Antimicrobial activity of Sida acuta, Phyllanthus amarus and Phyllanthus muellerianus against microorganisms implicated in urinary tract infections

Increasing level of antimicrobial resistance among bacterial pathogens causing Urinary Tract Infection (UTI) is one of the most significant public health challenges globally. Hence, the search for alternatives from medicinal plants. This study investigated the efficacy of Phyllanthus amarus (PA), Phyllanthus muellerianus (PM) and Sida acuta (SA) leaf extracts on microorganisms implicated in UTI. Mid-stream urine samples collected from 100 patients clinically diagnosed with UTI were cultured. The microorganisms isolated were identified using their morphological and biochemical characteristics. Methanol leaf extracts of the three plants were obtained by cold maceration in 60% methanol. Crude extract of PM was thereafter purified by solvent partitioning. Antibiotic susceptibility test was determined using the Kirby Bauer disc diffusion. Antimicrobial effects of the extracts and oil was ascertained using agar well diffusion. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentrations (MBC) were also determined. Rate of kill and mechanism of action of the purified extract of PM on isolates were investigated. Cytotoxicity of plant extracts were assayed on brine shrimps while synergism of the purified extract with ciprofloxacin was ascertained using overlay inoculum susceptibility disc method. Antioxidant and phytochemical analyses of the extracts were conducted using standard methods. Phytochemical analysis of the leaf extracts showed the presence of alkaloids, tannins, saponins and steroids. Antioxidant assay also indicated SA had the highest total flavonoids and phenol content of 339.86 mgQUE/g and 27.63 mgGAE/g. Microorganisms isolated include: Escherichia coli (24%), Proteus mirabilis (24%), Staphylococcus aureus (19%), Klebsiella pneumoniae (13%), Candida albicans (11%), Enterobacter sp. (5%) and Citrobacter sp. (4%). The crude extract of PA had zone of inhibition ranging from 16.7 ± 1.53 mm to 24 ± 1.00 mm while SA crude extract had 14.7 ± 1.53 mm to 27 ± 2.00 mm. PM crude extract had inhibition zones of 17 ± 1.00 mm to 22.3 ± 2.12 mm. The MIC and MBC ranged from 6.25 mg/ml to 50 mg/ml and 12.5 mg/ml to 50 mg/ml respectively. Ethyl acetate fraction of PM showed the highest percentage yield and had a zone diameter range from 13.5 ± 1.00 mm to 28 ± 1.53 mm with MIC and MBC ranges of 6.25 mg/ml – 12.5 mg/ml and 25 mg/ml to 50 mg/ml respectively. Synergism with ciprofloxacin was observed at 25% of the microorganisms, 50% antagonism and 25% additively. Toxicity analysis showed lethal dose concentrations of 19.05 mg/ml, 25.12 mg/ml and 130.11 mg/ml for PM, PA and SA respectively. The findings of this study suggest that the methanol extracts of the medicinal plants used in this study does possess a potent lead molecule in combating microorganisms causing UTI. Key words: Antimicrobial activity, Phyllanthus muellerianus, Phytochemicals, Toxicity, UTI,

Phytoremediation of Crude Oil Polluted Microbial Augmented Soil Using Cyperus esculentus and Phyllanthus amarus

