Antimicrobial activity of stigmasterol from the stem bark of Neocarya macrophylla

Antimicrobial resistance (AMR) driven by inappropriate use of antimicrobial agents has become a global health problem in recent years (Dryden 2017; Laxminarayan et al. 2016; Marston et al. 2016; WHO 2002). The problem of these multidrug resistance and undesirable side effects of most antibiotics necessitate the search for new antimicrobial agents of plant origin. Historically, natural products have been an important source of novel antimicrobial agents and folkloric medicinal use of these products inspired the discovery of several bioactive natural products (Li et al. 2016; Maxson et al. 2016; Simpson et al. 2016). In the last few decades, the interest of the industry in natural products has dwindled; however, in recent times, the need to tackle multidrug resistance and other emerging health problems has fuelled the rebirth of natural product drug discovery. In this regard, natural products may continue to play a vital role in antimicrobial drug discovery; therefore, there is an urgent need for novel natural product scaffolds with superior properties (Chen et al. 2016; Moloney 2016). Nature’s biodiversity represents a huge reservoir of bioactive chemical diversity and has been central in the identification and development of therapeutic agents (Siddiqui et al. 2013); hence, drug discovery approaches exploiting nature’s chemical diversity may uncover novel natural products with desired bioactivity (Pawar 2014).


Introduction
Antimicrobial resistance (AMR) driven by inappropriate use of antimicrobial agents has become a global health problem in recent years (Dryden 2017;Laxminarayan et al. 2016;Marston et al. 2016;WHO 2002).The problem of these multidrug resistance and undesirable side effects of most antibiotics necessitate the search for new antimicrobial agents of plant origin.Historically, natural products have been an important source of novel antimicrobial agents and folkloric medicinal use of these products inspired the discovery of several bioactive natural products (Li et al. 2016;Maxson et al. 2016;Simpson et al. 2016).In the last few decades, the interest of the industry in natural products has dwindled; however, in recent times, the need to tackle multidrug resistance and other emerging health problems has fuelled the rebirth of natural product drug discovery.In this regard, natural products may continue to play a vital role in antimicrobial drug discovery; therefore, there is an urgent need for novel natural product scaffolds with superior properties (Chen et al. 2016;Moloney 2016).Nature's biodiversity represents a huge reservoir of bioactive chemical diversity and has been central in the identification and development of therapeutic agents (Siddiqui et al. 2013); hence, drug discovery approaches exploiting nature's chemical diversity may uncover novel natural products with desired bioactivity (Pawar 2014).
Neocarya macrophylla (formerly Parinari macrophylla) known as Gawasa in Hausa language is widely distributed along coastal savannahs from Senegal to Liberia, and woody savannahs of Background: Natural products play a significant role in human therapy.They represent a huge reservoir of bioactive chemical diversity and help in understanding the cellular pathways that are essential component of drug discovery process.
Southern Mali, Niger and Northern Nigeria (Abonnier 2004;Burkill 1985).The plant belongs to the Chrysobalanaceae family which is composed of 17 genera, while about 525 species are found in the tropical and subtropical regions (Yakandawala, Morton and Prance 2010).N. macrophylla has been used in ethnomedicine for the treatment of a number of ailments and diseases including asthma, dysentery, diarrhoea, skin infections, cancer and ear and eye infections (Warra et al. 2013).The stem bark is extensively used in ethnomedicine in Northern Nigeria in the treatment of pain, inflammation, diarrhoea and snakebites (Amina Yusuf Jega, Department of Pharmaceutical and Medicinal Chemistry, Usmanu Danfodiyo University, Sokoto, Nigeria pers.comm.November, 2012).The antimicrobial activity of the methanol stem bark extract of the plant has been reported (Yusuf et al. 2015b).Recently, isolation and characterisation of stigmasterol (Figure 1) from the stem bark of N. macrophylla was reported (Yusuf et al. 2015a).In this study, antimicrobial activity of stigmasterol isolated from the stem bark of N. macrophylla investigated against representative gram-positive and gram-negative bacteria and two fungi is reported.

Isolation and structure elucidation of stigmasterol
Stigmasterol was previously isolated from the n-hexane soluble fraction of the stem bark of N. macrophylla as a white crystalline solid substance using a combination of silica gel and sephadex LH-20 column.The structure was established using one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopic analysis and by direct comparison of data obtained with those reported in the literature (Yusuf et al. 2015a).

