Abstract: Phytochemical and anti-microbial properties of the methanol extracts of Aspilia mossambicensis (family, Compositae) were studied. The plant has been used by traditional healers to treat skin diseases, wounds, gonorrhea, abdominal pains, respiratory problems and malaria. Bacteria used for antimicrobial analysis consisted of clinical strains of Streptococcus pyogenes (gram positive) and Salmonella typhi (gram negative) bacteria and one strain of fungi (Aspergillus niger). Phytochemical screening of the plant leaves and roots of A. mossambicensis revealed that the plant contains active compounds of flavonoids, alkaloids, saponin, steroids, carbohydrates and anthraquinones. The methanol extract showed antimicrobial activity against the three microorganisms at relatively lower concentrations. Leaves extracts showed higher level of growth inhibition of S. pyogenes, followed by S. typhi and A. niger, respectively. Root extracts showed higher growth inhibition for A. niger, followed by S. typhi and S. pyogenes in that order. In comparison leaves extracts showed better microbe growth inhibition than root extracts and was attributed to absence of aldehydes in the roots, or a more concerted synergistic activity of active compounds present in the leaves. The study has shown that species of Aspilia mossambicensis have considerable activity against gram positive bacterium Streptococcus pyogenes, gram negative bacterium Salmonella typhi and one fungal strain, Aspergillus niger. The study supports the previous claims by traditional healers of the plant to heal several diseases in traditional communities in Kenya. The study provides a strong evidence for new sources of antimicrobial drugs from this plant.
INTRODUCTION
People inhabiting different ecological zones use different plants and plant parts in their treatment arsenal (Bussmann et al., 2006; Njoroge and Bussmann, 2006). About 70-80% of people in the world depend on traditional herbal medicine for primary health care needs (Hamayun et al., 2006). While plant species used in traditional medicines continue to be reliable sources for discovery of useful compounds, the pharmaceutical value and concentration of active ingredients in each plant vary depending on climatic and edaphic factors (Rajakaruna et al., 2002; Adebooye and Opabode, 2004; Bussmann et al., 2006; Duraipandiyan et al., 2006). Few published studies on phytochemical compounds have aimed at elucidating the degree of specificity of the effect of the compounds among the various types of their bioactivity. Several factors such as phonological age of the plant, percent humidity of the harvested material and the method of extraction have been identified as possible sources of variation for the chemical composition, toxicity and bioactivity of the extracts (Rajakaruna et al., 2002). Moreover, these chemical compounds seem to possess distinct molecular formula, a certain molecular weight and certain physicochemical properties.
Aspilia plant (Aspilia mossambicensis) is widely spread in south, south west and west of Kenya from coast to Lake Victoria. As a result of variable ecological conditions, the chemical composition of the plant is known to vary and this may have been the reason as to why the plant has been used to treat different diseases in different places (Agnew and Shirley, 1994). The plant belongs to the family of Compositae (Asteraceae) and genus Aspilia. Few reports indicate that the plant possess antimalaria activity against Plasmodium falciparium (Offulla et al., 1996). The plant has been used traditionally by many African communities to treat several diseases including, cystitis and gonorrhea, abdominal pains and backache. Root decoction is normally given to breast feeding mothers to increase milk production. Pounded leaves decoction is drunk in order to treat intestinal worms including hookworms (Kokwaro, 1976). Among the Kamba community of Kenya the pounded leaves decoction is applied to circumcised young men for faster healing of the wounds (D.M Musyimi personal communication). Some phytochemical constituents have been reported to have medicinal or antimalarial properties (Kokwaro, 1976; Offulla et al., 1996). There were no reports in the literature of earlier studies carried out to test antimicrobial activity of A. mossambicensis, hence this study. The objective of this study was to isolate, identify and to evaluate the antimicrobial properties of the compounds present in the leaves and roots of Aspilia mossambicensis (oliv) wild species in Kenya.
MATERIALS AND METHODS
Field Collection and Extraction
Plant roots and the leaves were collected in February 2007, from areas surrounding
Maseno University, Kenya. Specimens were cleaned off adhered soil or dust in
the field by shaking and were placed inside polythene paper bags and taken to
the Maseno University Botany laboratory where the study was undertaken. The
specimens were cleaned of all dust and allowed to dry under shade. The voucher
specimens in duplicate were deposited in the herbarium of Maseno University.