Aim: To assess the phytoremediation potential of Cyperus esculentus and Phyllanthus amarus in crude oil polluted soil and ascertain the enhancement of augmented microbes (fungi). Study Design: The study employs experimental design, statistical analysis of the data and interpretation. Place and Duration of Study: Rivers State University demonstration farmland in Nkpolu- Oroworukwo, Mile 3 Diobu area of Port Harcourt, was used for this study. The piece of land is situated at Longitude 4°48’18.50” N and Latitude 6ᵒ58’39.12” E measuring 5.4864 m x 5.1816 m with a total area of 28.4283 square meter. Phytoremediation process monitoring lasted for 240 days, analyses were carried out weekly at 30 days’ interval. Methodology: Seven (7) experimental plots (two Control (Unpolluted and polluted soil) and five polluted amended/treated plots) employing Randomized Block Design (each having dimensions: 100 x 50 x 30 cm LxBxH) were formed and mapped out on agricultural soil and left fallow for 6 days before contamination on the seventh day; after which it was allowed for 21 days for proper contamination and exposure to natural environmental factors (to mimic soil crude oil spill site); thereafter bioaugmenting organisms were applied. Baseline studies were carried out on the top soil before and after contamination, major parameters monitored and assessed were Total Petroleum Hydrocarbon (TPH) uptake by plant roots and stem, Polycyclic Aromatic Hydrocarbon (PAHs) and TPH reduction in soil. Other physicochemical analyzed in the soil of different plots were pH, Electrical Conductivity, Moisture Content, Total Nitrogen, Available Phosphorus, Potassium, Total Organic Carbon, Plant Height, Iron, Lead at regular intervals; days 1, 60, 90, 120, 150, 180, 210 & 240. Application of augmenting organisms was to enhance phytoremediation by test plant Cyperus esculentus (Cyp) and Phyllanthus amarus (Phy). The rate of phytoremediation was estimated from percentage (%) uptake of Total petroleum hydrocarbon (TPH) in plant roots and stem from day 1 -240; while percentage (%) reduction of TPH and PAHs in soil was estimated from day 1 to the residual at day 240. Results: The test plants decreased significant amount of crude oil as revealed in TPH uptake in their roots and Stem. Mean amount and percentage Total Petroleum Hydrocarbon (TPH) uptake by Cyperus esculentus roots and stem were; 152.33±50.34mg/kg, 12.57±4.16% and 201.13±8.80mg/kg, 13.27±0.58% respectively; while that of Phyllanthus amarus roots and stem were 141.50±35.62mg/kg, 11.68±2.94% and 174.44±19.98mg/kg, 11.51±1.32% respectively. Similar trend was observed in the control plots were TPH uptake by Cyperus esculentus roots and stem were; 24.2mg/kg, 2.00% and 20.01mg/kg, 1.32% respectively while in control plot of Phyllanthus amarus TPH uptake by roots and stem were 23.19mg/kg, 1.91% and 19.80mg/kg, 1.31% respectively. Comparatively, uptake of TPH was higher in plant stem than roots. From the initial TPH contamination value of 5503.00mg/kg , Total Petroleum Hydrocarbon Reduction and % Hydrocarbon Reduction in soil at 240 days in the different treatment plots in a decreasing order were as follows: PS+AN+MR+SMS+Phy (5470.9mg/kg; 99.43%) >PS+MR+SMS+Phy (5460.60mg/kg; 99.23%) >PS+AN+MR+Phy (5451.30mg/kg; 99.06%) >PS+MR+Cyp (5448.30mg/kg; 99.01%) >PS+AN+MR+Cyp (5440.00mg/kg; 98.86%) >PS+AN+Phy (5422.905mg/kg; 98.54%) >PS+Cyp (no amendment) (5380.90mg/kg; 97.78%). Comparative evaluation revealed higher reduction of PAHs in soil (plot) planted with Phyllanthus amarus. Highest PAHs reduction in soil was seen in PS+AN+MR+SMS+Phy (31.3mg/kg; 65.89%) while least was recorded in PS+ Cyp (no amendment) (23.4mg/kg, 49.26%). Conclusion: it was observed that plots planted with Cyperus esculentus (TPH 5492.75±76.36mg/kg) showed higher reduction of TPH from soil than those planted with Phyllanthus amarus (TPH 5449.72±18.27mg/kg); while PAHs degradation/reduction in plots planted with Phyllantus amarus (PAHs 28.72±2.74mg/kg; 60.46±5.77%) was higher than plots planted with Cyperus esculentus (PAHs 25.77±2.12mg/kg, 54.24±4.47%). More so, plots amended with augmentating microbes showed significant higher percentage reduction in hydrocarbon in the polluted soil than unamended polluted soil. It is therefore recommended that Cyperus esculentus is a suitable plant species for phytoremediation of crude oil contaminated soil with high TPH value while Phyllanthus amarus is the best option for phytoremediation of polluted soil with high PAHs value, in combination with augmenting microbes.

