Antimicrobial Compounds and Antimicrobial Activity of Extracts of Thespesia garckeana F. Hoffm. on Candida albicans , Staphylococcus aureus and Escherichia coli Archives of Ecotoxicology

Plant products as antimicrobials have received serious attention in the recent years. Herbal medicines form a significant role in African ethnomedicine. Thespesia garckeana F. Hoffm. is an important herbal medicinal plant used to treat or manage human diseases and ailments such as chest pains, cough, infertility and sexually transmitted infections. However, its antimicrobial compounds and activity analysis has not been evaluated. This study aimed at evaluating the phytochemical and antimicrobial activities of compounds in Thespesia garckeana leaves and bark extracts on growth of C. albicans , S. aureus and E . coli . The plant leaves and bark were collected from Mwingi, Kitui County of Kenya and transported to Maseno University department of Botany laboratory, where they were dried separately and crushed to obtain a fine powder. Crude plant extraction was done using soxhlet method with methanol and water solvents. Thin layer chromatography was used to purify the extracts and phytochemical screening was done using standard methods to determine the compounds present in the extracts. Thin layer chromatography was carried on bark and leaves extracts of Thespesia garckeana . Thin layer chromatography plate surface was coated with silica gel, and n-Hexane and Ethyl acetate used as solvent systems in the ratio 2:3 and 3:7 respectively. Antimicrobial activity was performed on test microorganisms using different concentrations of the plant extracts; 0 (distilled water), 90, 180, 270 and 360 mg/ml of the extract. Paper disc method was used for inoculation with three replications. Diameter of inhibition zones was measured using a transparent ruler. The data on growth inhibition was subjected to analysis of variance (ANOVA), and the treatments were separated and compared using LSD at p<0.05. The phytochemical compounds screening of leaves and stem bark extracts of Thespesia garckean revealed the presence of Tannins, Saponins, Cardiac glycosides, and Alkaloids. TLC using n-Hexane and Ethyl acetate ratio of 2:3 methanol leaves extracts produced 9 spots with Rf values of 0.94, 0.9, 0.81, 0.71, 0.35, 0.25, 0.2, 0.125 and 0.075 respectively. The stem methanol extracts produced 4 spots with Rf values of 0.99, 0.96, 0.9, and 0.06 respectively. Leaf methanol extracts using n-Hexane and Ethyl acetate in the ratio of 3:7 produced 8 spots with Rf values of 0.95, 0.89, 0.78, 0.46, 0.36, 0.3, 0.19, and 0.1 respectively. The stem bark methanol extracts produced 2 spots with Rf values of 0.97 and 0.94 respectively. Increase in extracts concentration of the leaves and stem bark of Thespesia garckeana, significantly inhibited the growth of Candida albicans, Staphylococcus aureus and E. coli. Methanol leaf and bark extracts had more inhibitory activity compared to the water extracts. The results from this study confirm the use of Thespesia garckeana in herbal medicine by traditional herbalists.


Introduction
Plants convert simple substances into complicated entities producing chemicals that are essential for human health  et al., 2015). The emergence of pathogenic micro-organisms that are resistant or multi-resistant to major classes of antibiotics has increased in recent years due to the indiscriminate use of synthetic antimicrobial drugs. Emerging and re-emerging infections and microbial drug-resistance pose a challenge to the global public health ( Thespesia garckeana has long been used by Mbeere community in Eastern Kenya region to treat chest pains and cough (Keter and Mutiso, 2017). The plant is only regarded as a wild tree and its utilization in most parts of Kenya is poor hence the need for this study. Due to high cost of manufactured synthetic drugs in the market it's hard for local communities to access these drugs due to poverty. Besides, chemosynthetic drugs are associated with resistance, so more natural alternative drugs are required. There is need to investigate the potential antimicrobial activity and phytochemical constituents of plants. The essential values and uses of some medicinal plants has been worked out and published, but many of them remain unexplored in Kenya (Sujit and Amol, 2014). Despite the penetration of conventional medicines, traditional medicine continues to be a feasible health care alternative for the majority of the Kenyan population (Keter and Mutiso, 2017). Although Thespesia garckeana has been greatly tested on antimicrobial activity but very few studies have been conducted in Kenya especially on Candida albicans, Staphyllococcus aureus and Escherichia coli. There is scanty of information on the antimicrobial activity of Thespesia garckeana on Candida albicans, Staphylococcus aureus and Escherichia coli in Kenyan studies hence there is need to screen this plant against the three microbes. This study aimed at investigating the phytochemical and antimicrobial properties of leaf and bark extracts of Thespesia garckeana on Staphylococcus aureus, Escherichia coli and Candida albicans.

