THE PHYTOCHEMICAL AND ANTI-MICROBIAL POTENTIAL OF THE EXTRACTS FROM THE FRUIT PULP OF Landlphia owariensis

The phytochemical composition and antimicrobial potential of extracts of fruit pulp of Landolphia owariensis was investigated. L. owariensis mesocarp flour was obtained by oven drying at 50 0 C and pulverized while microwave assisted extraction was used to obtain hexane, chloroform, ethyl acetate and methanol extracts, which were concentrated in vacuo using microwave and thereafter air dried at ambient temperature. Antimicrobial sensitivity tests on fourteen (14) human pathogenic microorganisms were performed using diffusion method while minimum inhibitory concentration (MIC) and minimum bacterial/fungal concentration (MBC/MFC) tests were performed using dilution method. The oven dried L. owariensis fruit pulp flour contained 12.45±0.03 mg/100 g tannins and 6.39±2.78 mg/100 g total flavonoids but low levels of anthraquinones, alkaloids, cyanogenic glycosides, saponins, steroids and terpenes. The phytosterols consisted of cholesterol, cholestanol, campesterol, stigmasterol, beta sitosterol and tocopherol with stigmasterol being the most abundant (1.06±0.21 mg/100 g). The flavonoids constituents included orientin, isovitexin and keampferol. Microbiological analysis of hexane, chloroform, ethyl acetate and methanol extracts of L. owariensis fruit pulp showed antimicrobial activities against S. aureus, S. pyogenes, S. typhi, S. dysenteriae, K.pneumoniea, C. albicans, C. krusei, C. stellatoidea and M. rubrum. Chloroform extract exhibited the highest zones of inhibition, followed by hexane and ethyl acetate extracts while methanol extract had least diameter of zones of inhibition. Also ethyl acetate extract was more potent (2.5-5 mg/mL), followed by chloroform and methanol extracts while hexane extract had the least potency (5-10 mg/mL) level. Similarly, ethyl acetate extract was most effective (5-10 mg/mL) in inhibiting bacterial/fungal growth. L. owariensis fruit pulp flour and extracts contained important secondary metabolites that may account for its antimicrobial activities.


INTRODUCTION
In many tropical countries in Africa, rural people traditionally harvest wide range of fruits from the wild because of its taste, cultural uses and as food supplements.Labeled as famine or hunger food, wild plants have been recognized to have potential to meet household food and income security (Guinand and Dechassa, 2000;Kebu and Fassil, 2006).In rural communities of many developing nations, wild fruits are often consumed to complement cultivated commercial fruits.In Nigeria, the indigenous fruits collected from wild play significant role in the food and medicinal needs of rural poor.Scrutiny of plants of various tropical forest areas through constituent analysis may lead to selection of valuable wild species that can be taken through crop improvement and hybridization process to establish it as cultivated variety.Report has it that less than 10 plant species are meeting over 90% of the world food demand (Wilkes, 1981), however, there is huge diversity of wild plant species whose food and health potentials are underexploited and underutilized.The rural poor in developing nations use wild edible plant species to complement their food and economic resource base.The high rate of deforestation occasioned by urbanization and industrialization is a huge threat to the survival and sustained availability of these wild edible plant species in the nearest future for man's use.Therefore, conscious effort at conserving the economically important wild edible plant species before they really become extinct is required.
Landolphia owariensis P. Beauvis fruit (white rubber vine) is an under-utilized and underexploited wild food plant.Its sweet and sour stringy fruit pulp with numerous seeds are consumed in Southern and North-Central regions of Nigeria; where it is known variously as Nwalika or Eso (Igbo), mba (Yoruba), Ciwo (Hausa), Ana (Idoma) and Ipungwa (Tiv) (Nwaogu et al., 2008;Nwaogu et al., 2007).Oil of edible quality has been extracted from its seeds (Akubugwo and Ogbogu, 2007).The hexane, chloroform, ethyl acetate and methanol extracts of Landolphia owariensis fruit back had been reported to contain alkaloids, cardiac glycosides, saponins and terpenes (Iombor and Anyam, 2015).However, not much is known about the phytochemical composition and antimicrobial activity of the fruit pulp extracts.The study therefore, determined the phytochemical composition of L. owariensis fruit pulp and elucidated the health potentials of its extracts.

Fruits Collection and Drying
Fresh fruits of Landolphia owariensis were collected from the wild forests around Adikpo, Benue State, Nigeria (6° 50' 37.8 "N 9°15 '31.1"E).The plant was identified in the Department of Forest Products and Production, University of Agriculture, Makurdi, Nigeria.The mesocarp (pulp) together with the seeds were extracted after the fresh fruits were split open using a stainless kitchen knife; oven dried at 50 0 C to constant weight, followed by seed removal and pulverization of oven dried pulp using a domestic kitchen blender (Supper master, China).The pulverized pulp was stored in black polyethylene bags in vacuumed desiccators at ambient temperature (36±1 0 C) until needed for use.

