Bioactive compounds, antibacterial and antioxidant activities of methanol extract of Tamarindus indica Linn.

Tamarindus indica is one of the tropical medicinal plants that has been attributed curative potential of numerous diseases by many rural dwellers. This study was designed to evaluate the antioxidant, antibacterial activities and also to determine the various chemical constituents responsible for its pharmacological activities. The methanol extract of Tamarindus indica fruit pulp was analyzed by Gas Chromatography/Mass Spectrometer to determine the volatile compounds present. The antioxidant activities were performed using DPPH and FRAP method and the antibacterial activity was tested against some common pathogens by macro broth dilution method. The GCMS analysis shows the presence of 37 compounds, out of which 14 had their peak area percentages ≥ 1% and only two compounds had no reported pharmacological activities. Most of the bioactive compounds including 5-Hydroxymethylfurfural (31.06%)-3-O-Methyl-d-glucose (16.31%), 1,6-anhydro-ß-D-Glucopyranose (9.95%), 5-methyl-Furancarboxaldehyde (3.2%), Triethylenediamine (1.17%), 1-(2-furanyl)-1-Propcanone (2.18%), Methyl 2-furoate (3.14%), Levoglucosenone (3.21%), methyl ester-Hepta-2,4-dienoic acid, (8.85%), 2,3-dihydro-3,5-dihydrox-4H-Pyran-4-one (3.4%), O-α-D-glucopyranosyl-(1.fwdarw.3)-ß-D-fructofuranosyl-α-D-Glucopyranoside (2.18%), n-Hexadecanoic acid (1.38%), 2-Heptanol, acetate (1.29%), 5-[(5-methyl-2-fur-2-Furancarboxaldehyde (1.08%), 3-Methyl-2-furoic acid (1.05%) and cis-Vaccenic acid (2.85%)have been reported with different activities such as antibacterial, antifungal, antitubercular, anticancer, antioxidant and other prophylactic activities. The extract demonstrated inhibitory potential against all tested pathogen. However, Plesiomonas shigellosis ATCC 15903 and Bacillus pumillus ATCC 14884 are more sensitive with the MIC of 0.22 and 0.44 mg/ml respectively. The antioxidant activity was relatively low due to the low phenolic content of the extract. This shows that there is a strong correlation between antioxidant activities and phenolic content. GC-MS analysis revealed the presence of bioactive phytoconstituents with various biological activities and this justifies the rationale behind its usage as a curative therapy by many local dwellers.

Computational Insights and In Vitro Validation of Antibacterial Potential of Shikimate Pathway-Derived Phenolic Acids as NorA Efflux Pump Inhibitors.

The expression of the efflux pump systems is the most important mechanism of antibiotic resistance in bacteria, as it contributes to reduced concentration and the subsequent inactivity of administered antibiotics. NorA is one of the most studied antibacterial targets used as a model for efflux-mediated resistance. The present study evaluated shikimate pathway-derived phenolic acids against NorA (PDB ID: 1PW4) as a druggable target in antibacterial therapy using in silico modelling and in vitro methods. Of the 22 compounds evaluated, sinapic acid (-9.0 kcal/mol) and p-coumaric acid (-6.3 kcal/mol) had the best and most prominent affinity for NorA relative to ciprofloxacin, a reference standard (-4.9 kcal/mol). A further probe into the structural stability and flexibility of the resulting NorA-phenolic acids complexes through molecular dynamic simulations over a 100 ns period revealed p-coumaric acid as the best inhibitor of NorA relative to the reference standard. In addition, both phenolic acids formed H-bonds with TYR 76, a crucial residue implicated in NorA efflux pump inhibition. Furthermore, the phenolic acids demonstrated favourable drug likeliness and conformed to Lipinski's rule of five for ADME properties. For the in vitro evaluation, the phenolic acids had MIC values in the range 31.2 to 62.5 µg/mL against S. aureus, and E. coli, and there was an overall reduction in MIC following their combination with ciprofloxacin. Taken together, the findings from both the in silico and in vitro evaluations in this study have demonstrated high affinity of p-coumaric acid towards NorA and could be suggestive of its exploration as a novel NorA efflux pump inhibitor.

Chemical composition, antioxidant activities and antibacterial activities of essential oil from Erythrina caffra Thunb. growing in South Africa.

