ISOLATION AND ANTIMICROBIAL ANALYSIS OF A STEROIDAL TERPENE FROM THE BUTANOL FRACTION OF BYROPHYLLUM PINNATUM ( LAM . ) OKEN

Bryophyllum pinnatum (Lam.) Oken is a plant used in treatment/management of ear-ache, cough, gastro-intestinal disorders and inflammation. Prior to this study, reports of the isolation of cardiac glycosides from the ethyl-acetate fraction of the plant abound. However, very scanty or no literature exists on other organic fractions from where chemical constituents could also be obtained. Hence, the chemical and biological properties of the butanol fraction of the plant were studied. The silica gel column chromatography of the fraction led to a steroidal terpene whose identity has been revealed to be 3-hydroxy-(3β, 17β)-spiro(androst-5-ene-17,1ʹ-cyclobutan)-2ʹ-one using the MS and IR spectral techniques. This compound was strongly bacteriostatic against Staphylococcus aureus and Candida albican sbut recorded no activity against Escherichia coli.


Introduction
Bryophyllum pinnatum (Lam.)Oken syn, (Cotyledon pinnatum, Crassula pinnatum and Kalanchoe pinnatum) is known as miracle leaf or life plant grows as succulent perennial herb in the tropical climatic zones of Africa, Latin America and Asia.However, the plant is now cultivated on a large scale and sold to the pharmaceutical industry for economic benefits. 1The plant is used in the treatment of earache, cough, gastro-intestinal disorders and leucorrhoea.Furthermore, extracts of the plant are employed in the treatment /management of inflammations such as cardiac problems, wounds, sores, diabetes, liver problems, certain cancers and kidney troubles. 2Previous chemical investigations of the plant have led to the isolation of three cardiac glycosides namely, bryophillia A, bersaldegenin -3acetate and bryophillin C while the fractionation of ethylacetate marc yielded seven kaempferol rhamnosides. 3The present study was carried out to isolate chemical constituent(s) in the butanol fraction which showed a higher antimicrobial activity than that by the ethyl-acetate fraction 4 and also screen the compound(s) for possible antibacterial and antifungal activities.

Experimental Collection of plant material
The fresh leaves of B. pinnatum were collected in the month of July, 2016 from inside the University of Uyo Main Campus, Akwa Ibom State, Nigeria.The plant had previously been identified 4 and a voucher specimen of the plant (No.H 045) was deposited in the Herbarium Unit of the Faculty of Pharmacy.Immediately after collection, the leaves were dried in a laboratory oven at 40 0 C for 48 h and the resultant material powdered on an electric mill (Uniscope, England).

Antimicrobial tests
The micro-organisms used in this study were limited to three viz: one Gram(+), one Gram(-) and a fungus.Staphylococcus aureus (ATCC 21824), Escherichia coli (ATCC 23523) and Candida albicans (NCYC 106) were clinically isolated from specimens of diarrheal stool, abscesses, necrotizing fascitis, urine and wounds obtained from the Medical Laboratory, University of Uyo Health Centre, Uyo.The clinical isolates were collected in sterile bottles, identified and typed by convectional biochemical tests. 5,6These clinical microbes were then refrigerated at -5 0 C prior to use.The agar plates used were prepared by adhering to the manufacturer's instructions.The media and plates were sterilized in an autoclave at 121 °C for 15 min.The hole-in-plate agar diffusion method was used observing standard procedure with Nutrient Agar-CM003, Mueller-Hinton-CM037 (Biotech Limited, Ipswich, England) and Sabouraud Dextrose Agar (Biomark, India) for the bacteria and fungus respectively.The inoculum of each microorganism was introduced into each petri-dish (Pyrex, England).Cylindrical plugs were removed from the agar plates by means of a sterile cork borer (Simax, India) to produce wells with diameter of approximately 5 millimeters.The wells were equidistant from each other and the edge of the plate. 7,8 oncentrations of 20 mg mL -1 of crude extract, 10 mg mL -1 of butanol fraction, 2 mg mL -1 of KF-2 were introduced into the wells.0][11][12][13] The experiments were carried out in triplicates.The plates were labelled on the underside and left at room temperature for 2 h to allow for diffusion.The plates were then incubated at 37±2 0 C for 24 to 48 h.Zones of inhibition were measured in mm with the aid of a ruler.).FTIR cm -1 : 913, 856 (alkyl substitution), 1612 (-C=C) and 3219 (-OH).The chemical structure of KF-2 was established by a combination of spectroscopic techniques as highlighted above.These data were matched with those in the library data of organic compounds.Furthermore, the obtained data were found to be consistent with those reported in literature. 14,15 herefore KF-2 has been identified to be 3hydroxy-(3β,17β)-spiro(androst-5-ene-17,1ʹ-cyclobutan)-2ʹone.(Due to the nature of the matrix, many fragmented peaks appeared in the MS of the compound but those that are easily identifiable include [M] + at m/z 328 (3.14 %), while fragments at 285 (5.25 %), 273 (6.04 %) and 271(29.46%) represent the losses of methyl groups and a hydroxy unit and methyl groups and carbonyl units from the molecular ion respectively.Furthermore, the ion a 242 (11.24 %), in addition to the excisions of methyl and carbonyl units also indicates the removal of methylene groups from the molecular matrix.However, the base peak at 183 (100.00 %) reveals the removal of methyl, carbonyl, methylene and hydroxy units from the [M] + .The IR spectrum of KF-2 shows absorptions at 913, 856, 1612 and 3219 cm -1 indicating characteristic alkyl substitutions, endocyclic -C=C and -OH functional groups respectively.It is interesting to note that this steroidal terpene has been isolated from Saccharium spontaneum (L.) and Rauwolfia vomitoria (Afzel) using gas-chromatography/mass spectrophotometry. 14,15

Antimicrobial Screening
The spectrum of microbes employed in the sensitivity tests was narrowed down to one Gram positive (S.aureus), Gram negative (E. coli) bacterial strains and fungus (C.albicans).The results displayed in the Table 1 show that the crude extract, butanol fraction and KF-2 are remarkably bacteriostatic against S. aureus and C. albicans while no activity was recorded against E. coli.Furthermore, the butanol fraction also inhibited the growth of S. aureus and C. albicans but was inactive against E. coli.Gram negative bacteria are well known for their unique resistance to antimicrobial agents.This resistance is believed to be due to the nature of the cell envelope of these organisms which unlike Gram positive organisms possess asophisticated  16 Derivatization studies are currently ongoing in our laboratories with the aim of improving on the observed activities.

Conclusion
The isolation of 3-hydroxy-(3β, 17β)-spiro(androst-5-ene-17,1ʹ-cyclobutan)-2ʹ-one is being reported for the first time from the butanol fraction of the plant.Hence, the compound is expected to serve as chemotaxonomic marker for this species and the genus, Bryophyllumin general.Furthermore, the results of the antimicrobial sensitivity tests lend some justification to the use of this plant especially in the treatment/management of bacterial disease.However, the compound and derivatives expected to be obtained in another study will be further screened against other bacterial and fungal strains with the aim of obtaining improved activity and widening the spectrum of antimicrobial activity.

Table 1 .
Antimicrobial screening of crude extract, butanol fraction, KF-2 at different concentrations on test microbes.