Phytochemistry and Antimicrobial Properties of Gmelina arborea (Verberaceae) Ethanolic Leaf Extract and its Secondary Metabolites

Preparation of Gmelina arborea Leaf Extract

Seventy (70) g of sun dried leaves of G. arborea were ground and put in a soxhlet extractor fitted with a reflux condenser and extracted with 250 ml of ethanol for 6 hours. The ethanol extract was allowed to evaporate completely at room temperature to give a gel, which was dissolved in ethanol/water mixture (4:1) and filtered. The filtrate was used without further purification for the phytochemical screening, antimicrobial experiments and preparation of acidic, basic and neutral metabolites.

Preparation of Basic Metabolite

This metabolite was prepared as follows; the filtrate of G. arborea leaf extract was treated with dilute HCl and extracted with chloroform in a separatory funnel. The lower chloroform layer was removed and reserved for the preparation of neutral metabolite. The HCl layer was treated with NaHCO3 until the mixture became basic. The precipitate from the resultant solution was filtered out and washed well with distilled water. The ppt was dissolved in 40 ml of 95% ethanol and filtered. The filtrate was used for antimicrobial experiments without further purification [18].

Preparation of Neutral Metabolite

The chloroform layer obtained above was placed in a separatory funnel and treated with dilute NaOH solution. After equilibrating, the aqueous NaOH layer was removed and reserved for preparation of acidic metabolite. The chloroform layer was removed and allowed to evaporate completely at remove temperature to produce a gel, which was dissolved in 40 ml of chloroform and filtered. The filtrate was used without further purification for antimicrobial experiments [18].

Preparation of Acidic Metabolite

The aqueous alkaline layer obtained above was treated with dilute HCl until the solution became acidic. The precipitate of the mixture was filtered out and washed well with distilled water. The ppt was dissolved in 40 ml of 95% ethanol and filtered. The filtrate was used for antimicrobial experiments without further purification [18].

Phytochemical Screening of Extract and Metabolites

Preliminary phytochemical screening of the extract and metabolites was performed using standard methods [19] which showed that the crude extract contained alkaloids, flavonoids, tannins, saponins, cyanogenic glycoside, steroids, carbonyl compounds and carbohydrates whereas the basic metabolite showed presence of alkaloids, cyanogenic glycosides and saponins. The acidic metabolite showed the presence of flavonoids, saponins and tannins while the neutral metabolite gave positive results for presence of steroids, esters, carbonyl compounds and carbohydrates.

Antimicrobial Tests

The antimicrobial experiments were carried out in microbiology Department, Federal Medical Centre Owerri, Imo State of Nigeria. Test microorganisms used were Streptococcus spp, Staphylococcus aureus, Salmonella spp, Escherichia coli and Coliform bacilli and the method used was Agar disc diffusion method. An inoculating loop was touched on five isolated colonies of the pathogen on an agar plate and used to inoculate a tube of culture broth, which was incubated at 35-37°Ç until it became slightly turbid and was diluted to march the turbidity standard. Then a sterile cotton swab was dipped into the standardized bacterial test suspension and used to evenly inoculate the entire surface of the agar plate. After 5minutes, the appropriate antibiotic test disks were placed with a multiple applicator device, and the agar plate was incubated at 35-37°c for 16-18 hours. The inhibition zone diameter (areas showing little or no microbial growth) were measured to the nearest mm [20] and taken as a measure of the antimicrobial activity.

Determination of Minimum Inhibitory Concentration (MIC)

The determination of the minimum inhibitory concentration (MIC) was carried out to obtain an idea of the antibacterial activities of the plant extracts, since agar disc diffusion assay is a quantitative method based on the European Society of Clinical Microbiology and Infectious Diseases (2000) for evaluation of antimicrobial potentials. Standard solutions of the metabolites were prepared: 1.0 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.125 mg/ml and 0.0625 mg/ml, in agar nutrient and distributed into sterile test tubes. 1ml of the extract or metabolite dilution was separately added into the agar plate for the bacteria and poured into Petri-dishes. The test microorganism was spotted onto the surface of the solidified extract-agar mixture and the plates were inoculated, starting from the lowest concentration to the highest concentration. After inoculation the plates were allowed to dry for 30 minutes and incubated at 37°C for 18 hours, after which the samples were examined for microbial growth. The lowest concentration of the extract or metabolite which showed little or no visible growth of the microorganism was taken as the MIC [20].