Extraction of Fruit Pulp Material
Microwave Assisted Extraction was carried out using a domestic microwave oven according to method described by (Abuın et al., 2000) with modifications.The pulverized pulp (100 g) was introduced to extraction vessel [Winchester bottle (2.5 L)].Two hundred milliliters each of hexane, chloroform, ethyl acetate and methanol were separately added (a solvent at a time, in the sequence listed) to the pulverized pulp and the mixture bombarded with microwaves (70 Watts/Defrost Function) using a domestic kitchen microwave (Mio-star, Model 7173.295,Germany) for 30 minutes (3 minute microwaving with 15 minute cooling pauses).Pressure build up was vented after every two successive sessions by very slowly unscrewing cap of extraction vessel and agitating very gently.Watman number 1 (size: 24) filter paper was used to filter the extracts which were then concentrated in vacuo at 40 o C and air dried at ambient temperature (36±1 0 C).The dried extracts were stored in clean glass bottles at ambient temperature until required for use.

Quantification of Phytochemical Constituents
The method of Luthria et al. (1997) was used in quantifying the phytochemical constituents in methanol extract of oven dried fruit pulp of L. owariensis, with the aid of gas chromatography-mass spectrometry (GC-MS) (model 6890 gas chromatograph and model 5973 quadrupole mass spectrometer; Hewlett-Packard, Palo Alto, CA).Separations of the individual flavonoids and phytosterols were performed using a DB5 capillary GC column (30 × 0.25 mm internal diameter, 0.25μm film; J & W Scientific, Folsom, CA).Splitless injections of 2 μL were made and the column temperature was programmed from 150 (held for 3 min) to 230°C at 5°C/min with a final hold at 230°C for 30 min.Helium was used as the carrier gas with a flow rate of 0.7 mL/min and with an average linear velocity of 23 cm/min.Quantitative analysis was performed using 70-eV electron ionization and select-ion monitoring of fragment ions at a mass-to-charge ratio (m/z) of 355 with a dwell time of 100 m/s channel.

Antimicrobial Activities Collection of microbial isolates
The human pathogens; Staphylococcus aureus; Streptococcus pyogens, Escherichia coli, Salmonella typhi, Shigella dysenteriae, Pseudomonas aeruginosa, Klebsiella pneumonia (bacteria species), Candida albicans, Candida tropicalis, Candida krusei, Candida stellatoidea, Microsporum gypseum, Microsporum spp and Trichophyton rubrum (fungi species) used in the study were obtained from the stock culture of the Department of Medical Microbiology, Ahmadu Bello University Teaching Hospital, Zaria, Kaduna State, Nigeria.

Antimicrobial Sensitivity Test
The sensitivity of test organisms to n-Hexane, Chloroform, Ethyl acetate and Methanol extracts of Landolphia owerensis fruit mesocarp was carried out using the diffusion method described by Ebi and Ofoefule (1997).The extract (0.3 g) was weighed and dissolved in dimethyl sulfoxide (DMSO) (10 mL) to obtain a concentration of 30 mg/mL.Mueller Hinton agar was used as growth medium for bacteria while Sabouraud dextrose agar was used as growth medium for fungi.The media were prepared according to standard procedures and poured into sterile petri dishes, cooled and allowed to solidify.Sterilized media were fed with standard inocula (0.1 mL) of test microbes.Inocula were spread evenly over surface of media by use of a sterile swab.Using a standard cork bearer of 6 mm in diameter, a well was cut at the center of each inoculated medium.The standard solution of the extract (0.1 mL) of concentration 20 mg/mL was then introduced into each well on the inoculated medium.The Petri-dishes were allowed to stand for about 30 minutes at room temperature to allow for proper diffusion of extracts to take place.The plates were then incubated at 37 0 C for 24 hours for the bacteria and at 30 0 C for 1-7 days

Phytochemical and Anti-Microbial Potential of Landlphia owariensis Fruit Extracts
for the fungi.The zones of inhibition of growth in millimetres were measured and recorded.

