Phytochemical Investigation and Antimicrobial Activity of Hexane, Ethyl Acetate and Methanol Fractions from Stem Bark of Icacina Trichantha Oliv. (Icacinaceae)

Medicinal plants have been used to treat health disorders for centuries all over the world in order to develop low cost and readily available therapeutic compounds (Elisabetsky, 1991). These bioactive constituents from plant fashion are potent antimicrobial agents used for combating microorganisms such as bacteria, fungi and protozoa (Krishnaiah et al., 2009). People are becoming increasingly attracted to the use of these plants because of their good therapeutic performance, toxicity, and affordability. Many research efforts have been directed towards the provision of empirical proofs to back up the use of plants species in medicinal practices (Ojo et al., 2005). Researchers have examined the effects of plants used Phytochemical Investigation and Antimicrobial Activity of Hexane, Ethyl Acetate and Methanol Fractions from Stem Bark of Icacina Trichantha Oliv. (Icacinaceae)


Introduction
Medicinal plants have been used to treat health disorders for centuries all over the world in order to develop low cost and readily available therapeutic compounds (Elisabetsky, 1991). These bioactive constituents from plant fashion are potent antimicrobial agents used for combating microorganisms such as bacteria, fungi and protozoa (Krishnaiah et al., 2009). People are becoming increasingly attracted to the use of these plants because of their good therapeutic performance, toxicity, and affordability. Many research efforts have been directed towards the provision of empirical proofs to back up the use of plants species in medicinal practices (Ojo et al., 2005). Researchers  traditionally by indigenous healers to support treatment of various diseases; scientific validations are being made globally to get evidence for traditionally reputed herbal plants. However, there are several plants with tremendous medicinal potentials that have not been investigated. In many developed countries, 70-80% of the population have used some forms of alternative or complementary medicine e.g. acupuncture, while in most African countries 80% of the population depend on traditional medicine for primary health care (WHO, 2002).
Icacina trichantha Oliv. an Icacinaceae, is a droughtresistant shrub indigenous to West and Central Africa. Natives of indigenous tribes in Nigeria, commonly use it as a medicinal plant. Among the Igbos in Nigeria, it is known as "Urumbia" or "Eriagbo" especially with respect to its emetic effect while Yorubas in western Nigeria, refer to it as "Gbegbe" which means to cleanse (Asuzu & Abubakar, 1995). The plant is a shrub that can grow up to two meters high (Burkill, 1994). It is characterized by large, fleshy, yam-like underground tubers, as big as several kilograms in weight, which are often consumable. The tuber is said to be rich in starch and can be eaten fresh or processed into flour to make soup, pastes or porridges (Umoh & Iwe, 2014). It has been reported to serve as a source of emergency food energy during long periods of drought or famine. Both the nutrient composition and "anti-nutritional factors of the flour have been analyzed, showing the presence of not only carbohydrates (mostly starch), lipids and proteins, but also mineral elements such as potassium, sodium and calcium (Umoh, 2013;Umoh & Iwe, 2014;Udofia et al., 2014). The fruit of this plant is a drupe with a soft sweet outer pulp which is edible (Burkill, 1994).
Ethnomedicinally, I. trichantha is widely used as a common household medicine for emergency and first-aid treatment for food poisoning (Mbatchou & Dawda, 2012). Tubers and leaves of the plant are allegedly aphrodisiacs (Burkill, 1994& Quattrocchi, 2012. The leaves and seeds, when crushed and macerated in local gin, can be used for the treatment of hypertension and asthma (Ajibesin et al., 2008). Tubers are said to be used by traditional healers to treat various medical conditions including constipation, poisoning, malaria, rheumatism, toothache, as well as to induce emesis and abortion (Asuzu & Abubakar, 1995;Ariwaodo et al., 2012). Ubom (2010), reported that the tuber juice can be used for treating mumps. Leaf methanol extract have anti-convulsion effect in mice pretreated with pentylenetetrazole, as well as increased pentobarbitoneinduced sleeping time (Asuzu & Aforonwa, 1998). The anti-inflammatory activity of the Chloroform extract of the tuber in Laboratory mammals have been reported (Asuzu et al., 1999). In vitro screening of the leaf, wood, and root parts of I. trichantha have reportedly exhibited moderate levels of antioxidant activity with respect to 2, 2-diphenylpicryl-hydrzyl radical (Oke & Hamburger 2002;Udeh & Nwaehujor, 2011). The in vitro antimicrobial activity of I. trichantha leaf was first observed in Pseudomonas aeruginosa and Escherichia coli (Timothy and Idu, 2011); both ethanol and aquous extracts were reported to be active against Staphylococcus aureus, Candida albicans, and Klebsiella pneumonia (Shagal & Kubmarawa, 2013). Other reports have it that the hexane and ethyl acetate extracts were active against E. coli, P. aeruginosa and K. oxytoca (Otun et al., 2015).
In the light of the foregoing, this current research was focused on the stem bark of I. tricantha with the sole aim of evaluating its phytochemicals as well as screen different extracts (hexane, ethyl acetate and methanol) for its antimicrobial properties.

Collection of Plant Materials
Fresh stem bark of Icacina trichantha Oliv. was collected from the Forest Research Institute of Nigeria (FRIN), Ibadan Nigeria and authenticated in the Forest Research Herbarium, where voucher samples were deposited with specimen voucher number FIH-112033. The plant sample was sorted, cut and air-dried for eight weeks.