Phytochemical Screening of Extract and Metabolites

Results of phytochemical screening of the extract and various metabolites are presented (Table 1).

Table 1: Phytochemical Screening of Extract and Metabolites. +++= Strongly Positive; ++=Positive; +=Fair; -=not detected The crude extract showed presence of tannins, saponins, flavonoids, alkaloids, glycosides, Aldehydes, ketones, carbohydrates, Anthraquinones, phenols, carboxylic acids and esters. The acidic metabolite showed presence of tannins, saponins, flavonoids, phenols and carboxylic acids while the basic metabolite showed presence of saponins, alkaloids and glycosides. The neutral metabolite contained steroids/ terpenoids, Aldehydes, ketones, carbohydrate, Anthraquinones and esters.

These phytochemicals present are in accordance with previous work done by other researchers [21]. Luteolin (flavonoid) and gmelinoside (iridoid glycoside) have been found in the leaves of G.arborea which is responsible for its anti-inflammatory and anti-carcinogenic effect [9, 22]. Flavonoids and tannins compounds have been attributed to have an anti-oxidiant property which is why the plant is used as anti-cancer [23]. Quercetin (flavonoid), anthracene and tannins which are obtained from the G.arborea plant is used to treat hypertension [21]. These features can be attributed to the plant been used as a medicinal plant since the result obtained from these analysis contain these phytochemicals.

The presence of triterpenoids in natural products have been proved to possess anti-inflammatory and antimicrobial effect [24] also, medicinal plant containing saponins, glycosides, flavonoids, tannins and phenolics have demonstrated antidiabetic activities [25]. Anthraquinones in plant have been discovered to have therapeutic effects which include constipation, arthritis and multiple sclerosis [26] and can be trace to the application of Gmelina arborea as anti- inflammatory, antimicrobial, antidiabetic and therapeutic effect.

These phytochemicals contained in the metabolites are substances that control cell growth and division, reduce inflammation, promote formation of blood cells and fight infections [27]. We cannot say (for now) which of these bioactive compounds was responsible for the observed antimicrobial properties of the crude extract, acidic metabolite and/or neutral metabolite. However, five principal bioactive compounds such as alkaloids, saponins, steroids, flavonoids and glycosides were investigated in methanol and chloroform extracts of Gmelina arborea [10]. Thus, it may be speculated that the flavonoids, saponins, tannins, steroids/terpenoids and Anthraquinones which were found to dominate in the acidic and neutral metabolites may be responsible for the observed antimicrobial activity/ potential because they have been shown to possess bactericidal, fungicidal and medicinal properties [13].

Preliminary Antimicrobial Screening

The results of preliminary antimicrobial screening of the crude extract and various metabolites of Gmelina arborea are shown (Table 2) from which it was observed that only the crude extract, acidic and neutral metabolites possessed antimicrobial activity against the microorganisms tested to various extents whereas the basic metabolite showed no activity against the microorganisms.