INTRODUCTION: Essential oils from plants are recognized as one of the most promising secondary metabolites for the development of cheap and safer drugs. While Erythrina caffra has been prominently used in folk medicine for the treatment of microbial infections, there is dearth of information on the pharmacological effectiveness and chemical composition of its essential oil. The study, therefore, aimed at identifying the chemical composition and biological activities of the essential oil of Erythrina caffra. METHODS: In this study, the essential oil was extracted with all-glass Clevenger. The antioxidant activities of the essential oil and antibacterial susceptibility assay by agar well diffusion techniques were assessed while GC-MS analysis was performed to identify the chemical constituents of the essential oil. RESULTS: The study showed that the radical scavenging activity of the essential oil increases as the concentration of the essential oil increases. All bacterial isolates were susceptible to essential oil with the exception of Salmonella typhimurium and Pseudomonas aeruginosa producing inhibition zones ranging between 22 ± 1.3 and 35 ± 2.1 mm in the susceptible isolates. The GC-MS chromatogram indicated there are 35 bioactive compounds in the essential oil and Caryophyllene oxide (53.54%), [1S-(1α,7α,8aß)]-1,2,3,5,6,7,8,8a-octa-1 - hydro-1,8a-dimethyl-7-(1-methylethenyl)-Naphthalene (7.81%), Kauran-18-al (6.49%), 10,10-Dimethyl-2,6-dimethylenebicy clo[7.2.0]undecan-5.beta.-ol (5.83%), 10s,11s-Himachala-3(12),4-diene (4.51%), Caryophyllene (3.65%) and 1- Hexanol (3.31%) were the most prominent compounds. CONCLUSION: Excessive production of free radicals or reactive oxygen species (ROS) causes oxidative stress and disease. Oxidative stress resulting from imbalance between excessive generation of free radicals and inadequate antioxidant defense system has been linked to pathogenesis of many diseases. The essential oil of E. caffra stem bark extract possess antimicrobial and good antioxidant activities and its rich level of phytochemicals can be used as either dietary or complementary agents.

Molecular characterization, gas chromatography mass spectrometry analysis, phytochemical screening and insecticidal activities of ethanol extract of Lentinus squarrosulus against Aedes aegypti (Linnaeus).

Mosquito-transmitted diseases like zika, dengue, chikungunya, and yellow fever are known to affect human health worldwide. Numerous synthetic insecticides have been used as vector control for these diseases, but there is the challenge of environmental toxicity and vector resistance. This study investigated the medicinal and insecticidal potential of Lentinus squarrosulus against Aedes aegypti. The fruiting bodies were identified morphologically as well as using internal transcribed spacer (ITS) sequences for its molecular characterization. Genomic deoxyribonucleic acid (DNA) yield was confirmed with NanoDrop Spectrophotometer ND-1000 and amplified with ITSl and ITS4 primers. The amplicons were sequenced and the National Center for Biotechnology Information (NCBI) database identified the nucleotides. Its ethanol extract was subjected to phytochemical screening and gas chromatography mass spectrometry (GC-MS) analysis and tested against the pupa and fourth instar larva of Aedes aegypti with percentage mortality monitored. The Macrofungus was identified morphologically and confirmed with molecular characterization as Lentinus squarrosulus (LS). The gene sequence was deposited in GenBank (Accession number MK629662.1). GC-MS analysis showed that its ethanol extract has 25 bioactive compounds with 9,12-Octadecadienoic acid, ethyl ester having the highest percentage of 43.32% as well as methyl-2-oxo-1-pyrrolidine acetate and 17-octadecynoic acid having the lowest percentage (0.09%). The macrofungus contained varied concentrations of phytochemicals including phenols (159 mg/g GAE), tannins (1.6 mg/g TAE), and flavonoids (31.4 mg/g QE). The ethanol extract had significant potent effects on Aedes aegypti larva and pupa which could be due to the occurrence and abundance of 9,12-octadecadienoic acid in LS. The LC50 of the extract for larvicidal and pupicidal activities were 2.95 mg/mL and 3.55 mg/mL, respectively, while its LC90 were 6.31 mg/mL and 5.75 mg/mL respectively. Lentinus squarrosulus had insecticidal effects against the Aedes aegypti larva and pupa and possessed great potential as a source of alternative medicine and eco-friendly insecticides.