Leaves and roots of Aspilia mossambicensis (oliv) wild species were air
dried at room temperature for 2 weeks. The roots and leaves were subsequently
ground into fine powder using an electric blender yielding 300 and 500 g, respectively
and cold extracted with methanol then filtered under vacuum using a 0.2 mm Nalgene
filter unit. The resulting extracts were concentrated to remove the methanol
using the rotary vapour at 40°C. Roots and leaves extracts were dried and
subsequently weighed and yielded a crude extract of 31.8 and 19.7 g, respectively.
Extracts were then stored in refrigerator until they were needed for analysis.
All work was carried out in accordance with the general guidelines for methodologies
on research and evaluation of traditional medicine (WHO, 2000).
Phytochemical Screening
The qualitative chemical analysis of the powders was carried out for the
determination of the presence of anthraquinone, saponins, steroids and alkaloids,
Aldehydes, ketones and carbohydrates. Phytochemical screening was done according
to Akinyemi et al. (2005).
Isolation of Pure Compounds
Methanol extract (15 g) was chromatographed on silica gel column eluting
with n-hexane:ethyl acetate (90:10) according to Kaberia et al. (1999).
Most of the chemicals used were of analytical grade. Silica gel column (60 g,
si 60, 40-63 μm) was from Merck, Germany and commercially prepared TLC
plate (GmbH and co. D-3440) from Eschwege, Germany. The Rf-values
of the bioactive fraction in the sample and the standard were determined (El-Olemyl
et al., 1994).
Test Microorganisms
The clinical isolates were obtained from biomedical school of Maseno University,
Kenya. The microorganisms consisted of one gram positive bacteria (Streptococcus
pyogenes) and one gram negative bacteria (Salmonella typhi) and one
fungal strain (Aspergillus niger).
Preparation of the Culture Media
Nutrient agar pH 7.4 and Sabouraud dextrose agar pH 5.4, all products of
oxoid laboratories, England were used in this study. Thirty mine gram of Sabouraud
dextrose agar and 28 g of nutrient agar were dissolved in 1 L of water and heated
to dissolve the contents completely. The nutrient agar and Sabouraud dextrose
agar petri dishes were prepared by pouring 15 mL of molten media into sterile
petri dishes and allowed to solidify for 5 min. The media, distilled water and
petri dishes were sterilized separately by autoclaving at 120°C at 1 bar
pressure for 20 min.
In vitro Antibiosis
Extracts from the two plant parts (leaves and roots) were diluted in hexane
(2 g of the dried filtrate was reconstituted with 10 mL of 100% hexane to prepare
stock solution). Different concentrations of the plant extracts were prepared
by diluting the stock extract to hexane according to the following ratios 1:9
(10%), 1:18 (5%) and 1:32 (2.5%). Control experiment had n-hexane alone without
any extract to nullify the effect of the solvent on the test organisms. Two
methods were used: pit and disc methods according to Murthy and Nagodra (1977).
Susceptibility testing was carried out by measuring the inhibitory zone diameters
on the Nutrient Agar (NA) and Sabouraud dextrose agar using conventional paper
disc method and pit method. The inhibitory zone distances and rounded off to
the nearest whole numbers (mm) for analysis.
Pit Method
A standard cork borer of 8 mm diameter was used to cut uniform wells on
the surface of the agar media, into which 0.1 mL of the prepared plant extracts
of various concentrations were added. The microorganisms were introduced on
the media around the pit using applicator sticks bearing cotton wool at the
tips. Three replicates for each microbe and plant extract concentration were
used. The petri dishes inoculated with bacteria and fungi were kept for incubation
for 24 h at 37 and 25°C, respectively. The clear zone of growth inhibition
formed around each pit was measured to the nearest millimeter using a transparent
ruler.
Disc Method
Circular paper discs 8 mm diameter were cut out from Whatman No. 1 filter
paper using a paper punch and each dipped in a known concentration of the plant
extracts for about 2 min, then gently transferred at the center of the inoculated
agar media. Petri dishes inoculated with bacteria and fungi were kept for incubation
for 24 h at 37 and 25°C, respectively. The petri dishes inoculated with
bacteria and fungi were kept for incubation for 24 h at 37 and 25°C, respectively.
The diameter of inhibition zones were measured with a ruler and compared with
the n-hexane control disk to nullify the effect of the solvent on the growth
of the test organisms.
Data Analysis
Data collected were subjected to analysis of variance (ANOVA) using SAS
statistical package. Means were separated and compared by Duncan’s multiple
range test at (p<0.05).
