Phytochemical analysis and anthelmintic activity of Combretum mucronatum leaf extract against infective larvae of soil-transmitted helminths including ruminant gastrointestinal nematodes.

BACKGROUND: Soil-transmitted helminths (STH) infect more than a quarter of the world's human population. In the absence of vaccines for most animal and human gastrointestinal nematodes (GIN), treatment of infections primarily relies on anthelmintic drugs, while resistance is a growing threat. Therefore, there is a need to find alternatives to current anthelmintic drugs, especially those with novel modes of action. The present work aimed to study the composition and anthelmintic activity of Combretum mucronatum leaf extract (CMLE) by phytochemical analysis and larval migration inhibition assays, respectively. METHODS: Combretum mucronatum leaves were defatted with petroleum ether and the residue was extracted by ethanol/water (1/1) followed by freeze-drying. The proanthocyanidins and flavonoids were characterized by thin layer chromatography (TLC) and ultra-high performance liquid chromatography (UPLC). To evaluate the inhibitory activity of this extract, larval migration assays with STH and GIN were performed. For this purpose, infective larvae of the helminths were, if necessary, exsheathed (Ancylostoma caninum, GIN) and incubated with different concentrations of CMLE. RESULTS: CMLE was found to be rich in flavonoids and proanthocyanidins; catechin and epicatechin were therefore quantified for standardization of the extract. Data indicate that CMLE had a significant effect on larval migration. The effect was dose-dependent and higher concentrations (1000 µg/mL) exerted significantly higher larvicidal effect (P < 0.001) compared with the negative control (1% dimethyl sulfoxide, DMSO) and lower concentrations (≤ 100 µg/ml). Infective larvae of Ascaris suum [half-maximal inhibitory concentration (IC50) = 5.5 µg/mL], Trichuris suis (IC50 = 7.4 µg/mL), and A. caninum (IC50 = 18.9 µg/mL) were more sensitive to CMLE than that of Toxocara canis (IC50 = 310.0 µg/mL), while infective larvae of Toxocara cati were largely unaffected (IC50 > 1000 µg/mL). Likewise, CMLE was active against most infective larvae of soil-transmitted ruminant GIN, except for Cooperia punctata. Trichostrongylus colubriformis was most sensitive to CMLE (IC50 = 2.1 µg/mL) followed by Cooperia oncophora (IC50 = 27.6 µg/mL), Ostertagia ostertagi (IC50 = 48.5 µg/mL), Trichostrongylus axei (IC50 = 54.7 µg/mL), Haemonchus contortus (IC50 = 145.6 µg/mL), and Cooperia curticei (IC50 = 156.6 µg/mL). CONCLUSIONS: These results indicate that CMLE exhibits promising anthelmintic properties against infective larvae of a large variety of soil-transmitted nematodes.

Towards the development of analytical monograph specifications for the quality assessment of the medicinal plant Phyllanthus urinaria.

Many people in developing countries rely on herbal remedies for their primary healthcare needs. The challenge however is that several of these products lack proper documentation of quality and safety. To ensure consistent quality, validated methods are needed to establish and control quality attributes associated with identity, purity, and levels of bioactive constituents of the respective herbal materials. The present study focused on Phyllanthus urinaria (PU), a widely used medicinal plant in Ghana and West Africa that lacks the necessary quality control standards. The study aimed to develop an HPTLC identification method, which together with UPLC-ESI-Q-TOF-MS/MS analysis established the identity of PU samples and differentiated PU from other closely related Phyllanthus species. Quantitative UPLC and HPTLC methods were developed to assess the contents of selected active markers in the PU samples, which invariably led to the proposal of acceptance criteria for the active markers. Prior to the content analyses, the sample extraction procedure was optimized through the use of Design of Experiment method. The effects of harvest time and geographic origin on the content of active compounds were demonstrated in the investigations. PU samples were also found to be contaminated with higher levels of pesticides like chlorpyrifos and folpet. Essentially, this study provides analytical protocols, insights into the quality status of PU samples in Ghana, and analytical specifications contained in a drafted monograph for future consideration in regional and subregional African pharmacopoeias.

Anthelmintic Activities of Extract and Ellagitannins from Phyllanthus urinaria against Caenorhabditis elegans and Zoonotic or Animal Parasitic Nematodes.