Antibacterial Activity of Defatted and Nondefatted Methanolic Extracts of Aframomum melegueta K. Schum. against Multidrug-Resistant Bacteria of Clinical Importance.

The antibacterial activity of the extracts of Aframomum melegueta including n-hexane extract (NHE), nondefatted methanol extract (NDME), and defatted methanol extract (DME) was investigated in this study. The NHE exhibited no antibacterial activity. The DME showed higher antibacterial activity than the NDME against the different isolates. At the highest concentration of 10 mg/mL in agar diffusion, NDME produced inhibition zones ranging from 11 to 29 mm against the microorganisms while DME produced inhibition zones ranging from 20 to 40 mm with the concentration of 10 mg/mL against the microorganisms. 0.1 mg/mL of the DME produced inhibition zones ranging between 12 and 14 mm in Aeromonas hydrophila ATCC 35654 and Pseudomonas aeruginosa ATCC 15442, respectively, while none of the isolates were inhibited by the NDME at a concentration of 1 mg/mL or less. In the agar dilution assay, the MICs of the NDME and DME ranged between 0.31 and 10 mg/mL, but more isolates were inhibited at 0.31 mg/mL of DME than those in NDME. In macrobroth assay, the MICs of the NDME ranged between 0.15 and 5.0 mg/mL and the MBCs ranged between 0.63 and 5.0 mg/mL, and the MICs of the DME ranged between 0.08 and 5.0 mg/mL and the MBCs were between 0.31 and 5.0 mg/mL. This study indicated that DME was more active with higher antibacterial activity than the NDME of this plant, and extracting the fatty portion of plant materials prior susceptibility testing would allow plant extracts to be more effective as well as justifying the use of Aframomum melegueta in traditional medicine for the treatment of bacterial infections.

Interaction of Ziziphus mucronata subsp. mucronata Methanol Extract and First-Line Antibiotics is Synergistic In Vitro through Production of Reactive Oxygen Species.

With the increased incidence of antibacterial resistance in microorganisms, combining natural products from plants with antibiotics may be considered interesting alternatives for synergy to attain multitarget effects. In this study, the antioxidant activity of the methanol extract of Ziziphus mucronata and its interactions with antibiotics against bacteria of clinical importance were investigated. While its phytochemicals and antioxidant activities were determined by free radical scavenging assays, the antibacterial activities of the extract and its interactions with the antibiotics were determined by macrobroth dilution and the checkerboard methods. From the results, total phenolic content was 29.67 ± 1.90 mg GAE/100 g, total flavonoid content was 8.72 ± 0.08 mg QE/100 g, and total proanthocyanidin content was 1.94 ± 0.00 mg CE/100 g of dry plant material. The inhibition concentration 50% (IC50) of DPPH, BHT, and ascorbic acid was equal to 0.04 ± 0.02 mg/ml, respectively. Those of the ABTS, BHT, and ascorbic acid were equal to 0.02 ± 0.02, 0.04 ± 0.03, and 0.04 ± 0.02 mg/ml, respectively. The checkerboard assay showed that combining the extract with different antibiotics resulted in synergistic (38.75%), indifferent (30%), additive (28.75%), and antagonistic (2.5%) interactions. The interactions between the extract and antibiotics resulting in enhanced antibacterial activities could have resulted from the antioxidant activities of the extract mopping up the ROS generated by the antibiotics or the ability of both extract and antibiotics simultaneously producing reactive oxygen species with deleterious effects resulting in synergistic antibacterial effects.

Vancomycin intermediate resistant Staphylococcus aureus in the nasal cavity of asymptomatic individuals: a potential public health challenge.