Antimicrobial Activities of Extract and Metabolites

The antimicrobial activities of the crude extract and metabolites were presented (Table 3) from which it was seen that the antimicrobial activities of the extract and metabolites against the organisms were close to each other and their inhibition zone diameters ranged from 15-20 mm (for crude extract), 25-35 mm (for acidic metabolite) and 22-30 mm (for neutral metabolite) at 1.0 mg/ml. The data showed that the crude extract exhibited its greatest activity against Streptococcus spp, Staphylococcus aureus and Escherichia coli with inhibition zone diameter of 20 mm at 1.0 mg/ml. The least activity was against Salmonella spp with inhibition zone diameter of 15 mm. The acidic metabolite exhibited its greatest activity against Escherichia coli with inhibition zone diameter of 35 mm at 1.0 mg/ml. The least activity was against Staphylococcus aureus and Salmonella spp with inhibition zone diameter of 25 mm at 1.0 mg/ml respectively. The neutral metabolite exhibited its greatest activity against Salmonella spp and Coliform bacilli with inhibition zone diameter of 30 mm at 1.0 mg/ml. the least activity was against Staphylococcus aureus with inhibition zone diameter of 22 mm at 1.0 mg/ml. the data presented showed that the acidic metabolite and the neutral metabolite exhibited almost the same activity against the test microorganisms especially against Coliform bacilli where they have the same inhibition zone diameter of 30 mm at 1.0 mg/ml respectively. It was also observed that the basic metabolite showed no activity against the microorganisms tested. These differences in bioactivity arose probably from differences in phytochemical composition of the various metabolites [18, 28, 29]. The result of the control experiment with the following drugs Augmentin, Ciprofloxacin, Gentamycin, Cefuroxine and Ceftriaxon against the test microorganisms were presented (Table 3b) showing their different inhibition zone diameters.

Table 3b: Control Drugs.

Minimum Inhibitory Concentration of Extract and Metabolites

The MIC of the extract and metabolites were presented (Table 4) from which it was observed that the crude extract and the neutral metabolite exhibited MIC of 0.5 mg/ml respectively against the entire test microorganism. The acidic metabolite exhibited MIC of 0.5 mg/ml against Staphylococcus aureus and Salmonella spp while the MIC against Streptococcus spp, Escherichia coli and Coliform bacilli was 0.25 mg/ml respectively.

Over the centuries, man made use of medicinal plants/ herbs even though he was unable to find a rational explanation for their effect, it wasn’t until the 19th century and the rapid development of organic chemistry and pharmacology, that man determine which active principle are responsible for a given therapeutic effect. The outcomes of this, is that there has been a marked renewal of medicinal interest in classic physiotherapy [30]. Man has also searched for remedies to problems of infections and diseases by the use of chemical substances that can eradicate or at least minimize the effects of the microorganisms without harming the host. Many antibiotics have also been used as preservatives for raw foods, especially protein foods like meat, fish, poultry, etc but microorganisms soon became resistant to these antibiotics and new strains of these organisms developed WHO Report.

Hence, the use of medicinal plant extracts and metabolites became necessary [31].

The crude leaf and stem bark extracts of G. arborea have being reported to show significant antimicrobial activities against Gram positive and Gram negative organisms and the activity is due to presence of bioactive compounds such as alkaloids, saponins, carbohydrates, phenolics, tannins and anthraquinone in leaf while the stem back possessed saponins, carbohydrates, alkaloids, tannins and anthraquinone but no phenolics [32]. In vitro studies of both extract of leaf and stem bark shown significant activity against E. coli, K. pnemoniae, P. dysentria and S. typhi. It have been suggested that the presence of saponins, tannins, flavonoids, phenolics, alkaloids and anthraquinone in the leaf extract of G. arborea are responsible for the antimicrobial action of the metabolites[33, 34, 35, 36]. The crude extract had been reported to facilitate wound- healing due to the presence of tannins in the extract because tannins and tannic acids owe their astringent action to their ability to precipitate proteins and this forms a protective coating over the wound, prevents external irritation and promotes healing [12].

Hence, the observed antimicrobial activities of the crude extract, acidic metabolite and neutral metabolite against the tested microorganisms may be due to tannins or other phenolics, saponins, flavonoids, steroids and anthraquinone found in this study to be present in the metabolites or due to alkaloids present in the basic metabolite that was found in this study to lack antimicrobial activity against the tested microorganisms. Nevertheless, more work is to be done to isolate and characterize the active principle responsible for the observed antimicrobial potential of the ethanolic leaf extract.