The Chemical Constituents and Bioactivity of the seed (Fruit) extracts of Buchholzia Coriacea Engler (Capparaceae).

: Buchholzia coriacea Engler ( Capparaceae) is important in traditional medicine because many parts of the plant have been reported to possess therapeutic potentials. The seeds of the fruits of Buchholzia coriacea were extracted with petroleum ether, chloroform and methanol respectively, and the crude extracts were analyzed using GC-MS. The result indicated presence of cyclooctasulphur, oleic acid, (Z,Z)-9,12-octadecadienoic acid, n-hexadecanoic acid, methyl ester (Z,Z)-9,12-octadecadienoic acid, 5-hydromethylfurfural, (E)-9-octadecanoic acid, 1-methyl-pyriolidine-2-carboxylic acid, undecane, palmitoleic acid, stigmasterol, beta-sitosterol and methyl ester 3,5-dicyclohexyl-4-hydroxy-benzoic acid amongst other constituents. Bioactive test of the crude extracts and fractions from column chromatography showed great potential as antifungal agent than antibacterial agent, when tested against isolated fungi and Gram-positive and Gram-negative bacteria. © JASEM http://dx.doi.org/10.4314/jasem.v19i4.30


INTRODUCTION
In traditional medicine, the efficacy of herbs is tested and proven and their application has been of great assistance in treatment and prevention of diseases as well as development of new plant derived drugs.Buchholzia coriacea, a perennial plant, which belongs to the family Capparaceae, grows as a forest tree and it is one of the plants used in herbal medicine due to its medicinal potential.The crushed seeds are used on skin eruption.The bark decoction is also used to wash persons with small-pox (Burkill, 1985).The young leaves are used as a poultice for boils and the seeds used as condiments or as cough medicine (Dalziel, 1937).Previous studies on the plant reported isolation of cyclooctasulphur which had significant antifungal activity on Aspergillus fumigates, Mucor mucedo and Penicillium nigricans (Ojinnaka et al., 1992).The leaves had anthelmintic effects on Fasciola hepatics (Ajaiyeoba et al., 2001).The ethanolic extract of Buchholzia coriacea was also shown to have antitrypanosomial activity in mice experimentally infected with Trypanosoma brucei (Nweze et al., 2009).Mbata et al., (2009) reported that the methanolic extract of Buchholzia coriacea seed exhibited good antibacterial activity on some gastrointestinal bacteria; Bacillus cereus, Escherichia coli, Salmonella typhimurium, Staphylococcus aureus and Vibrio cholera.The aim of present work is to detect the chemical constituents of crude extracts of Buchholzia coriacea using GC-MS and to determine the bioactivity of the constituents.To the best of our knowledge, present study reports, for the first time, detection of bioactive constituents from the fruit of Buchholzia coriacea.

MATERIALS AND METHOD
General: Evaporation was carried out with Rotavapor R110, (Buchi, Laboratoriums Technik AG CH-9230 FLAWIL/SCHWEIZ).The Gas Chromatography-Mass Spectroscopy (GC-MS) analyses were performed on a Gas Chromatography (GC) ailment 7890A with Mass Spectrometer system 5975.Capillary column HP-5 was used with column length 30m and diameter 0.32.The oven temperature was programmed from 60-240 O C and helium was used as the carrier gas at 1ml/min.The components of different extracts were identified based on the names of the compounds, retention time, area percentage, peak numbers and mass spectra obtained using the data base library of National Institute of Standard and Technology (NIST) Integrator-Chemstation. CC was on a column (length 60cm, diameter 2.5cm) packed with silica gel (60 -120 mesh).
Plant material.The fruits of Buchholzia coriacea were collected from Ojinnaka's compound in Akpulu, Ideato-North LGA, Imo State, Nigeria and were authenticated by Dr. Edwin Wosu, a staff of University of Port Harcourt Herbarium.