BACKGROUND: The potential of transmitting multidrug resistant Staphylococcus aureus from asymptomatic individuals to healthy individuals could constitute a great challenge to antimicrobial therapy. METHODS: The antibiograms of the S. aureus from asymptomatic individuals were determined by disk diffusion and agar dilution assay techniques with different antibiotics and vancomycin. RESULTS: Of the 152 S. aureus isolated, (59)38.8% isolates were multi-drug resistant strains. Streptomycin was the most effective and inhibited (135)88.82% of the isolates while ceftazidime inhibited (24)15.8% of the isolates. While (82)54.0% of the isolates inhibited by cefuroxime had resistant colonies within their inhibition zones (Rc) and ofloxacin inhibited (100)65.8% of the isolates without having resistant colonies within the inhibition zones, ceftazidime inhibited (7)4.6% of the isolates with resistant colonies within the inhibition zones. Subjecting the isolates to vancomycin showed that (27)17.8% were resistant to 2 µg/ml, (43)28.3% were resistant to 4 µg/ml and (27)17.8% of the isolates were simultaneously resistant to both concentrations of vancomycin. Although (100)65.8% of the isolates had MARindex ≥0.2, (52)34.2% of the isolates had MARindex ≤ 0.2 and (65)428% of the isolates were considered multidrug resistant strains. CONCLUSION: The isolation of multi-drug and vancomycin intermediate resistant strains of S. aureus in high percentage, in this study, presents a great threat to clinicians and general populace. The vancomycin intermediate resistant S. aureus (VISA) in asymptomatic individuals could be a critical concern to the therapeutic dilemma to be added to the presence of multi-drug resistance. A more sustainable therapy must be in place to prevent its dissemination or the outbreak of its infection.

Pharmacological Evaluation of Selected Medicinal Plants Used in the Management of Oral and Skin Infections in Ebem-Ohafia District, Abia State, Nigeria.

Oral and skin infections contribute significantly to the global health challenges responsible for the current trend of increased morbidity and premature death. The purpose of this study was to document medicinal plants used in the management of oral and skin infections in Ebem-Ohafia Local Government Area (LGA), Abia State, and to characterize the in vitro antioxidant and antibacterial activity. The thin layer chromatography (TLC) profiling of ten of the selected folklore medicine was carried out using a various solvent system of different polarity index. The antioxidant capacity of the plant extracts was evaluated using chemical-based methods, and its antibacterial effect was investigated using disc diffusion and microdilution methods. Sixty-one plant species belonging to 26 families were discovered, and the most frequently cited species are Euphorbiaceae (18.03%), Fabaceae (11.47%), and Asteraceae (11.47%). All the plant extracts showed a promising free radical scavenging activity and efficient ferric reducing antioxidant power in a concentration-dependent manner possibly due to their richness in polyphenol with TLC profiling showing maximum three bands of phytochemicals. Also, the plant extracts exhibited a mild to weak antibacterial activity against our panel of bacterial strains having MIC values ranging from 256 to > 512 µg/ mL reflected in their zone of inhibition at 10 µg/disc. The data obtained for Breynia nivosa (BN), Eleusine indica (EI), Cassia alata (CA), Chromolaena odorata (CO), and Acalypha hispida (AH) extracts substantiate the traditional use of these herbal remedies in the region and open the possibility for the development of cheaper and affordable drugs in the treatment of oral and skin infections. Further studies are needed to identify active ingredient with strong antibacterial and antioxidant capacities along with their molecular mechanisms.

Effects and time-kill assessment of amoxicillin used in combination with chloramphenicol against bacteria of clinical importance.

With the emergence of multidrug-resistant organisms in an era when drug development faces challenges causing pharmaceutical companies to curtail or abandon research on anti-infective agents, the use of combined existing antimicrobial agents may be an alternative. This study evaluated the effects of combining amoxicillin and chloramphenicol, to which many bacteria have become resistant, in vitro against Gram positive and Gram negative bacteria by agar diffusion, checkerboard and time-kill assays. The test isolates were susceptible to amoxicillin with minimum inhibitory concentrations (MICs) ranging between 0.448 and 500 µg/ml and between 1.953 and 31.25 µg/ml for chloramphenicol. Upon combining these agents, there was a drastic reduction in their MICs indicating an increased antibacterial activity that showed synergistic interaction against all the bacteria. At the highest concentrations, the inhibition zones ranges were 20.33-38.33±0.58 µg/ml for amoxicillin, 27.67-37.67±0.58 µg/ml for chloramphenicol and 31.67-39.33±0.58 µg/ml for the combined agents. The fractional inhibitory concentration indices (FICIs) showed synergy ranging from 0.129 to 0.312 while FICIs for additive interaction were between 0.688 and 1.0. There was no antagonistic interaction. At the 1/2MICs of the combined antibiotics, all the tested bacteria, except for Klebsiella pneumoniae ATCC 4352, Proteus vulgaris CSIR 0030 and Enterococcus cloacae ATCC 13047 were eliminated before 24 h. At the MICs, all the tested bacteria were eliminated except Enterococcus cloacae ATCC 13047 which was almost totally eliminated. Post-antibiotic assessment after 48 h showed that all the cultures were sterile except for that of Enterococcus cloacae ATCC 13047. The lack of antagonism between these antibacterial agents in checkerboard and time-kill assays suggested that combining amoxicillin with chloramphenicol can provide an improved therapy in comparison to the use of each antibiotic individually. The study indicates the potential beneficial value of combining amoxicillin and chloramphenicol in the treatment of microbial infections in clinical settings.

