Chemical Composition, Antibacterial and Antifungal Activities of Anthocleista vogelii Planch Root Bark Extracts

This study sought to investigate the chemical composition, antibacterial and antifungal activities of ethanol extract and fractions of the root bark of Anthocleista vogelii Planch. The GCMS analysis of the n-hexane fraction (nHF) of A. vogelii revealed the presence of 12 volatile compounds. While the classes of compounds profiled in terpenoid rich fraction (TRF) using LCMS were majorly terpenoids (tarennoside, 3-Hydroxybenzaldehyde, 12-hydroxyjasmonic acid, 5-megastigmen-7-yne-3,9-diol 9-glucoside, gibberellin A2 O-beta-D-glucoside, 15-Acetoxyscirpene-3,4-diol 4-O-a-D-glucopyranoside), organic/carboxylic acids and fatty acids. The results revealed good antibacterial activity of ethanol extract (EEx) against S. aureus at MIC = 2 mg/mL, but moderate activity by TRF (MIC = 8 mg/mL) although, the TRF had better antibacterial activity than EEX against E. coli (MIC = 8 mg/mL) and S. paratyphi (MIC = 8 mg/mL). The TRF had similar antifungal activity with EEX against T. rubrum (MIC = 4 mg/mL) and T. mentagrophytes (MIC = 8 mg/mL), but displayed better activity against C. albicans (MIC = 4 mg/mL). The nHF showed no antibacterial or antifungal activities. The antimicrobial activities of extract (EEX) and fraction (TRF) of A. vogelii root back validates its use in traditional medicine for treatment of typhoid, urinary tract infection, food poisoning, diarrhea, stomach aches and skin diseases.


INTRODUCTION
Basic bacterial and fungal infections remains a challenge in developing countries even though incidence of some infections could be prevented by maintaining high standards of hygiene. According to WHO (2004), mortality rate due to infectious diarrhoea could be as high as 56% in developing countries. Diarrhea can be caused by bacterial organisms, such as Escherichia coli, Campylobacter spp., Shigella spp. and Salmonella spp. There are far more infectious diseases caused by bacterial organisms such as E.coli (urinary tract infection and food poisoning), Staphylococcus aureus (food poisoning, impetigo cellulitis, boils, abscesses, wound infections, pneumonia and toxic shock syndrome), Salmonella typhi (typhoid, vomiting and diarrhea) and Salmonella paratyphi (paratyphoid) to mention a few of interest in this study.
In Africa, clinically diagnosed skin diseases are commonly caused by fungal infections (Havlickova et al., 2008), and fungal infections significantly contribute to human morbidity and mortality (Brown et al., 2012). Trichophyton rubrum and Trichophyton mentagrophytes 2 are among the causative agents for fungal skin infections like tinea cruris (ringworm of the groin), tinea pedis (athlete's foot), tinea unguium (onychomycosis, nail infections), while Candida albicans is the major causative agent of various forms of candidiasis. In Sub-Saharan Africa, the overall incidence of tinea was estimated to be 78 million in 2005 (Hay et al., 2006). A study revealed T. mentagrophytes and T. rubrum in second and third positions respectively out of 5 fungal causative agents of mycotic infections among school children in Ekpoma, Nigeria (Enweani et al., 1996).
Traditional healers in Nigeria use various herbal preparations to manage/treat a diversity of diseases, including many microbial infections, such as diarrhea, sore throat and gonorrhea (Olukoya, 1993). Medicinal plants are used by a relatively large proportion of the population due to non-availability of orthodox medicines in rural settlements (Olukoya, 1993), poverty, high cost of antimicrobial drugs, traditional practice handed-over from older generations, and availability of medicinal plants in the immediate environment.
The investigated medicinal plant, Anthocleista vogelii Planch has been extensively reviewed under the Anthocleista species (Anyanwu et al., 2015). The bark, root and seed of A. vogelii are used as traditional medicines for the treatment of stomach troubles, wounds, inflammations and venereal diseases (Burkill, 1985;Jiofack et al., 2010). The leaves of A. vogelii are used in Igala traditional medicine for the treatment of typhoid (Musa et al., 2010), while the roots are traditionally used for treating throat problems (Omobuwajo et al., 2008). Few studies have validated the antibacterial properties of crude extracts of the leaves and stem bark of A. vogelii (Olukoya et al., 1993;Musa et al., 2010) and little or no visible work has been done on the antifungal properties of the plant. This study sought to investigate the chemical composition, antibacterial and antifungal activities of ethanol extract and fractions of the root bark of A. vogelii.

Collection of plant material
Fresh plant material was collected from Ovuakali, Ngor-okpala, Imo State, Nigeria. The plant was identified and authenticated by Dr. Jerome Ihuma of the Biological Sciences Department, Bingham University. Voucher specimen (GA134-7421) for Anthocleista vogelii Planch was deposited in the Department of Biological Sciences, Bingham University, Nigeria.

Preparation of extract and fractions
The fresh root bark of A. vogelii was shade-dried and ground to powder form. The plant powder (1.5kg) was extracted with absolute ethanol using Soxhlet extractor for 48h in batches. The solution was filtered with Whatman No. 1 filter paper and the filtrate was concentrated under reduced pressure using a rotary evaporator at 40 o C to give ethanol extract (EEX). A portion of EEX was dissolved in n-hexane solvent for 12 hr three times, then filtered and filtrate was concentrated to give n-hexane fraction (nHF). The marc was extracted with acidified water (2 M H 2 SO 4 ) and partitioned with chloroform three times (3 x 150ml) using a separating funnel (Harborne, 1998). The partitions of chloroform were combined and concentrated to yield terpenoid rich fraction (TRF).