Test organisms
The antimicrobial activity of stigmasterol was determined using selected pathogenic microbes.The microbes were obtained from the Department of Medical Microbiology, Ahmadu Bello University Teaching Hospital Zaria, Kaduna State, Nigeria.All bacterial cultures were checked for purity and maintained in a blood agar slant, while the fungi were maintained on a slant of Sabouraud dextrose agar (SDA).The microbes include methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), S. aureus, Streptococcus faecalis, Escherichia coli, Salmonella typhimurium, Pseudomonas fluorescens, Klebsiella pneumoniae, Candida albicans and Candida krusei.

Susceptibility test
The antimicrobial activity of the compound was carried out using stock concentration of 100 µg/mL.Mueller Hinton agar was used as the growth medium for the microbes.The medium was prepared according to the manufacturer's instructions and sterilised at 121°C for 15 min.It was poured into sterile petri dishes and then allowed to cool and solidify.The sterilised medium was seeded with 0.1 mL of standard inoculum of the test microbe; the inoculum was spread evenly over the surface of the medium using a sterile swab.A standard sterile cork borer of 6 mm diameter was used to bore a well at the centre of each inoculated medium.The wells were filled with 0.1 mL of the solution of the compound and allowed to diffuse for 1 h.Incubation of the inoculated medium was made overnight at 37°C and 25°C for bacteria and fungi, respectively, after which the medium was observed for the zone of inhibition of growth; the tests were conducted in duplicates and the zone of inhibition was measured with a transparent ruler.The mean of the results was recorded in millimetres (mm) (Vollekova, Kostalova and Sochorova 2001).

Minimum inhibitory concentration
The minimum inhibitory concentration (MIC) of the compound was determined using the broth dilution method (Vollekova et al. 2001).Twofold serial dilutions of the compound in the sterile broth were made to obtain the concentrations of 100 µg/mL, 50 µg/mL, 25 µg/mL, 12.5 µg/mL, 6.25 µg/mL and 3.13 µg/mL.An amount of 0.1 mL suspension of the standard inoculum of the test microbe was then inoculated into the different concentrations of the compound.The tubes were incubated at 37°C for 24 h and 25°C for 48 h for bacteria and fungi, respectively, after which the plates were observed for turbidity (growth).The MIC was defined as the lowest concentration of the compound inhibiting the visible growth of each micro-organism.

Minimum bactericidal and /fungicidal concentrations
Minimum bactericidal and fungicidal concentration was carried out to determine whether the test microbes were killed or only their growth was inhibited.Mueller Hinton agar broth was prepared, sterilised at 121°C for 15 min and transferred into sterile Petri dishes to cool and solidify.The contents of the MIC in the serial dilution were subcultured into the prepared medium and incubated at 37°C for 24 h; the plates were observed for colony growth; the MBC/MFC was the plate with lowest concentration of the compound in serial dilution without colony growth (Vollekova et al. 2001).

Results
The compound, stigmasterol, exhibited varying degrees of antimicrobial activity against the test microbes.Susceptibility test result showed inhibition ranging from 23 mm to 30 mm against all the organisms, with the exception of VRE, S. typhimurium and K. pneumoniae.The compound recorded highest sensitivity against MRSA (30 mm) and the least sensitive organism was P. fluorescens (23 mm).Ciprofloxacin (5 µg/mL), the standard antibacterial drug, had inhibitory effect against all the test organisms except VRE and P. fluorescens with zone of inhibition of 32 mm-41 mm, while the standard antifungal drug, fluconazole (5 µg/mL), exhibited activity against the two fungi species tested with inhibition range of 34 mm-35 mm (Table 1).