Extraction
and identification of bioactive constituents.The seeds were deseeded from the fruits, air-dried, at room temperature, for three (3) weeks, then cut into small pieces and milled into fine powder using hand mill.Extraction was done by cold maceration using different solvents (petroleum ether The Chemical Constituents and Bioactivity of the seed (Fruit) 7961* CHUKWUNONYE M. OJINNAKA, 1 KELECHI I. NWACHUKWU, 2 MARYCOLETTE N. EZEDIOKPU 60-80 0 C, chloroform, 60-62 0 C and methanol, 64.6 0 C) successively.The milled seeds (1.65kg) were soaked with 2.6L petroleum ether for 72hrs in a 5L aspirator bottle with intermittent shaking, and later soaked again with 1.0L of petroleum ether for complete extraction.This process was repeated using chloroform (2.5L) and methanol (2.5L).The filtrate of different solvents were concentrated using rotary evaporator at 40 0 C. The petroleum ether crude extract was filtered and the precipitated solid washed with cold petroleum ether to obtain a light-yellow solid crystal of cyclooctasulphur (2.165g) and oil (15.276g).The Chloroform and Methanol extracts gave viscous oil 3.477g and 151.075g respectively.The petroleum ether, chloroform and methanol extracts were screened, using gas chromatographymass spectrometry (GC-MS) technique to obtain the constituents in Table 1, Table 2 and Table 3 respectively.
Chromatographic analysis.The crude methanol extract (3g), which was eluted with a mixture of petroleum ether in chloroform and then methanol, yielded fractions A (0.139g), B (0.038g), C (0.433g), D (0.495g), E (1.160g), and F (0.0465g) after concentration under reduced pressure (45 o C).The antimicrobial activity of fractions A, C, D and E were tested against the microorganisms of interest, together with petroleum ether, chloroform and methanol crude extracts (Tables 4-9).
Isolation and identification of bacteria.The bacteria used for the bioassay test were clinical isolates collected from the laboratory of the Medical Microbiology Department of Braithwaite Hospital, Port Harcourt, Rivers state, Nigeria.The isolates were cultured on both blood agar and chocolate agar and thereafter further purified by subculturing each isolates on a fresh plate of Nutrient Agar (NA) and incubated at 37 O C for 24 hours.The isolates were characterized and identified based on the results of gram stain and other biochemical test as Escherichia coli, Staphylococcus aureus, Klebsiella pneumonia, Proteus mirabilis and Pseudomonas aeuroginos with reference to the Bergey's Manual of Determinative Bacteriology, (1994).
Isolation and identification of dermatophytes.This was done using modified method by Grodmadzki et al., 2003.Fungi used were isolated from scrapings obtained from affected feet of a student from the University of Port Harcourt, Rivers state, Nigeria by standard method.Samples were prepared using standard 10fold serial dilution method in buffered saline.Prepared samples were placed on Sabouraud Dextrose Agar plates containing cyclohexamide 4mg/ml, penicillin 20µ/ml, streptomycin 40µ/ml and bromocresol purple 1.6mg/ml.Plates were incubated for 7days at 37ºC.Colonies that exhibited a colour change from light green to purple were purified and identified using Mycology online (2001).Isolates (Bacteria and Dermatophytes) were maintained on Nutrient Agar (NA) and on Sabouraud Dextrose Agar (SDA) slants respectively at 4 0 C in refrigerators for further identification.Preparation of crude extracts.A stock concentration of 100mg/ml of solid crystal and oil from petroleum ether crude extract, chloroform, methanol crude extracts and fractions A, C, D and E from the column chromatography respectively were prepared by dissolving 1g of each crude extract in 10ml Dimethyl sulphoxide (DMSO), which was used for the susceptibility test.
Antibacterial assay.The Kirby-Bauer diffusion method on Mueller-Hinton Agar was used for the antibacterial activity for the different crude extracts.A standard concentration of bacteria strain was prepared using 0.5M McFarland standard under aseptic condition.A loopful of colony (bacteria) on a sterile inoculating loop was inoculated into a test tube containing 5ml of freshly prepared sterile physiologic saline (i.e.prepared by dispensing 8.5g of sodium chloride salt in 1000ml of distilled water).The broth was left to stand for 5-10 minutes and its turbidity compared with the 0.5M McFarland standard.A sterile non-toxic swab stick was dipped into the adjusted suspension and pressed several times on the wall of the test tube, then inoculated into the Mueller -Hinton Agar media by spreading the plates.The sterile disc was impregnated with crude extracts and placed into agar plates and allowed to stand for 15 minutes to dry.The inoculated plates were incubated at 37 0 C for 24 hours.A control test was also carried out using dimethyl sulphoxide (DMSO) only.Antibacterial activity of the extracts was determined by measuring the Zone of inhibition.(Coyle, 2005) Antifungal assay.This was done using the modified method of Rahman et al., (2001).Sabouraud Dextrose Agar (SDA) was prepared by mixing 4% of glucose agar and agar in distilled water, stirred with a magnetic stirrer to dissolve it and 3ml amount was dispensed into bijou bottles, upon sterilization (at 121 o C for 15mins).Bijuo bottles were allowed to cool to 50 o C and the test sample (crude extracts) of 0.1ml concentration pipetted from the stock solution into the non-solidified Sabouraud agar media and allowed to solidify at room temperature, each bijou bottle was inoculated with 4mm diameter of innocullum removed from a seven day old culture of fungi.A control test was also carried out using dimethyl sulphoxide (DMSO) only.

