Pharmacological activity of selected medicinal plants with potential for preventing and treating infectious bovine mastitis, and identification of compounds from active fractions of Kalanchoe gunniae

Abstract

Infectious mastitis is an inflammation of the udder usually associated with increased somatic cells and presence of microorganisms. It is challenging to the health of cattle due to antibiotics resistance resulting in huge financial losses. Inflammation is a complex reaction of living tissues to microbial infection or injury. Normally it promotes healing, but when uncontrolled it can result in cell damage or death. In cattle and humans, inflammation is a major feature of infectious mastitis which has significant health and financial challenges. Excess reactive oxygen species accompanying uncontrolled inflammation are also harmful. Staphylococcus aureus is increasingly becoming resistant to antibiotics for the management of infectious bovine mastitis. This is often coupled with significant financial losses for the farmers. Plant secondary metabolites like flavonoids, phenolic compounds and their synergetic activity have been reported to provide alternative treatment due to their antibacterial, antioxidant and anti-inflammatory activities. In this study, the antimicrobial activity of 15 plants selected on the basis of preliminary activity of related plant species in the same family (chemotaxonomic selection), or relevant traditional uses of the species for treatment of infections in humans (ethnopharmacological selection) was initially investigated. A broth microdilution assay using acetone and ethanol extracts of these plants was used to determine activity against an ATCC strain of Staphylococcus aureus (S. aureus ATCC 29213). Among these 15 plants, extracts of two plants from chemotaxonomy selection and two plants from ethnopharmacological selection, namely Maytenus undata and Maurocenia frangula, and Kalanchoe gunniae and Bryophyllum pinnatum (synonym Kalanchoe pinnata), respectively, with promising minimum inhibitory concentration (MIC) values ranging from 0.02 to 0.31 mg/ml were selected for further assay. These extracts were then tested against six multiple-drug resistant S. aureus isolates from clinical bovine mastitis cases. The four plant extracts were further analysed for cytotoxicity and anti-quorum sensing activities. Two of the plants with good results were assessed for anti-biofilm activity. The anti-inflammatory and antioxidant activities of acetone and ethanol leaf extracts of Maurocenia frangula, Maytenus undata, Kalanchoe gunniae and Bryophyllum pinnatum were further investigated. Antioxidant activity was evaluated using radical scavenging 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and electron reducing 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays. Anti-inflammatory activity was determined via inhibition of 15-lipoxygenase (15-LOX) and cyclooxygenase (COX) enzymes, as well as inhibition of nitric oxide (NO) production by lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. Anti-inflammatory activity through regulating cytokine production was further investigated using ELISA kits. Kalanchoe gunniae consistently gave good results for all the above-mentioned assays so K. gunniae 80% methanol crude extract was fractionated with hexane, dichloromethane, ethyl acetate, butanol and water. Antibacterial activity of crude extracts and fractions of K. gunniae were investigated against S. aureus (ATCC 29213) and six S. aureus bovine mastitis isolates using a broth microdilution assay. Cytotoxicity of the crude extract and fractions were further investigated against Vero cells and their selective indices were determined. The total flavonoid and total phenolic contents of the fractions were determined using the aluminium chloride spectrometric method and the Folin-Ciocalteu method respectively. Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) analysis of the crude extract and active fractions were performed. Minimum Inhibitory Concentration (MIC) values of the extracts against S. aureus isolates ranged from 0.02 to 0.63 mg/ml. Kalanchoe gunniae extracts were the least cytotoxic with selectivity index (SI) ranging from 12 to 25. Significant biofilm inhibition against S. aureus mastitis isolates was observed at time zero (0 h), however there was no eradication of developed biofilm. Kalanchoe gunniae extracts had the best anti-quorum sensing activity with minimum quorum sensing inhibition concentration (MQSIC) and MIC values of 0.04 mg/ml and 0.63 mg/ml respectively on Chromobacterium violaceum ATCC 12472. From the results of antioxidant and anti-inflammatory assays, K. gunniae extracts had the best antioxidant potency with IC50 values ranging from 0.06 to 0.42 μg/ml. K. gunniae extracts also had the best 15-LOX inhibitory activity with IC50 values of 1.25 and 2.03 μg/ml for acetone and ethanol extracts respectively. At the highest concentration (100 μg/ml), the acetone extract of B. pinnatum had the best NO inhibition of 80.48% and cell viability of 96.75%. From COX-2 assay it was observed that K. gunniae extracts and B. pinnatum extracts were able to significantly inhibit COX-2. B. pinnatum extracts had the best cytokine activity. MIC values of the 80% methanol crude extract and fractions of K. gunniae against S. aureus ATCC 29213 ranged from 0.03 mg/ml to 0.63 mg/ml. MIC values against the six S. aureus bovine mastitis clinical isolates ranged from 0.02 mg/ml to 2.50 mg/ml. Total flavonoid content of fractions and crude extract ranged from 2.12 mg QE/g to 140.30 mg QE/g. The total phenolic content of fractions and crude extract ranged from 252.75 mg GAE/g to 943.09 mg GAE/g. From the crude extract, the major compounds 1-docosene, 1-nonadecene and benzenedicarboxylic acid and butyl 2-ethylhexyl ester were identified from GCMS. The compounds genistin, myristicin, apigenin-7-o-α-l-rhamnose (1→4)-6” o-acetyl-β-d-glycoside and mudanpioside H, were identified from UPLC-MS analyses. From active fractions, the major compounds 1-docosene, butane, 2,2,3,3-tetramethyl, cetene, tetradecane, nonadecane and heptadecane, 2-methyl- nonadecane were identified from GCMS-MS and the compounds genistin, mudanpioside h, apigenin-7-o-α-l-rhamnose (1→4)-6” o-acetyl-β-d-glycoside, myristicin, kaempferol-3-o-rhamnoside and safflor yellow A were identified from UPLC-MS analyses.