Gas chromatography mass spectrometry (GCMS) analysis of fractions of A. vogelii
The GCMS analysis of n-hexane fraction of A. vogelii was done using Agilent GC 7890B, MS detector MSD 5977A (Agilent Technologies, USA) which is equipped with a library software (MassHunter: NIST 14.L software). Briefly, the GCMS detection involved an electron ionization system with 70 eV ionization energy and Helium gas as the carrier gas at 3 1 mL/min constant flow rate. The inlet temperature was fixed at 250 o C, while oven temperature was set at 100 o C for 1.5 min and then raised to 270 o C at the rate of 5 o C per min. Exactly 1 mL of diluted samples were injected and scan range was selected as 40-600.

Analysis of Terpenoid Rich Fraction (TRF) of A. vogelii by Liquid Chromatography Mass Spectrometry (LCMS-MS)
Sample of TRF was diluted 10X with methanol and analyzed using Agilent 1290 Infinity LC system coupled to Agilent 6520 Accurate-Mass Q-TOF mass spectrometer with dual ESI source. The solvents 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) were used with injection volume of 1.0uL. A column of Agilent Zorbax Eclipse XDB-C18, 2.1x150mm narrow-bore, 3.5 micron and 25⁰C column temperature and 23⁰C autosampler temperature at 0.5 mL per min flow rate were used for the separation. The ESI-MS analysis for TRF sample was run in negative polarity for 25 mins and 5 mins as post run time, mass range was 100 -3200 m/z, and 119.03632 and 966.000725 reference ions were used. Agilent MassHunter Qualitative Analysis B.05.00 (Method: Metabolomics-July2015.m) was used to process the data.

Preparation of inoculum
Bacteria isolates were cultured on Mueller Hinton agar (MHA) plates at 37°c for 24hrs and fungi isolate were cultured on Saboraud dextrose agar (SDA) plate at 37°c for 48hrs and were sub-cultured into Muller Hinton broth (MHB) or Saboraud dextrose broth (SDB). All the media were prepared according to the manufacturer's instruction.

Minimum Inhibitory concentration determination (MIC)
The micro dilution method according to Shanmugapriya et al., (2012) was employed. The 96microtiter well was prepared by dispensing 50 L of SDB (fungi) and MHB (bacteria) and left for 15 minutes before adding 5 L of the bacterial or fungal suspension into each well. One hundred microlitres from the stock solution of extracts was added into the first well, then followed by two fold serial dilution down the remaining wells. The concentration of the extract/fractions were 16, 8, 4, 2, 1, 0.5, 0.25, 0 mg/mL. The last row of wells did not contain the extract thus serving as organism viability control. The plate was shaken for 20 seconds and then incubated at 37 o C for 24 h (bacteria) and 30 o C for 48 hours (fungi). After incubation, the plates were observed for the absence or presence of growth. The bioassay was performed in triplicate. MIC was the lowest concentration of the extract/fraction showing no turbidity after incubation, where the turbidity was interpreted as visible growth of the micoorganisms.

Antimicrobial Studies
The antimicrobial activities of A. vogelii and the reference drug presented as MIC are represented on Table 3.

DISCUSSION
The groups of phytochemicals found in the leaf, stem-bark and root bark extracts of A. vogelii were reported to include alkaloid, carbohydrates, saponins, flavonoids, tannin, terpenes,  (Jegede et al., 2011;Anyanwu et al., 2013). This study gives insight to the specific compounds and their classes found in the nHF (Table 1) and TRF (Table 2) (Giannini et al., 2004). However, the compounds in TRF appeared to have acted synergistically to exhibit its antimicrobial activity.
Our study revealed good antibacterial activity of ethanol extract against S. aureus at MIC = 2 mg/mL, but moderate activity by TRF and no activity by nHex fractions of A. vogelii. The TRF had better antibacterial activity than EEX against E. coli and S. paratyphi, which indicated that fractionation of extracts might have led to increased concentration of active principles in the fraction. Although, Olukoya et al. (1993) reported no activity for ethanol extracts of A. vogelii against Escherichia coli, Staphylococcus aureus and Salmonella typhimurium, the extract concentration used in the study was undefined, hence it is difficult to compare with this study. However, in corroboration with this study, Musa et al. (2010) had reported antibacterial activities for ethanol, aqueous and chloroform leaf extracts of A. vogelii against S. typhi corroborating its traditional medicinal use in the treatment of typhoid fever.
The TRF had similar antifungal activity with EEX against T. rubrum and T. mentagrophytes, but displayed better activity against C. albicans. Although, we found no studies on the antifungal activities of extracts of A. vogelii, except by Tene et al. (2008) on the antifungal activities of xanthone compounds (1-hydroxy-3,7-dimethoxyxanthone and 1-hydroxy-3,7,8trimethoxyxanthone) isolated from A. vogelii stem bark against Candida parapsilosis. The nHF showed no antibacterial or antifungal activities indicating that the active principles necessary for activities were absent in the fraction.

CONCLUSION
The antibacterial activities of extract (EEX) and fraction (TRF) of A. vogelii root back against E. coli, S. aureus and S. paratyphi validates its use in traditional medicine for the treatment of diarrhea, sore throat, and stomach aches. Also, the antifungal activities of the EEX and TRF of A. vogelii provides scientific credence to its traditional use against wounds and skin diseases. The overall display of better antimicrobial activities by TRF of A. vogelii compared EEX might due to the increased concentration of active principles in fraction. Thus further studies on elucidating the bioactive compounds in A. vogelii and screening for their antimicrobial activities is necessary.