Tricophyton interdigitale
The susceptibility test carried out on the Gram-positive and Gram-negative bacteria and the dermatophytes (fungi) using the crude extracts of the seeds of the fruit of B.coriacea indicated that all the bacterial isolates showed resistance to the crude extracts with no observed zones of inhibition (Table 5).

Key: -++= No growth (high Susceptibility) -= Resistant
The fungi gave positive results by responding differently to the various extracts.Isolate 1 (E.floccosum) was shown to be susceptible to crude extracts of methanol, oil and fraction D. Isolate 2 (T.rubrum) was found to be susceptible to chloroform extracts, oil and fractions A, C whereas isolate 3 (T.interdigitale) were found susceptible to crude extracts of methanol, chloroform, solid crystal (cyclooctasulphur), oil and fraction A (Table 6).The control test (performed with DMSO) showed no inhibition on isolates.The results obtained for the MIC assay on the 3 crude extracts showed that E. floccosum, T.rubrum, and T.interdigitale were most susceptible to the oil (petroleum ether crude extract) by showing a wider range of susceptibility as far as 1:32 (3.125mg/ml) concentration (Table 7-9).The methanol crude extract had a poor activity on E.floccosum with MIC value of 1:4 (25mg/ml) concentration (Table 7) and on T.interdigitale with MIC of 1:16 (6.25mg/ml,Table 9).Also chloroform extract had MIC value of 1:8 (12.5mg/ml) concentration on T.rubrum (Table 8) and 1:16 (6.25mg/ml) concentration on T.interdigitale (Table 9) respectively.The GC-MS analysis of the crude extracts showed the presence of some major compounds, which contributed to the antimicrobial activity of the seeds.These include oleic acid, (Z, Z) -9, 12-octadecadienoic acid, n-hexadecanoic acid, methyl ester (Z, Z)-9, 12-octadecadienoic acid and (E)-9-octadecenoic acid, which are known to have antifungal potential (Seidel andTaylor, 2004, Hong-Wei et al., 2014).Palmitoleic acid, 5hydromethylfurfural, cyclooctasulphur (Ojinnaka et al., 1992), 3-amino-8-(prrolidine-1-sulfonyl)dibenzofuran, 1-ethyl-3-methyl-benzene (Sujathal et al., 2011), methyl ester 3, 5-dicyclohexyl-4-hydroxybenzoic acid and 1-methyl-pyriolidine-2-carboxylic acid, which have been shown in literature to possess antifungal and antibacterial potential were identified as constituents of crude extracts of Buchholzia coriacea.The result of present study shows that the seeds of the fruit of Buchholzia coriacea possess medicinal and pharmacological properties.The variety of the medicinal and pharmacological potential possessed by this plant may be traced to some of the identified compounds acting either singly or in synergy.Compounds having antimicrobial, wound healing and anti-inflammatory potential were identified.This result encourages the continuous use of the seed of Buchholzia coriacea in traditional medicine and also the possibility of the use of the identified constituents as lead compounds in the development of new useful drugs.The presence of the constituents might have contributed to the use of the plant extracts as antimicrobial agent in traditional medicine and in the treatment of various diseases.

Table 2 :
Chemical composition of the chloroform crude extract fraction

Table 3 :
Chemical composition of methanol crude extract fraction

Table 4 :
Identification of Dermatophytes

Table 5 :
Susceptibility result of crude extracts on Bacterial isolates.

Table 6 :
Susceptibility result of crude extracts on Dermatophytes isolates.

Table 7 :
Minimum inhibitory concentration of crude extracts on E. floccosum.

Table 8 :
Minimum inhibitory concentration of crude extracts on T.rubrum.