Minimum Inhibitory Concentration (MIC) Test
The broth dilution method described by (Forbes et al., 2007) was used to determine the minimum inhibitory concentration.Muller Hinton and Sabouraud dextrose broth were prepared; 10 mL of the broth was dispensed into test tubes and were sterilized at 121 0 C for 15 minutes and allowed to cool.McFarland turbidity standard scale number 0.5 was prepared to give turbid solution.Normal saline (10 mL) was prepared and dispensed into sterile test tubes; test microbes were inoculated and incubated at 37 0 C for 6 hours.Dilution of test microbes in normal saline was made until turbidity reached the Mcfarland scale by visual comparison; at this point test microbes had a concentration of about 1.5×10 8 cpu/mL.Two-fold dilution of the extract in sterile broth was made to obtain the concentrations of 20, 10, 5, 2.5 and 1.25 mg/mL.The initial concentration was obtained by dissolving of the extract (0.3 g) in the sterile broth (10 mL).Standard inocula of test microbes in normal saline (0.1 mL) were then inoculated into the different concentrations, onto Mueller Hinton and Sabouraud dextrose broth, incubated at 37 0 C for 24 hours and 30 0 C for 1-7 days for bacteria and fungi respectively.Thereafter, each test tube of broth was observed for turbidity (growth).The lowest concentration of extract in broth which showed no turbidity was recorded as minimum inhibition concentration.

Minimum
Bacteria/Fungi Concentration (MBC/MFC) test Minimum bacteria and fungi concentrations were carried out to determine whether test microbes were killed or their growth was inhibited.Mueller Hinton and Sabouraud dextrose broths were prepared using standard procedures (Forbes et al., 2007), poured into sterilized petri dishes and allowed to cool and solidify.Content of minimum inhibition concentration in serial dilution was sub cultured onto the media; bacteria on Mueller Hinton and fungi on Sabouraud dextrose agar.Incubation was made at 37 0 C for 24 hours and 30 0 C for 1-7 days respectively.Thereafter, each plate was observed for colony growth.MBC/MFCs were plates with lowest concentration of extract without colony growth.

RESULTS AND DISCUSSION
Quantitative Phytochemistry Quantification of phytochemical constituents of ethyl acetate extract of Landolphia owariensis fruit pulp revealed it contained 0.03 mg/100g anthraquinones, 0.12 mg/100g alkaloids, 6.39 mg/100g flavonoids, 0.08 mg/100g cyanogenic glycosides, 0.15 mg/100g saponins, 0.55 mg/100g steroids, 12.45 mg/100g tannins and 0.67 mg/100g triterpenes (Table 1).The high level of flavonoids in fruit pulp of L. owariensis is suggestive of its anti-oxidative properties (Okonkwo & Osadebe, 2013;Owoyele et al., 2001;Middleton et al., 2000).Anti-oxidant property of flavonoids has been employed in protection against cancer (Lee and Shibamoto, 2002).Flavonoids have been implicated in numerous studies to have, among a host of other medicinal properties, diuretic, laxative, anti-spasmodic, anti-hypertensive, antimicrobial, anti-inflammatory and anti-radical properties (Abdelrazig, 2013; Tor-Anyiin and Anyam, 2013; Guarize et al., 2012;Nogueira and Lopes 2011;Macdonald et al., 2010;Kubmarawa and Ajoku, 2007).The high tannin content of the fruit mesocarp may confer wound healing, antibacterial properties and other numerous medicinal implications upon consumption (Liu et al., 2014;Banso and Adeyemo, 2007;Lim et al., 2006;Osadebe and Ukwueze, 2004;Tan et al., 1991).Tannins have equally been reported to act as alphaglucosidase and alphaamylase (digestive enzymes) inhibitors thereby helping to retard postprandial blood glucose increase (Boath et al., 2012).The presence of saponins, tannins and alkaloids in the fruit pulp of L. owariensis is suggestive of anti-nutritional tendency that can cause haemolysis, nutrient malabsorption and abnormal hematopoiesis (Balogun and Akinloye, 2012).The alkaloids and saponins content of the fruit pulp however, may be employed dietetically in management of headache associated with hypertension; as well as anti-fungal agent in treatment of some fungal infections (Mensah, 2013).The cardiac glycosides levels of the mesocarp may also be used in treatment of congestive heart failure and cardiac arrhythmia (Mensah, 2013).They have also proven to be useful when used in therapeutic doses in inhibiting or preventing the growth and spread of tumors or malignant cells (Seigler, 1991;Conn, 1981;Jones, 1972).
Daily intake of certain terpenoids might be useful for the management of obesity-induced metabolic disorders, such as type 2 diabetes, hyperlipidemia, insulin resistance, cardiovascular diseases, and a lower prevalence of metabolic syndrome (Goto et al., 2010).Other health benefits include reduced incidences of coronary heart disease and inflammation.Further analysis of its flavonoid composition revealed that it contained 0.02 mg/g orientin (a flavone subclass of flavonoid), 0.06 mg/g isovitexin (a flavone subclass of flavonoid) and 0.09 mg/g kaempferol (a flavonol subclass of flavonoid).The isolation of orientin from L. owariensis fruit pulp is of health significance in the food industry.Lin et al. (2004) and Li et al. (2002) have demonstrated that orientin has moderate or potent antiviral activity against Para 3 virus.Apart from antiviral effects, orientin also expresses antibacterial effects.The antibacterial significance of orientin has been expressed in a study by (Ali and Dixit, 2012), in which orientin and vicenin together as flavonoids, synergistically inhibited the growth of Escherichia coli, Staphylococcus aureus, Staphylococcus cohnii, Klebsiella pneumoniae, and Proteus, while the individual flavonoids were found to be less effective than the combined flavonoids (Ali and Dixit, 2012).Similarly, orient in and vitexin in Trollius chinensis together has been reported to show evidence of antibacterial (Lin et al., 2004).Vitexin (a flavone) has been reported to lower blood pressure and exert antiinflammatory effect (Prabhakar et al., 1981), protect against cardiac hypertrophy (Lu et al., 2013), inhibit platelet aggregation (Piccinelli et al., 2008), vascular smooth contractility (Je et al., 2014) and apoptosis (Wang et al., 2015;An et al., 2015).Kaempferol a gastrointestinal lipase inhibitor hinders fat digestion and absorption (Sergent et al., 2012).Kaempferol (a flavone) also has a potent antioxidant property towards chemical induced hepatic injury (Bigoniya et al., 2013;Sahu and Gray, 1996) and has an anti-lipid peroxidative effect.The phytosterol composition of ethyl acetate extract of L. owariensis fruit pulp showed it contained 0.28 mg/kg, 0.81 mg/kg, 0.21 mg/kg, 1.06 mg/kg, 0.98 mg/kg and 0.86 mg/kg cholesterol, cholestanol, campesterol, stigmasterol, beta-sitosterol and to copherol, respectively.High intake of campesterol, sitosterol and stigmasterol has been reported to protect against obesity and atherosclerosis (Schonfeld, 2010) and decrease serum total and LDL-cholesterol levels (Izar et al., 2011).Mechanistically, phytosterols compete with cholesterol for micelle formation in the intestinal lumen and inhibit cholesterol absorption (Izar et al., 2011).Their influence on intestinal genes and transcription factors make phytosterols key regulators in metabolism and cholesterol transport in the expression of liver genes (Gupta et al., 2011, Jesch et al., 2008).

