Antimicrobial and Wound Healing Properties of Leaf Extracts, Fractions and Ointment Formulations of Spermacoce Verticillata Linn (Family: Rubiaceae)

Abstract

This study investigated the antimicrobial and wound healing properties of leaf extracts and ointment formulations of Spermacoce verticillata Linn (Family: Rubiaceae). Three different solvents: acetone, ethanol and water were used in the extraction of phytochemicals from the leaves of Spermacoce verticillata. The bioactive constituents of acetone, aqueous and ethanol extracts were determined using standard phytochemical analytical methods. The cup plate agar diffusion method was used to evaluate the antimicrobial activities of the three crude extracts at 100 µg/ml, 200 µg/ml and 400 µg/ml concentrations, in comparison with the control- gentamycin at 40 µg/ml concentration. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the extracts were obtained using agar dilution method for determination of the most efficacious extract. Acetone extract was observed to be the most potent. Accelerated gradient chromatography (AGC) was used to fractionate the acetone extract into two; fractions A and B. Phytochemical analysis of fractions A and B were carried out using standard laboratory methods. The cup plate agar diffusion method was used to assess the antimicrobial activities of the fractions at 25 µg/ml, 50 µg/ml, 100 µg/ml, 200 µg/ml and 400 µg/ml concentrations in comparison with the control -gentamycin at 40 µg/ml concentration. Fraction B which had better antimicrobial properties was formulated into ointments of 0.1% w/w, 0.2%w/w, 1%w/w, 2%w/w and 5%w/w concentrations and assessed for wound healing studies on wounds inflicted on albino rats. The relationship between types of microorganisms, concentrations of ointment, and wound healing time were studied. Acute oral toxicity studies for the determination of the medium lethal dose (LD50) of the acetone extract was calculated using the Kerber's method. Histological studies that would indicate toxic effect(s) of the acetone extracts on probable target organs (skin, kidney, liver, and heart) were also undertaken. Data generated were subjected to analysis using analysis of variance (ANOVA) which was arranged in a randomized complete block design (RCBD) and means were compared or separated using least significant difference (LSD) at 5% confidence interval. Correlation and regression analysis was also used in which case coefficient of simple determinant (r2) was obtained which was used to measure the relationships that existed between inhibition zones/wound diameters and various levels of concentrations of extracts. Descriptive statistics was employed to measure the level skewness or variability of data that were obtained in the study. The following phytochemicals: balsam, terpenes and steroids were present in all the three leaf extracts. Acetone and ethanol extracts both had alkaloids, phenols and volatile oils. The acetone extract had glycosides and saponins in addition; all extracts however lacked flavonoids and resins. All the extracts possessed antibacterial activity but lacked antifungal activity. Minimum inhibitory concentration (MIC) was 50 µg/ml for acetone leaf extract on S. aureus and E. coli, which was the least MIC; ethanol extract had an MIC of 100 µg/ml concentration on S. aureus and E. coli; while the aqueous extract had an MIC of 200 µg/ml on B. subtilis and E. coli. The aqueous extract had a minimum bacterial concentration (MBC) of 400 µg/ml on S. aureus, while the ethanol extract had an MBC of 400 µg/ml on E. coli. The best result was however obtained by acetone extract with 200 µg/ml of MBC on E. coli and P. aeruginosa. The acetone extract was seen to have more pronounced antibacterial activity than the other extracts. Antibacterial studies of the extracts (A and B) fractions on the test organisms showed that the B fraction had better antibacterial activity. All the strengths of the ointments promoted wound healing on rats infected with test microorganisms with best result achieved on S. aureus, followed by E. coli, B. subtilis and lastly, P. aeruginosa. Comparing the progress of healing of the infected wounds treated with the blank ointment and those treated with ointments of fraction B, suggests that the wound healing effect of fraction B was attributed to its antibacterial property especially when the extract ointment at 2 % concentration promoted wound healing better than 0.1% gentamycin ointment. Complete healing was achieved on the 10th day (on S. aureus, E. coli and P. aeruginosa infected wounds); however, all the concentrations achieved complete healing on the 14th day, except the blank ointment. The toxicity study of fraction B indicated that 100 µg/g dose did not kill or adversely affect any animal, while 250 µg/g dose killed just one rat. Since the MICs of fraction B were between 50-100 µg/g, a safe dose for administration would be between 100 µg/g – 250 µg/g. From the histopathological study, the photomicrographs of organs from rats administered with LD50 acetone extract, food and water, showed no histological abnormalities.