Physicochemical Indices of Phyto Remediated Hydrocarbon Polluted Soil and the Effect of Bio-nutrient Amendment

Aim: To assess the Physicochemical indices of Phytoremediated Crude Oil polluted amended soil using grass plant Cyperus esculentus (Cyp) and Phyllanthus amarus (Phy). Study Design: The study employs experimental design, statistical analysis of the data and interpretation. Place and Duration of Study: Rivers State University demonstration farmland in Nkpolu- Oroworukwo, Mile 3 Diobu area of Port Harcourt, was used for this study. The piece of land is situated at Longitude 4°48’18.50” N and Latitude 6ᵒ58’39.12” E measuring 5.4864 m x 5.1816 m with a total area of 28.4283 square meter. Phytoremediation process monitoring lasted for 240 days; analyses were carried out monthly at 30 days’ interval. Methodology: The study was carried out on Crude Oil Polluted soil (PS) amended with bio-nutrient supplements (Spent Mushroom Substrate (SMS) and selected fungi (Aspergillus niger(AN) andMucor racemosus (MR)) used to stimulate and augment the indigenous microbial population present in a crude oil polluted soil thereby enhancing hydrocarbon reduction in pari per sue with phytoremediation (uptake of Crude oil by test plants) over a period of 240 days. Ten (10) experimental plots (two Control (Unpolluted and polluted soil without amendment) and eight polluted amended/treated plots) employing Randomized Block Design (each having dimensions: 100 x 50 x 30 cm LxBxH); formed and mapped out on agricultural soil and left fallow for 6 days before contamination on the seventh day; after which it was allowed for 21 days for proper contamination and exposure to natural environmental factors (to mimic soil crude oil spill site); thereafter nutrients/organics (biostimulating agents) and bioaugmenting organisms were applied. Baseline studies were carried out on soil profile before and after contamination, major parameters monitored and assayed were Total Petroleum Hydrocarbon (TPH) uptake by plant roots and stem, Polycyclic Aromatic Hydrocarbons (PAHs) and TPH reduction in soil. Other physicochemical properties analyzed in the soil from different plots were pH, Electrical Conductivity, Moisture Content, Total Nitrogen, Available Phosphorus, Potassium, Total Organic Carbon, Plant Height, Iron, Lead and Zinc at regular intervals; days 1, 60, 90, 120, 150, 180, 210 & 240. The rate of phytoremediation was estimated from percentage (%) uptake of Total petroleum hydrocarbon (TPH) in plant roots and stem from day 1 -240; while percentage (%) reduction of TPH and PAHs in soil was estimated from day 1 to the residual at day 240. Results: The test plants decreased significant amount of crude oil as revealed in TPH uptake in their roots and Stem. Mean amount and percentage Total Petroleum Hydrocarbon (TPH) uptake by Cyperus esculentus roots and stem were; 152.33±50.34mg/kg, 12.57±4.16% and 201.13±8.80mg/kg, 13.27±0.58% respectively; while that of Phyllanthus amarusroots and stem were 141.50±35.62mg/kg, 11.68±2.94% and 174.44±19.98mg/kg, 11.51±1.32% respectively; revealing higher Uptake of TPH in plant stem than roots. From the initial TPH contamination value of 5503.00mg/kg, it was observed that plots planted with Cyperus esculentus (TPH 5492.75±76.36mg/kg) showed higher reduction of TPH from soil than those planted with Phyllanthus amarus(TPH 5449.72±18.27mg/kg); while PAHs degradation/reduction showed a reverse trend with plots planted with Phyllanthus amarus (PAHs 28.72±2.74mg/kg; 60.46±5.77%) higher than plots planted with Cyperus esculentus s (PAHs 25.77±2.12mg/kg, 54.24±4.47%). Conclusion: Plots planted with Cyperus esculentus showed higher reduction of TPH from soil than those planted with Phyllanthus amarus while PAHs degradation/reduction in plots planted with Phyllanthus amarus was higher than plots planted with Cyperus esculentus. TPH uptake was higher in plant stems than roots; more so, plots amended with nutrient supplements showed significant higher percentage reduction in hydrocarbon in the polluted soil than unamended polluted soil. It is therefore recommended that Cyperus esculentus is a suitable plant species for phytoremediation of crude oil contaminated soil with high TPH value while Phyllanthus amarusis the best option in phytoremediation of polluted soil with high PAHs value, both in combination with bio-nutrient supplement.