Plant collection, identification and preparation
Plant bark and leaves of Thespesia garckeana were collected from Mwingi, Kitui County-Kenya on July 2021 (plate 1). The leaves and stem bark of Thespesia garckeana were cleaned off the soil or dust by shaking, and were packaged inside carrier bags then transported to Maseno University, department of Botany for identification by the taxonomists. The leaves and stem bark were air-dried separately under shade for two weeks and pulverized using a wooden mortar and pestle to obtain a fine powdered-like texture (plates 2 and 3). According to WHO (1998), this is done to enhance the penetration of the extracting solvents into the plant cells, thus facilitating the release of the active principles. The pulverized plant samples were then stored in amber bottles and kept in a cool and dried environment under room temperature until it is was required for usage.

Phytochemical extraction
All work was done in accordance to the general guidelines and methodologies on research and evaluation of traditional medicine (WHO, 1998). The dried bark and leaves were extracted in Soxhelt apparatus by using 25ml of water and methanol for 48 hours and then were concentrated by evaporation. The extracts were used for phytochemicals analysis.

Preliminary phytochemical profiling
The bioactive constituents present in the methanol and aqueous extracts were screened for flavonoids, alkaloids, tannins, saponins, steroids, terpenoids and cardiac glycosides using Test for Tannins 0.5g of dried powdered sample was boiled in 20ml of water in a test tube and filter. A drop of 0.1% ferric chloride was then added to the filtrate. An observation of brownish green of blueblack coloration would indicate a positive test.
Test for Saponins 2g of powdered sample was mixed in 20ml of distilled water in a boiling tube then placed in a hot water bath for 5 minutes and then filtered. 10ml of the filtrate was mixed with distilled water and shaken vigorously for a stable persistent froth. The frothing was mixed with three drops of olive oil and shaken vigorously and formation of an emulsion observed.
Test for flavonoids About 5g of powdered plant sample was heated in a boiling tube with 10ml ethyl acetate over a steam bath for three minutes. They were then filtered and 4ml of the filtrate taken and shaken with 1ml of dilute ammonium solution. A yellow colouration observed would indicate a positive test for the flavonoids.
Test for Terpenoids To 0.5g each of plant extract in a test tube, 2ml of chloroform was added followed by 3ml of 0.1M Sulphuric acid to form a layer. A reddish brown colouration of the interface formation would indicate a positive test for terpenoids.
Test for Steroids 2ml of acetic anhydride was added to 0.5g ethanoic extract of each sample with 2ml of 0.1M Sulphuric acid in a test tube. The colour change from violet to blue or green in the sample would indicate the presence of sterols.
Test for Cardiac glycosides 5ml of each extract in a test tube was exactly treated with 2ml glacial acetate acid containing one drop of 0.1% ferric chloride solution. To this, concentrated hydrochloric acid was added. A brown ring of the interface would indicate a deoxy-sugar characteristic of cardinolides thus determine a positive test. A violet ring would also appear below the rings while in the acetic acid layer or greenish ring may just gradually throughout the thin layer.
Test for alkaloids A sample of 2g of dried plant extract was mixed with 40ml of 0.1M hydrochloric acid in a boiling tube and heated in a water bath for 10 minutes. The mixture was cooled and then filtered. To a portion of the filtrate, a few drops of Mayor's reagent were added. A slight turbidity of heavy precipitate was assumed to indicate the presence of alkaloids.

Thin layer chromatography (TLC)
Thin layer chromatography was carried on the two extracts (stem bark and leaves) of Thespesia garckeana according to Musa et al. (2017). Using a glass TLC plate whose surface was coated with silica gel, the TLC plate was cut into a size of 5cm by 10cm; a mark was made in pencil about 1.5cm from the lower edge of the end of the plate which was the origin. The dissolved extracts were spotted on the line creating a distance of about 0.5 to 1cm between each spot, the distance prevent the spots from overlapping or mixing while separating. Solvent was prepared in the development chamber which makes the solvent system by mixing n-hexane and ethyl acetate in the ratio of 2:3 and 3:7 (v:v) respectively in which the spotted plate was placed in such that the liquid solvent does not touch the origin (line marked in pencil) and spotted with dissolved extract. The solvent which is the eluent moved through the plate and went up by capillary action of the plate carrying the compounds present in the extract which separates and appears as spots on the plate. The compounds that were closer to the origin showed less movement and are polar solvent was marked in pencil which marks the solvent front. The spots would be visible but if not, the spots were seen by use UV lamp to enhance visualization at different wavelengths of 365nm and 254nm.Then the retention factor was calculated using the formula proposed by Mueller Hinton Agar (Oxford, UK) was prepared according to manufacturer's instructions, autoclaved and dispensed at 20ml per plate in 12 by 12cm petri dishes. Set plates were incubated overnight to ensure sterility before use.