In vitro pharmacological interaction of caffeine and first-line antibiotics is antagonistic against clinically important bacterial pathogens.

The in vitro antibacterial activity of pure caffeine powder and its interaction with first line antibiotic against bacterial isolates were investigated with the macrobroth dilution and the checkerboard assay methods. This study showed that caffeine and the antibiotics exhibited various degrees of antibacterial activities. While caffeine had MICs ranging between 67.19 and 268.75 µg/ml, chloramphenicol was characterized by MICs between 0.98 and 31.25 µg/ml, kanamycin - 15.63-62.5 µg/ml, nalidixic acid - 0.49-250 µg/ml, erythromycin - 0.49-62.5 µg/ml, tetracycline - 1.99-62.5 µg/ml and metronidazole - 15.63-31.25 µg/ml. Combining ½ MICs and MICs of caffeine with the antibiotics as well as direct combination of caffeine and the antibiotics resulted in significant reduction of antibiotics' effectiveness. The fractional inhibitory concentration index (FICI) for the combination of ½ MICs of caffeine with different antibiotics showed antagonistic interactions with the antibiotics except kanamycin which had additive and indifferent interactions with caffeine. The FICI of the MICs of caffeine combined with antibiotics showed a reduction in the number of antagonistic interactions as chloramphenicol, nalidixic acid and erythromycin showed some indifferent interactions while kanamycin was the only antibiotic that showed indifferent interaction against all the bacterial isolates. The direct combination of caffeine and the antibiotics resulted in significant antagonistic interactions higher than in the case when caffeine, at the ½ MICs and MICs, was combined with the antibiotics. Although caffeine demonstrated significant antibacterial activity against the selected bacterial isolates, its combination with the selected antibiotics resulted in significant antagonistic interactions. Caffeine should not be combined with antibiotics as this could result in serious therapeutic failure and, possibly, drug toxicity in vivo.

Alpha-Amylase Inhibition and Antioxidative Capacity of Some Antidiabetic Plants Used by the Traditional Healers in Southeastern Nigeria.

Oxidative stress plays a significant role in the pathogenesis of metabolic syndrome including diabetes mellitus (DM). The inhibition of alpha-amylase is an important therapeutic target in the regulation of postprandial increase of blood glucose in diabetic patients. The present study investigated the alpha-amylase inhibitory and antioxidant potential of selected herbal drugs used in the treatment of DM by the traditional healers in Isiala Mbano and Ikwuano regions of southeastern Nigeria. Antioxidant activity was evaluated in terms of free radical scavenging, reducing power, and total phenolic (TPC) and flavonoid content (TFC) in consonance with the TLC profiling. The results showed that methanol crude extracts from Anacardium occidentale (AO) and Ceiba pentandra (CP) recorded higher TPC and TFC, potent free radical scavenging, and efficient reducing power (RP) as compared with other plant samples. All the plant extracts exhibited a relative alpha-amylase inhibition apart from Strophanthus hispidus (SH) extract with a negative effect. We discovered a mild to weak correlation between alpha-amylase inhibition or antioxidative capacity and the total phenol or flavonoid content. At least in part, the results obtained in this work support the traditional use of certain plant species in the treatment of patients with DM.

Antimicrobial Activity, Phenolic Content, and Cytotoxicity of Medicinal Plant Extracts Used for Treating Dermatological Diseases and Wound Healing in KwaZulu-Natal, South Africa.