Antimicrobial Activity
Microbiological analysis of hexane, chloroform, ethyl acetate and methanol extracts of Landolphia owariensis fruit pulp showed antimicrobial activities against S. aureus, S. pyogenes, S. typhi, S. dysenteriae, K. pneumonia, C. albicans, C. krusei, C. stellatoidea and M. rubrum.The extracts however showed no activity on E. coli, P. aerogenosa, C. tropicalis, M. gypseum and T. rubrum (Table 2).The results of diameters of zones of inhibition of extracts on growth of pathogenic organisms indicate that chloroform extract had highest zones of inhibition, followed by hexane and ethyl acetate while methanol extract had least diameters of zones of inhibition.The minimum inhibition concentration (MIC) of extracts on bacterial growth showed that effect of extracts is dose dependent (Table 3).Meanwhile, extracts had minimum inhibition concentrations of 5 mg/mL (chloroform and methanol), 5 to 10 mg/mL (hexane) while ethyl acetate ranged between 2.5 mg/mL and 5 mg/mL; implying that ethyl acetate extract was more potent even at lower concentrations.The extracts ability to inhibit bacterial/fungal growth was 20 mg/mL (hexane), 10 mg/mL to 20 mg/mL (methanol and chloroform) and 5 mg/mL to 10 mg/mL (ethyl acetate).The results showed that ethyl acetate extract would be more effective in treating bacterial and fungal infections.The microbiological result of this study corroborates previous findings by Galadima et al. (2010) and Nwaogu et al. (2007) that methanolic and ethanolic extracts of leaves and roots of the plant inhibited growth of Staphylococcus aureus and Salmonella typhi, respectively.It further laid credence to the use of the leaves and roots of the plant by traditional medicine practitioners in Nigeria in treating venereal diseases.Iombor T.T. and Gbeyonron F.M.

Table 3 :
Anti-microbial activities and Diameter of Zone of inhibition (DZI) of Landolphia owariensis fruit pulp extracts

Table 4 :
Minimum inhibition (MIC) and Bactericidal/Fungicidal concentrations (MBC/MFC) of Landolphia owariensis fruit pulp extracts against test microbes (mg/mL) Please include GC-MS chromatogram of L. owariensis The full meaning of DMSO solvent.