Bar-cas12a, a novel and rapid method for plant species authentication in case of Phyllanthus amarus Schumach. & Thonn

Rapid and accurate species diagnosis accelerates performance in numerous biological fields and associated areas. However, morphology-based species taxonomy/identification might hinder study and lead to ambiguous results. DNA barcodes (Bar) has been employed extensively for plant species identification. Recently, CRISPR-cas system can be applied for diagnostic tool to detect pathogen’s DNA based on the collateral activity of cas12a or cas13. Here, we developed barcode-coupled with cas12a assay, “Bar-cas12a” for species authentication using Phyllanthus amarus as a model. The gRNAs were designed from trnL region, namely gRNA-A and gRNA-B. As a result, gRNA-A was highly specific to P. amarus amplified by RPA in contrast to gRNA-B even in contaminated condition. Apart from the large variation of gRNA-A binding in DNA target, cas12a- specific PAM’s gRNA-A as TTTN can be found only in P. amarus. PAM site may be recognized one of the potential regions for increasing specificity to authenticate species. In addition, the sensitivity of Bar-cas12a using both gRNAs gave the same detection limit at 0.8 fg and it was 1,000 times more sensitive compared to agarose gel electrophoresis. This approach displayed the accuracy degree of 90% for species authentication. Overall, Bar-cas12a using trnL-designed gRNA offer a highly specific, sensitive, speed, and simple approach for plant species authentication. Therefore, the current method serves as a promising tool for species determination which is likely to be implemented for onsite testing.

Pharmacognostical Study, Phytochemical and Physicochemical Evaluation and Establishment of Quality Standards for Certain Traditional Antidiabetic Medicinal Plants

Aim: The present work was aimed at evolving pharmacognostical and phytochemical quality standards for certain traditional herbs like Phyllanthus amarus, Glycerrhiza glabra and Piper nigrum. These three plants are reported to possess antidiabetic activity. Study Design: The plants were collected, authenticated and Macro-morphological, qualitative and quantitative microscopic features as well as physicochemical, fluorescence analysis, phytochemical properties, and thin layer chromatography (TLC) Place and Duration of Study: The study was carried out at College of Pharmacy, Dr. APJ Abdul Kalam University, Indore, during 2019-20 Methodology: The material were collected, authenticated and Macro-morphological, qualitative and quantitative microscopic features as well as physicochemical, fluorescence analysis, phytochemical properties, and thin layer chromatography (TLC) profile of Phyllanthus amarus, Glycerrhiza glabra and Piper nigrum were determined using standard methods. Results: The macroscopical and microscopical studies revealed useful diagnostic features. Phytochemical screening reveals the presence of secondary metabolites, physicochemical including fluorescence analysis of powdered drug proved useful to differentiate the powdered drug material. Thin layer chromatography analysis showed the presence of important phytoconstituents such Phyllanthin, hypophyllanthin, glycerrizin and piperine Conclusion: The data generated from this study would serve as useful gauge for determining the quality of Phyllanthus amarus, Glycerrhiza glabra and Piper nigrum thereby correct identification and authentification of these plants. It would also help scientists to utilize such needful information regarding the plants identity and characteristics in building new polyherbal formulations.