Effects of selected medicinal plant extracts on bacterial activity, cytotoxicity, rumen fermentation, nutrient utilization, and methane mitigation.

Doctoral Degree. University of KwaZulu-Natal, Pietermaritzburg.This research was conducted to address two important areas of global interest in livestock production: (i) the contribution of ruminants to climate change through the emission of methane gas produced in the rumen during microbial fermentation of feedstuffs (ii) the effect of the subtherapeutic use of antibiotic and other chemicals as rumen manipulator/ growth promotant on antibiotic resistance and chemical residue in animal products, respectively. This research was designed to explore one of the identified natural alternatives to antibiotics; plant extracts rich in phytochemicals. Inquest for finding an alternative to antibiotics use in ruminant production that can manipulate rumen microbes towards effective fermentation that will mitigate methane production, some plants species that have been documented for their traditional medicinal value were exploited for their bioactivity. Hence, the broad objective of this study was to evaluate and identify medicinal plant extracts that can manipulate rumen fermentation and improve animal productivity while mitigating methane emission, serving as natural alternative to antibiotic ionophore use in ruminant production. To achieve the objective, series of studies were conducted: Firstly, the plant species used in this study were selected based on previous report of their nematocidal activities in ruminants by some researchers in our research group (Fomum 2018; Ahmed et al., 2012) and also evidenced by other literature and WHO documentation of medicinal plants (WHO monographs of medicinal plants). The selected plants were identified, the plants material/parts were collected, and extracted using 80% ethanol. Rumen bacteria belong to both the gram positive and gram negative groups, hence a perturbation in the activity of any of these groups work affect methane emissions; hence the study used bacteria in the two gram nature. Ethanolic extract of 22 plant materials were investigated for their ability to inhibit the growth of gram-positive (S. aureus and S. faecalis) and gram-negative (E. coli and S. typhimurium) bacteria using the disc diffusion and microdilution methods. Seventeen plant extracts displayed antibacterial activity against at least one of the bacterial strains. Zones of inhibitions ranged from 10.00±0.00 to 21.33±1.50 mm for the diffusion assay. The minimum inhibitory concentration (MIC) ranged from 0.391- 3.125 mg ml-1. E. coli was the least affected bacterial strains tested. Results of both assays revealed that plant extracts showed more antibacterial activity in the microdilution relative to the agar disc diffusion assay. This suggests that the disc diffusion method should be used along with the microdilution assay when testing bacterial susceptibility to plant extracts. Most of the selected plants have pronounced antibacterial effects on gram-positive bacteria. Secondly, all the plant extracts were evaluated for their basic phytochemical compounds, quantitatively. The study showed that Psidium guajava had the highest concentration of alkaloids (219.06±11.50 mg/g). In contrast, Acacia nilotica leaf extract had the highest level of flavonoids of 191.60±9.07 mg/g. Vernonia amygdalina (79.84±1.35 mg/g) and Moringa oleifera (70.45±3.87 mg/g) contained more steroids than the other plant extracts while condensed tannin was highest in Carya illinoinensis (21.72±0.84 mg/g). Coffea arabica, Acacia nilotica leaf, Vernonia amygdalina, Carya illinoinensis and Psidium guajava had more saponins of 32.57±4.27, 31.68±2.75, 30.55±2.06, 30.18±3.59 and 30.13±2.86 mg/g extracts, respectively, than other plant extracts. Crude fat was higher in Carica papaya (640.9±14.21 mg/g) and Acacia sieberiana (624.22±4.00 mg/g) leaf extracts, whereas Acacia nilotica pod extract had the lowest concentration of 0.37±0.02 mg/g. Our findings provide evidence that most of the medicinal plant extracts evaluated were good sources of phytochemicals that have the potential to improve rumen efficiency towards reduced methane production. Cytotoxicity study was carried out to determine the safety of the selected plant extracts against normal mammalian cells, using the in vitro colorimetric cytotoxicity assay. Since all the 22 plant extracts evaluated for their antibacterial activity inhibit at least one of the bacteria (microdilution assay), hence all of them were determined for their cytotoxicity. Results