RESULTS
Phytochemical screening of the plant (Table 1) indicated
a number of constituents were present and included flavonoids, alkaloids, steroids
and anthraquinones, ketones and aldehydes. Root extracts did not have aldehydes
unlike the leaves which had both ketones and aldehydes. Flavonoids were only
present in the roots. There were a lot of sterols in the roots extract than
in the leaves extract. Higher concentrations of alkaloids were present in both
root and leaves extracts. Meta and Ortho hydroxyls groups were
present in both roots and leaves extracts while Para hydroxyl was only
present in leaves, confirming the presence of carbohydrates, particularly the
monosaccharides. Elution of the column with n-hexane and ethyl acetate led to
isolation of fifteen Rf values of uncharacterized active compounds
(data not shown). Seven active compounds were isolated from the leaves while
nine active compounds were from the roots. All plant extracts were found to
possess microbial growth inhibitory activities (Table 2 and
3). Leaves extracts caused the higher degree of growth inhibition
on the test microorganisms as compared to the root extracts (Table
2 and 3). The zones of inhibition obtained in respect
to the leaf extract and test organisms were higher for S. pyogenes, followed
by S. typhi and least in A. niger. Root extracts
inhibited the growth of A. niger at a greater degree, followed by S.
typhi and least inhibition was experienced by S. pyogenes.
The two bioassay methods also influenced the growth inhibition results of the
microbes. There were significant (p<0.0001) interactions between the plant
parts used, extract concentration, microorganisms and bioassay methods used
(Table 4).
| Table 1: | Phytochemical screening of secondary metabolites present in the plant parts extracts of aspilia mossambincensis |
| -: Absent, +: Present, ++: Low concentration, +++: Moderate concentration, ++++: High concentration | |
| Table 2: | Effect of different concentrations of leaf and root extracts of aspilia mossambincensis on the micro-organisms. Data presented are means of three replicates±SE |
| *: Diameters zone of inhibition (mm) | |
| Table 3: | General comparison of method and growth inhibitory effects of root and leaf extracts of aspilia mossambincensis on the micro-organisms. Data presented are means of three replicates±SE |
| *: Diameters zone of inhibition (mm) | |
| Table 4: | Analysis of variance of microbial growth inhibition and interactions between plant parts extracts and methods used |
| Coeff Var: 30.40712; SF: Significant, NSF: Not significant at p<0.0001 | |
DISCUSSION
Medicinal plants have been the subject of human curiosity and need (Bussmann et al., 2006; Khalil and Dababneth, 2007). The medicinal value of plants lies in some chemical substances that produce a definite physiological action on the human body. Phytochemical screening of the leaf and root extract of the plant (Table 1) revealed that the plant contains flavonoids, saponins, anthraquinones, alkaloids, carbohydrates, aldehydes and ketones in agreement with studies reported for some other plants (Weimann and Heinrich, 1997; Atindehou et al., 2002; Oomah, 2003; Edeoga et al., 2005). The most important of these bioactive compounds are alkaloids, flavonoids and phenolic compounds (Atindehou et al., 2002; Edeoga et al., 2005), since are plant metabolites known for exhibiting physiological and antimicrobial activity. The zone of inhibition using leaf extracts was higher for S. pyogenes, a gram positive bacterium than for the other two microbes. Interestingly, S. pyogenes experienced the least inhibition with the root extracts compared to the other two microbes (Table 2 and 3). It is possible that the growth of the microbes was inhibited by different chemical compounds. For instance, roots lacked aldehydes hence aldehydes found in the leaves extracts could be responsible for greater growth inhibition of S. pyogenes. Generally methanol leaves extracts were more active than methanol roots extracts; probably because of higher concentration of the active chemical compounds in the leaves. The greater growth inhibition of A. niger by methanol root extracts, may be due to absence of aldehydes in the roots as evidenced from the study (Table 1). Interestingly, root extracts had higher number of active compounds determined by thin layer chromatography (data not presented); perhaps the comparatively higher activity of the leaves than roots was due to active constituents in the leaves acting in a more synergistic manner. It is worth noting that leaves phytochemical compounds had greater inhibitory activity on A. pyogenes than the other organisms. The antimicrobial activity of the methanol extracts appears to have broad spectrum of activity, especially since both gram positive and gram-negative bacterial and fungi were sensitive to the extracts. The antimicrobial activity of the methanol extract appears to have broad spectrum of activity, especially since both gram positive and gram-negative bacterial and fungi were sensitive to the extracts. Phytochemical compounds are heterogeneous mixtures of single substances and biological actions are primarily due to these components in a very complicated concert of synergistic or antagonistic activities. Mixtures of chemicals show a broad spectrum of biological effects and pharmacological properties (Robinson, 1967; Coelho-de-Souza et al., 1998; Atindehou et al., 2002).