Medicinal plants used for wound healing and skin diseases are the key to unlocking the doors to combating problematic skin diseases as resistance of pathogens to pharmaceuticals and allopathic management continues to increase. The study aimed at investigating the antimicrobial efficacies, phenolic content, and cytotoxicity effects of 11 medicinal plant extracts commonly used for treating skin conditions and wound healing in traditional medicine within KwaZulu-Natal. Eleven plant species were separated into different plant parts (bulbs, roots, leaves) and extracted with different solvents. The extracts were assessed for antimicrobial activity against six Gram-positive and seven Gram-negative bacterial strains and four fungi commonly associated with skin conditions using disc diffusion and microdilution techniques. The aqueous methanolic extracts were screened for phenolic content while cytotoxicity tests were performed on all extracts using the brine shrimp lethality and tetrazolium-based colorimetric (MTT) assays. Extracts from Aloe ferox, A. arborescens, and Hypericum aethiopicum were the most active against almost all of the tested bacterial and fungal strains. All plant species exhibited some degree of antimicrobial activity. Total phenolic levels, flavonoids and tannins were also higher for A. ferox, followed by A. arborescens and H. aethiopicum, respectively. The cytotoxicity results of all plant extracts were in the range of 90-100% survival after 24 h in the brine shrimp assay. Extracts considered lethal would demonstrate >50% shrimp death. The MTT cytotoxicity test yielded LC50 values of >1 mg/mL on all extracts indicating that they are not cytotoxic. The observed antimicrobial efficacy demonstrated by some plant species and the general lack of cytotoxic effects on all the tested extracts presents some promising and beneficial aspects of these medicinal plant extracts in the treatment of skin diseases and wound healing. The two Aloe species and H. aethiopicum were among the best extracts that exhibited consistently good antimicrobial activity and warrants further investigations and possible isolation of bioactive principles.

Influence of First-Line Antibiotics on the Antibacterial Activities of Acetone Stem Bark Extract of Acacia mearnsii De Wild. against Drug-Resistant Bacterial Isolates.

Background. This study was aimed at evaluating the antibacterial activity of the acetone extract of A. mearnsii and its interactions with antibiotics against some resistant bacterial strains. Methods. The antibacterial susceptibility testing was determined by agar diffusion and macrobroth dilution methods while the checkerboard method was used for the determination of synergy between the antibiotics and the extract. Results. The results showed that the susceptibility of the different bacterial isolates was concentration dependent for the extract and the different antibiotics. With the exception of S. marcescens, the inhibition zones of the extract produced by 20 mg/mL ranged between 18 and 32 mm. While metronidazole did not inhibit any of the bacterial isolates, all the antibiotics and their combinations, except for ciprofloxacin and its combination, did not inhibit Enterococcus faecalis. The antibacterial combinations were more of being antagonistic than of being synergistic in the agar diffusion assay. From the macrobroth dilution, the extract and the antibiotics exerted a varied degree of inhibitory effect on the test organisms. The MIC values of the acetone extract which are in mg/mL are lower than those of the different antibiotics which are in µg/mL. From the checkerboard assay, the antibacterial combinations showed varied degrees of interactions including synergism, additive, indifference, and antagonism interactions. While antagonistic and additive interactions were 14.44%, indifference interaction was 22.22% and synergistic interaction was 37.78% of the antibacterial combinations against the test isolates. While the additivity/indifference interactions indicated no interactions, the antagonistic interaction may be considered as a negative interaction that could result in toxicity and suboptimal bioactivity. Conclusion. The synergistic effects of the herbal-drug combinations may be harnessed for the discovery and development of more rational evidence-based drug combinations with optimized efficiency in the prevention of multidrug resistance and therapy of multifactorial diseases.

A comparative study of three Aloe species used to treat skin diseases in South African rural communities.

OBJECTIVES: Aloe species have been noted to be a miracle cure used by indigenous people of southern Africa. Geographically, each of the three Aloe species-Aloe arborescens, Aloe excelsa, and Aloe ferox-has a specific habitat. Although some species overlap in geographical regions, the species most abundant in a region is most often utilized by indigenous people. All three species display similar curative properties, aiding in wound healing, cures against other skin ailments, and some systemic conditions. RESULTS: All three Aloe species indicated high inhibitory activity against all gram-positive bacteria under investigation. The ethanol extract was most effective and inhibited all gram-positive bacteria and two gram-negative bacteria (i.e., Proteus vulgaris and Escherichia coli). CONCLUSIONS: All fungal species under investigation were successfully inhibited by both the boiled water as well as the ethanol extract, substantiating the traditional usage of this species.