Extraction and Phytochemical Screening
The root bark was air-dried and crushed to a coarse powder. The powdered root bark (200g) was successively fractionated with hexane, ethyl acetate and methanol till exhaustion. All fractions were evaporated in a rotary evaporator at 25 0 C under reduced pressure (Kokate et al., 2007) and screened. All the plant extracts were screened for the presence of various bioactive compounds using standard methods (Sofowora, 1993;Trease, 1989;Harbone, 1973).

Antibacterial and Antifungal Screening
The antibacterial and antifungal activities of the fractions were assayed as described by (Adekunle, 2009). The standardized suspensions were used to inoculate the p.9 surfaces of Muller Hinton agar plates (90 mm in diameter) using a sterile cotton swab. 6 mm diameter wells were bored using sterile cork borer in agar and filled with the desired concentrations of the plant extract of 20 mg/mL, 10 mg/mL, 5 mg/mL, 2.5 mg/mL and 1.25 mg/mL. Commercial antibiotics (ciprofloxacin 5μg/disc and fluconazol 5μg/disc) were used as a reference standard to determine the sensitivity of the isolates. The plates were allowed to stand for 5 hours at roomtemperature for extracts to diffuse into the agar and then incubated at 37 º C over night. The zone of inhibition was measured using a transparent ruler.       (Yahaya et al., 2012). Tannins have been reported to have antibacterial potential due to their basic character that allows them to react with proteins to form stable water soluble compounds thereby killing bacteria by directly damaging its cell membrane. Steroids have been reported to have antibacterial properties and they are very important compounds especially due to their relationship with compounds such as sex hormones. The antibacterial activities of alkaloids have been reported by a number of authors (Hassan et al., 2005;Aliero et al., 2008;Yesmin et al., 2008). Generally, the antimicrobial properties observed in plant are attributed to the secondary metabolites (Okoli & Iroegbu., 2005;Akinyemi et al., 2005). Table 2 shows the activity of each plant extracts against the microorganisms. The result revealed that MRSA, S. pyrogenes, E. coli, K. pneumonia, S. dysenteriae, C. albicans and C. krusei were sensitive to the fractions whereas P. aeruginosa, S. tyhi and C. tropicalis were found to be resistant to all the fractions. This is a clear indication that the plant could be used to treat dysentery, coughs, stomach aches, diarrhea and new wounds. The success of treating diarrhoea and stomach aches attributed to S. aureus and E. coli could be due to the antibacterial effects of alkaloids, saponins and steroids (Vieira et al., 2001). The result showing zone of inhibition table 3 revealed that the n-hexane extract had significant zone of inhibition between 16 mm to 20 mm, the methanol extract had considerable zone of inhibition of 21 mm to 23 mm while the ethyl acetate fraction showed the highest zone of inhibition of between 23 mm to 26 mm. All the fractions showed lower but comparable zone of inhibition against the microorganism with the standard drugs used (Ciprofloxacin and Fluconazol). The standard drugs ciprofloxacin and fluconazol are higher in performances with greater zone of inhibition than the fractions; this is possibly because of impurities which interfere with their various activities. However, when the active compounds are isolated the fractions may give higher activity as the standard drugs. The plant showed good in-vitro antimicrobial profile especially against MRSA, S. pyrogenes and E. coli which are known to cause infections that are difficult to treat due to their multiple drug resistance. Furthermore, the extracts revealed activity against gram positive and gram negative bacteria as well as some fungi species, these findings indicate that the extracts had broad spectrum activity against a wide range of infectious agents that could be the basis for the folkloric use of the plant. The result of minimum inhibitory concentration (MIC) Table 4 shows that n-hexane had MIC of 10 mg/mL against MRSA, S. pyrogenes, E. coli, K. pneumonia, C. albicans and C. krusei except for S. dysenteriae with MIC of 5 mg/mL. The ethyl acetate fraction showed MIC of 5mg/mL against all the organisms except P. aeruginosa, S. typhi and C. tropicalis. The methanol fraction had MIC of 5 mg/mL against all the microorganisms except K. pneumonia with MIC at 10 mg/mL. The result of the MBC/MFC

Conclusion
Phytochemical screening on fractions revealed the presence of saponins, alkaloids, steroids, tannins and glycosides. Agar diffusion assay on fractions showed growth inhibitory effects on all the organisms except Pseudomonas aeruginosa, Salmonella typhi, and Candida tropicalis. The MIC revealed that n-hexane fraction was active against MRSA, S. pyrogenes, E. coli, K. pneumonia, C. albicans and C. krusei at 10 mg/mL while S. dysenteriae was active at 5 mg/mL. The ethyl acetate fraction was active against all the organisms at a concentration of 5 mg/mL except P. aeruginosa, S. typhi and C. tropicalis. Methanol fraction showed activity of 5 mg/mL against MRSA, S. pyrogenes, E. coli, S. dysenteriae, C. albicans and C. krusei except for K. pneumoniae with activity at 10 mg/mL. Minimum bactericidal concentration/ fungicidal concentration MBC/MFC evaluated on the n-hexane fraction revealed that MRSA, S. pyrogenes, E. coli, K. pneumoniae, S. dysenteriae, C. albicans and C. krusei were active at 20 mg/mL, while the ethyl acetate fraction had MBC/MFC of 10 mg/mL against all the organisms except P. aeruginosa, S. typhi, and C. tropicalis. Methanol extract had MBC/MFC of 10 mg/mL against MSRA, E. coli and S. dysenteriae whereas S. pyrogenes, K. pneumoniae, C. albicans and C. krusei had MBC/MFC at 20 mg/mL. The result presented a broad spectrum of reliable and p.11 non-toxic phytochemicals in Icacina trichantha which could be useful in the therapeutic intervention of multi-resistant microorganisms.