Discussion
The low MIC value (6.25 µg/mL-25 µg/mL, Table 2) and MBC/MFC (12.5 µg/mL-50 µg/mL, Table 3) indicated that the compound has very good antimicrobial activity against the susceptible organisms considering the fact that compounds with MICs less than 100 µg/mL are regarded as having strong antimicrobial property (Tang et al. 2003).The methanol stem bark extract of N. macrophylla (20 mg/mL) was reported to possess good antimicrobial activity against Streptococcus pyogenes, Bacillus subtilis, Bacillus cereus, E. coli and C. albicans, with inhibition range of 22 mm-34 mm (Yusuf et al. 2015b); the isolated compound from this extract displayed an improved inhibitory effect and significantly lower MIC value when compared to the extract.The isolation and purification of stigmasterol from the stem bark extract of N. macrophylla has therefore allowed for an accurate activity evaluation of the compound, which reveals that the antimicrobial activity observed with the extract might be attributed to its stigmasterol content.
The broad-spectrum antimicrobial activity of stigmasterol observed in this study is in good agreement with previous studies.For example, Abdissa, Legesse and Delelegn (2015) reported the in vitro antibacterial activity of stigmasterol isolated from the roots of Caylusea abyssinica against S. aureus, E. coli, Pseudomonas aeruginosa and Salmonella typhimurium with zone of inhibition ranging from 11 mm to 18 mm at a higher concentration (50 mg/mL).A similar study conducted on stigmasterol gave a lower zone of inhibition (9 mm-10 mm) against S. aureus and E. coli at a concentration of 250 µg/mL (Tong-cun et al. 2012).Yinusa et al. (2014) (20 mm).This variability in antimicrobial activity observed for stigmasterol could be attributed to the difference in concentration of the compound used in the studies.Kanokmedhakul, Kanokmedhakul and Phatchana (2005) attributed the mechanism of action of steroids to their ability to inhibit bacterial cell surface protein 'sortase', thus preventing transpeptidation.Membrane disruption has been suggested as one of the likely mechanisms of action of sterols on microbes (Tamokou et al. 2011).
Importantly, this study represents the first antimicrobial evaluation of stigmasterol from the stem bark extract of N. macrophylla and therefore highlights an important natural source of bioactive stigmasterol in Nigeria.This study has also identified microorganisms susceptible to stigmasterol which were not previously reported.The highest activity (30 mm) exhibited by the compound on MRSA is worthy of mention as MRSA is a substantial public health problem worldwide causing morbidity and mortality and elevated health care costs.It is a leading cause of skin and soft tissue infections in patients reporting to emergency departments for treatment with a rising rate in primary care clinics and intensive care units (Green et al. 2012).MRSA has also been implicated in pneumonia and surgical site infections (Bush 1989;CDCP 2015).
The compound exhibited 29 mm as zone of inhibition against S. aureus, which is the most dangerous of all the many common staphylococcal bacteria and has been implicated as a causative agent in skin infections, pneumonia and breast and other fatal infections (Baorto et al. 1994).Enteropathogenic E. coli has been implicated in a number of prevalent infectious diseases including diarrhoea, dysentery (Ochoa et al. 2008) and urinary tract infection (Linhares et al. 2013).The good antibacterial activity against E. coli (24 mm zone of inhibition)   displayed by the isolated stigmasterol is hence very encouraging.
Alternative therapy with herbal medicinal products and supplements has continued to increase globally, with a significant proportion of the population in developing countries relying on herbal products for health care needs.In this regard, N. macrophylla has been widely used in West Africa for the treatment of a number of diseases including dysentery, diarrhoea and skin, ear and eye infections (Warra et al., 2013).The finding of this study which showed the broad-spectrum antimicrobial activity of stigmasterol isolated from N. macrophylla establishes the efficacy of stem bark preparations of N. macrophylla in the treatment of those infections.However, further studies are required to address possible safety and toxicity issues with the use of N. macrophylla.Analysis of the yields of stigmasterol from stem bark of N. macrophylla from different regions and in different seasons should be performed.This may allow the quantification of stigmasterol content in crude extracts, which in turn may provide guidance on the posology of the herbal preparations from this plant.

Conclusion
Stigmasterol isolated from the stem bark of N. macrophylla exhibited broad-spectrum antimicrobial activity, highlighting its potential as a candidate in the development of novel antimicrobial drugs.The overall result of this study can be considered as promising with respect to discovering novel drugs from plant sources, especially when the medical importance of the tested microorganisms is considered.The results of this study have established the efficacy of the stem bark preparation of N. macrophylla in the management of bacterial and fungal infections.

TABLE 1 :
Susceptibility of stigmasterol to various test organisms.

TABLE 2 :
Minimum inhibitory concentration of stigmasterol on the test organisms.

TABLE 3 :
Minimum bactericidal and fungicidal concentration of stigmasterol on the test organisms.