showed that Allium cepa and Tulbaghia violacea with concentration killing 50% of cells (LC50) values of 0.5182±0.40 and 0.4909±0.034 mg/ml, respectively, were the safest of all the plant extracts. Acacia nilotica pod and Camelia japonica leaf extracts were the most cytotoxic with LC50 values of 0.0101±0.016 and 0.0151±0.005 mg/ml, respectively, which are well below the recommended toxic cut-off point (0.03 mg/ml). The best selectivity index (SI) value of 1.061 was obtained with Aloe ferox against S. aureus, which is a Gram-positive bacterium. The majority of plant extracts evaluated were relatively non-cytotoxic, although the low SI values obtained for almost all the plant extracts indicate more significant cellular toxicity than activity. Furthermore, a study was conducted to determine the effect of all the selected medicinal plant extracts on rumen fermentation parameters in vitro using the in vitro gas production techniques. In the study, plant extracts and monensin were incubated as additives to hay at 16 and 48 h incubation period. Fermentation parameters were determined at both hours of incubation. The results showed that plant extracts influenced gas production (GP) in a varied way relative to control at both hours of incubation. Microbial protein yield (MY) was not significantly affected at 16 h (P> 0.05), but it was at 48 h (P<0.01). Higher MY were recorded for all treatment except for A. sativum and C. intybus at the early incubation stage (16 h) relative to 48 h incubation. Compared to control group at 48 h, all plant extracts had higher MY. After 48 h of incubation, the results showed that plant extracts influenced ruminal degradation, gas production, microbial protein yield and partitioning factor. C. papaya leaf extract had the highest true degradation and NDF-degradation, although this does not differ significantly from all other treatment except C. illinoinensis. Plant extracts of P. americana and M. nigra (40 and 44 ml respectively) reduced gas production significantly relative to control whereas C. limon (140 ml) and nine others increased total gas production, which did not differ from the control (104 ml). Partitioning factor which, is a measure of rumen fermentation efficiency in vitro differed significantly (p< 0.05) P. americana (14.32 mg ml-1) had the highest partitioning factor, which varied significantly from all other treatments and control except M. nigra, C. japonica, and A. nilotica pods. All the plant extracts improved the MY which is the major source of amino acids to ruminants and has significant importance to animal performance. Some of the plant extracts studied were promising and will improve digestibility and utilization of poor forages which may eventually reduce methane production from animals fed poor forages. However, promising plant extracts need to be logically selected for further study in vivo to verify in vitro observation. For the in vivo study, two trials were conducted; in the first trial, 36 ewes were randomly placed in 6 treatments consisting 4 ethanolic plant extracts of C. japonica, C. papaya, P. americana, C. illinoinensis, monensin and control. They were used to determine the effect of the treatments on feed intake, growth performance, ruminal pH, NH3-N and methane and hydrogen gas concentration in the rumen. In the second trial (digestibility trial), 12 sheep were stratified (2 groups) by live weight, randomly placed in the same 6 treatment for 3 periods in a planned crossover design, to have 2 animals per treatment every period. All animals were offered Themeda triandra hay ad-libitum, supplemented with 500 g of Lucerne hay and maize mix, and animals on each treatment were offered 100g of treatment marsh containing 2g of the plant extracts having active ingredient and 5mg of monensin for animals on MON group while the control had no active ingredient. The result showed that the plant extracts influenced total dry matter and organic matter intake. All plant extracts tended to have higher total dry matter intake (% body weight), total weight gain, average daily gain, and feed efficiency relative to the control group, although these were not significant (p> 0.05). Ruminal pH, NH3-N, and apparent digestibility of organic matter, crude protein and fibre did not differ (p> 0.05). C. llinoinensis and monensin reduced methane gas concentration in the rumen headspace by 27% and 29%, respectively, relative to the control. However, monensin and all plant extracts except C. japonica reduced dissolved methane gas concentration in the rumen fluid significantly (p