 0 μg/ml were added to cells in 96 well micro titre plates. The plates were labelled respective of the plant extracts. Distilled water was added to give a final volume of 300 μl in each well. The negative controls contained nutrient broth only and the positive controls contained cells only. Miconazole, a known antifungal and erythromycin, an antibiotic were also tested under the same conditions. The plates were covered with a parafilm and incubated at 37°C overnight. Viability of cells was then determined by adding 25 μl of a tetrazolium salt, MTT at 2 mg/ml. Plates were left to stand for three hours and colour change was observed. MTT assesses the viability of live cells through a reduction reaction of the yellow tetrazole to give purple formazan with the intensity depending on the concentration of the cells present. This reaction is catalysed by oxidoreductase enzymes in viable cells. Thus, the intensity of the colour was measured using a spectrophotometer at 540 nm. The lowest concentration of the extract that inhibited growth was recorded as the MIC. Minimum bactericidal concentration and minimum fungicidal concentration were determined by re-culturing cells from wells that had no viable cells present from the MTT assay. Cells were streaked onto nutrient agar and incubated at 37°C overnight. Growth of the cells on agar shows presence of viable cells in the well. The lowest extract concentration which showed no growth of bacteria or fungi on the media was the MBC or MFC value.

Haemolytic assay

A volume of 5 ml of sheep blood in EDTA was centrifuged at 1000 rpm and 4°C for 10 minutes. The supernatant and buffy coat layer was carefully discarded. The pellet collected was washed 3 times with phosphate buffered saline (PBS). The erythrocyte pellet (100 μl) was diluted using 900 μl of PBS and 50 μl of the diluted sample was mixed with 100 μl of plant extract. This was done for plants that showed antimicrobial activity against both S. aureus and C. albicans, at their MIC values. For the positive control, 50 μl of diluted erythrocytes were mixed with 10% Sodium Dodecyl Sulphate (SDS). The negative control had 100 μl of PBS. The contents were then incubated at 37°C for 60 minutes. After incubation 850 μl of PBS were added to each reaction vessel and thoroughly mixed. The tubes were centrifuged at 300 rpm for 3 minutes and the supernatant was put into respective wells of a 96 well ELISA plate. The absorbance was read at 545 nm. Percentage lysis was determined using the positive control as 100% lysis. Percentage haemolysis of the extracts was calculated by the formula:

(Absorbance of red blood cells with plant extracts/Absorbance of red blood cells with SDS)*100%

Results

Plant extractions

The total mass of the pounded Erythrina abyssinica bark powder was 37 g. The methanol extract obtained was 5.6 g and 75% of it was further partitioned by solvent–solvent extraction. The solvents used in solvent-solvent extraction were hexane, dichloromethane and ethyl acetate. The yields of plant extracts obtained are shown in Table 1.

Table 1: Yields of different extracts of Erythrin abyssinica.

Inhibition of growth of S. aureus and C. albicans by E. abyssinica extracts

The effects E. abyssinica extracts on the growth of S. aureus and C. albicans is shown by zones of inhibition in Table 2. All the extracts inhibited both species with C. albicans being inhibited the most. Although all the extracts inhibited both species, the effects were less than that of positive controls, erythromycin and miconazole. The ethyl acetate extract showed the highest zone of inhibition of 25 mm and dichloromethane showed the least with 12 mm against C. albicans. On the other hand, the hexane extract showed the highest inhibition zone of 23 mm and dichloromethane showed the least with 11 mm against S. aureus.

Table 2: Effects of Erythrin abyssinica bark extracts by disc diffusion assay on S. aureus and C. albicans.

MIC and MBC determination

The dichloromethane and hexane extracts of E. аbyssinicа were found to have MICs of 62.5 μg/ml whilst methanol and ethyl acetate had 125 μg/ml against C. albicans (Table 3). The MBC values were found to be 500 μg/ml for all the extracts except for ethyl acetate which had 250 μg/ml (Table 4). Dichloromethane and hexane extracts were also found to have the MIC values of 15.6 and 31.25 μg/ml respectively against S. aureus (Table 3). The MBCs values were more than the highest concentration tested, >500 μg/ ml for all the extracts except for hexane which had 31.25 μg/ml (Table 4).

Table 3: Minimum inhibitory concentrations for Erythrinа аbyssinicа bark extracts against S. aureus and C. albicans.

Table 4: Minimum fungicidal concentrations and Minimum bactericidal concentrations for Erythrinа аbyssinicа bark extracts against S. aureus and C. albicans.

Toxicity effects

Haemolysis assay was carried out to assess the effect of the active extracts on mammalian (sheep) red blood cells. The haemoglobin released by red blood cells when they lyse was measured at 545 nm against a positive control of 10% SDS. The MICs of plant extracts obtained against both species were used to determine haemolytic activity. Table 5 shows that all tested extracts had haemolytic activity less than 50%.

Table 5: Effect of Erythrinа аbyssinicа on sheep erythrocytes.

Discussion

A wide range of active compounds were extracted from the bark of E. abyssinica using solvents of increasing polarity. All the plant extracts showed antimicrobial activity against both S. aureus and C. albicans by the agar disc diffusion assay. The highest inhibition zone was 25 mm for ethyl acetate extract and the lowest inhibition zone was 12 mm for dichloromethane extract against C. albicans. Hexane showed the highest zone of inhibition of 23 mm and dichloromethane the lowest of 11 against S. aureus. Minimum inhibition concentrations and the minimum fungal concentrations of the extracts were determined using the broth dilution assay. The technique is quick, sensitive, and quantitative with reproducible results. The MICs for all the plant extracts ranged from 62.5 μg/ μl to 125 μg/μl and the MFCs for most extracts were 500 μg/μl against C. albicans. However, results showed that most of S. aureus grew when re-cultured suggesting that Erythrina abyssinica extracts can only inhibit the Staphylococcus aureus bacteria, but does not kill. Only the hexane extract showed an MBC of 31.25 μg/ml. Variation in the degree of activity were because of the different constituents and structures of natural products present [6].

Haemolysis assay was carried out to investigate the toxicity of the E. abyssinica extracts. The assay is based on the destruction of the membrane lipid bilayer of red blood cells exposing haemoglobin. The haemoglobin absorbs light at 545 nm and is detected by a spectrophotometer [7]. Haemolysis is related to the concentration and potency of the extract, the more the active biomolecules absorbed the more the lysis [8]. Coloured extracts absorb light at 545 nm and may lead to false positives [9]. Methanol extract showed the greatest haemolysis of 39.7% at the MIC of 125 μg/ ml. The toxicity of the methanol suggests the extract may not be suitable for drug development. The other three extracts showed tolerable haemolysis at 62.5 μg/ml as the MIC and may be suitable for drug development. Haemolysis for other extracts was hexane 15.5%, ethyl acetate 15.4% and DCM with the lowest at 9.1%.

Conclusions

The results of the study showed that E. abyssinica may have antimicrobial activity against C. albicans and S. aureus. This therefore verifies the use in the treatment of the bark by traditional healers. The extracts may not be toxic at minimum inhibitory concentrations.

Acknowledgment

This study was sponsored by the Department of Biochemistry at the University of Zimbabwe, and the University of Zimbabwe Research Board (Harare, Zimbabwe) is also acknowledged.

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