Antimicrobial bioassays
Bioassays were prepared according to Prescott et al. (1999) and Bonjar (2004). Plant extracts (stem bark and leaves) were diluted in distilled water and in methanol respectively to make a stock solution. Different concentrations of the plant extracts were prepared by diluting the stock extracts in water and methanol to make solutions of the following concentrations in triplicates: 0, 90, 180, 270 and 360 and a positive control of fluconazole on fungi and Amoxicillin on bacteria. Each labeled medium plate was uniformly inoculated with a test organism by using a sterile cotton swab rolled in the suspension to streak the plate surface in a form that lawn growth can be observed. Antimicrobial activity of the test agents was determined by measuring the diameter of the zone of inhibition. The experiment was arranged in a completely randomized design.

Disk method
Circular discs, 6mm diameter each was cut from a laboratory grade filter paper punch and each dipped in a known concentration of the plant extracts for about 2 minutes. According to Musyimi et al. (2008). The diameter of inhibition zones was measured using a ruler and compared with the control paper disc.

Data Analysis
The data obtained from the study was subjected to analysis of variance (ANOVA) using SAS statistical package. Means were separated and compared at (p<0.05) for any significant difference between treatments and the micro-organisms.

Phytochemical screening
Phytochemical screening of the leaves and stem bark extracts (table 1) showed that the leaves and stem bark extracts of Thespesia garckeana contained alkaloids, cardiac glycosides, saponins, flavonoids, and tannins. However, steroids and terpenoids were absent. It was evident that phytochemicals were more in the leaves than in the barks. Table 1 Phytochemical screening of secondary metabolites present in leaves and bark extracts of Thespesia garckeana.

Thin Layer Chromatography (TLC)
Elution of the column with the n-hexane and ethyl acetate in 2:3 ratio led to isolation of nine fractions or spots of uncharacterized active compounds in leaves extracts (table 2). The leaf extracts recorded highest number of spots compared to bark extracts. The leaf extracts of ratio 3:7 recorded eight spots. The bark extracts of ratio 2:3 recorded four spots, and the bark extracts recorded least number of spots.

Antimicrobial Assays
Water and methanol leaf and bark extracts of Thespesia garckeana plants represented good activity against the test microbes (table 3). There was no significant differences (p>0.05) between the water extracts and methanol extracts (table 4). There was no significant difference (p>0.05) between the plant part used either the leaves or the barks. There was a significant difference (p<0.05) between the extract concentration whereby the more concentrated extracts showed a greater diameter of growth inhibition. The antibiotics (positive control) exhibited a greater zone of diameter inhibition. There was a significant difference (p<0.05) between the two microorganisms, Candida albicans and E. coli and Staphylococcus aureus. Whereby diameter growth of Staphylococcus aureus was more inhibited compared to Candida albicans and E. coli. Table 3 Mean growth inhibition zones of the microbes using methanol and water extracts Thespesia garckeana leaves and stem bark extracts.

Discussion
Searching for antibacterial and antifungal activity of the secondary metabolites from medicinal plants is a spotlight to date (Zuo et al., 2008). In Kenya, traditional medicines play a major role in primary healthcare and upkeep of rural communities (Kokwaro, 2009;Kisangau and Kokwaro, 2004). Plants are rich in a wide variety of secondary metabolites such as tannins, alkaloids, phenolic compounds, and flavonoids, which have antimicrobial properties (Duraipandiyan et al.,  2006). The medicinal value of plants lies in some chemical substances that produce a definite physiological action on human body (Musyimi et al., 2008). The most important of these bioactive compounds are alkaloids, flavanoids, and tannins  , 2015). The type of extraction method, duration of extraction, temperature and polarity of the solvent used influence the quality and concentration of bioactive components isolated from the raw materials (Nantitanon et al., 2010). Both

E. coli
Methanol extract 0mg/ml 6.00 ± 0.00 90 mg/ml 8.  . Phenolic compounds act as antimicrobial agents. Since methanol and water extracts had the same activity on the test microbes, the two solvents can be used for extraction of the phytochemicals from the leaves and the stem barks of this plant. Secondary metabolites have the potential as bactericidal, bacteriostatic, or fungicidal effect against human pathogens (Dholaria and Desai, 2018; Motaleb, 2011). Data from our results reveal the great potential of Thespesia garckeana plants for therapeutic treatment, in spite of the fact that they have not been completely investigated. Further studies should be undertaken to unveil the exact mechanism of action and reveal the drug targets of the microorganisms tested.

Conclusion
From this study the following it can be concluded that the leaves and bark of Thespesia garckeana are rich in phytochemicals especially the tannins, saponins, flavonoids, cardiac glycosides and alkaloids, thus are of medicinal value. TLC revealed that leaves methanol extracts had more spots compared to bark methanol extracts. Antimicrobial testing showed that both methanol and water extract of leaves and bark of Thespesia garckeana had a broad spectrum activity against E. coli, S. aureus and C. albicans. This study has confirmed the antimicrobial potency of the plant extracts, and explains why the plant is used by the traditional healers and the possibility to use the plant as a suitable alternative to synthetic antimicrobial agents.