The antimicrobial properties exhibited by the plant could be traced to its possession of alkaloids (Shelton, 1991; Akinyemi et al., 2005), flavonoids (Narayana et al., 2001) and Meta and Para-hydroxyl compounds present in the plant extracts (Mostahar et al., 2007). The presence of hydroxyl groups in methanol extracts enhances the likelihood of intramolecular reactions such as the formation of cyclic acetals and anhydroethers (Mann, 1994). Carbohydrates could have facilitated the growth of some of the microorganisms and hence antagonized the antibacterial activity of the active compounds in the root extracts. Many alkaloids have pharmacological effects and could be associated with inhibition of nucleic acid, protein and membrane phospholipids biosynthesis (Shelton, 1991). Alkaloids have been used to treat diseases like malaria, pain killers and managing heart diseases (Oomah, 2003). Alkaloids are derivatives of amino acids and include morphine, atropine, quinine and nicotine. Flavonoids (such as, rutin and anthocyanins), allelopathic compounds (such as, caffeic acids, ferulic acid, other phenolics and polyphenolic compounds) and saponins (such as, protodioscin) are known to have biological and pharmacological activities on human health (Hartung et al., 1990; Akinyemi et al., 2005). Major flavonoid compounds such as rutin and its aglyconequercetin, have been reported to have beneficial biological effects, such as antagonizing the increase of capillary fragility associated with hemorrhagic disease (Griffith et al., 1944) and anti-carcinogenic activity (Yang et al., 2000). Biological activities of many other flavonoids, polyphenols or phenolics compounds were also reported (Edenharder and Grunhage, 2003; Wang et al., 2003).
The susceptibility of S. pyogenes, S. typhi and A. niger to the cold methanol extracts is an indication that the plant possess a great potential as a source of both antibacterial and antifungal compounds which may be comparable to those of commercially available drugs. The antimicrobial activities demonstrated by crude extracts of A. mossambicensis may, explain some of the previous claims by traditional healers, about this plant curing of diseases like dysentery and respiratory tract infections (Kokwaro, 1976). The antimicrobial properties exhibited by the plant could be attributed to presence of alkaloids and flavonoids which have been noted for numerous biological activities such as anti-inflammatory, vasoprotective and antithrombitic effects (Oomah, 2003; Mostahar et al., 2007). There are no reports on toxic effects of A. mossambicensis used in different African communities including Kenya for various ailments, hence phytochemical studies should be intensified to isolate, characterize and identify the specific active compounds in this plant responsible for the antimicrobial activity. We are in progress to find out the active compounds in the fractions of A. mossambicensis. Research is therefore needed to determine optimal doses and concentrations of the preparations and identify side effects of the traditional remedies.
CONCLUSIONS
Present study has provided evidence of antimicrobial activity of A. mossambicensis against gram negative bacterium S. typhi and gram positive bacterium S. pyogenes and fungal pathogen A. niger. The leaves extracts seem to have higher degree of growth inhibitory activity on S. pyogenes, followed by S. typhi and A. niger. On the other hand, roots extracts had higher growth inhibitory activity on A. niger, followed by S. typhi and S. pyogenes. Roots had the higher number of active compounds isolated by thin layer chromatography. The major compounds present in the methanol extracts includes, Flavonoids, saponins, anthraquinones and alkaloid compounds and seem to be responsible for the antimicrobial activity. The study has shown that methanol extracts of A. mossambicensis could be used as another source of cheaper conventional drugs since the plant is quite abundant in the forests. The study revealed that the plant contain chemicals that could help to control some of the problematic diseases caused by the microorganisms investigated. The results provide a background for rigorous screening of this plant for antimicrobial properties.
ACKNOWLEDGMENTS
The authors would like to thank the Department of Botany and Horticulture and the Department of Chemistry both of Maseno University for their financial support. They also appreciate the assistance accorded to them by Laboratory technicians from chemistry department and Mr. John Odera of Botany department during the study period.