The aerial parts of Phyllanthus urinaria are used in traditional medicine in West Africa against helminthiasis, but their anthelmintic potential has not been evaluated until now. Within the current study, a hydroacetonic extract (AWE) and fractions and isolated ellagitannins from P. urinaria were, therefore, tested in vitro against Caenorhabditis elegans and the larvae of the animal parasites Toxocara canis, Ascaris suum, Ancylostoma caninum, and Trichuris suis. Compounds 1:  - 13: , mainly representing ellagitannins, were isolated using different chromatographic methods, and their structures were elucidated by HR-MS and 1H/13C-NMR. AWE exerted concentration-dependent lethal effects (LC50 of 2.6 mg/mL) against C. elegans and inhibited larval migration of all animal parasites tested (T. suis L1 IC50 24.3 µg/mL, A. suum L3 IC50 35.7 µg/mL, A. caninum L3 IC50 112.8 µg/mL, T. canis L3 IC50 1513.2 µg/mL). The anthelmintic activity of AWE was mainly related to the polar, tannin-containing fractions. Geraniin 1: , the major ellagitannin in the extract, showed the strongest anthelmintic activity in general (IC50 between 0.6 and 804 µM, depending on parasite species) and was the only compound active against A. caninum (IC50 of 35.0 µM). Furosin 9: was least active despite structural similarities to 1: . Among the parasites tested, Trichuris suis L1 larvae turned out to be most sensitive with IC50 of 0.6, 6.4, 4.0, 4.8, and 2.6 µM for geraniin 1: , repandusinic acid A 3: , punicafolin 8: , furosin 9: , and phyllanthusiin A 10: , respectively.

Development of an Analytical Workflow to Support the Establishment of Monographs in African Pharmacopoeias - Combretum mucronatum Leaves as Example.

Herbal medicines are invaluable in African medicine, but quality and safety are not documented in many cases. Besides controlled farming, validated quality control methods are needed to ensure identity, purity, and content. Analytical specifications within modern monographs are needed for consistent batch quality. Combretum mucronatum leaves are widely used in West Africa, but state-of-the-art quality control methods and specifications are non-existent. The aim of the following study was the development of ICH-validated chromatographic protocols for identity, purity, content assay, and analytical specifications for consideration into pharmacopoeial monographs. UPLC-ESI-Q-TOF-MS/MS was used for untargeted phytochemical information on composition. Optimisation of extraction was based on phytochemical profiling. HPTLC was used for differentiation of C. mucronatum from other Combretum species and UPLC for simultaneous determination of 7 marker compounds. C. mucronatum batch analyses (n = 49) investigated the influence of harvest time and geographical origin. Pesticides screening from a 349-compound panel were carried out. 30 compounds, identified by LC-MS, were used for characterization of the plant material. Orietin, isoorientin, vitexin and isovitexin were used as specific marker compounds for qualitative and quantitative HPTLC purposes, while UPLC quantified additionally epicatechin, procyanidins B2 and C1. Influence of harvest time and geographic origin on the content of marker compounds was observed. Differences in the metabolite profiles of C. mucronatum compared to related Combretum species were established for quality control purposes. Contamination with high amoounts of chlorpyrifos, and folpet (sum of folpet and phtalimide, expressed as folpet) were also observed.The study provides analytical protocols, analytical specifications and a drafted monograph for consideration for African pharmacopoeias, and reveals potential challenges in the quality of C. mucronatum.

Metabolite profiling, antifungal, biofilm formation prevention and disruption of mature biofilm activities of Erythrina senegalensis stem bark extract against Candida albicans and Candida glabrata.

The antifungal activity of the 70% ethanol stem bark extract of Erythrina senegalensis (ESB) against different strains and drug resistant clinical isolates of Candida albicans and Candida glabrata were evaluated in the study. The effect of ESB on biofilms as well as its activity in combination with fluconazole, nystatin or caspofungin against the Candida strains were also evaluated. We then evaluated the antifungal activity of a microemulsion formulation of ESB against planktonic and biofilms of the Candida species. UPLC-QTOF-MS2 analysis was then undertaken to identify the phytoconstituents of the extract and UPLC fingerprints developed for the routine authentication as part of quality control measures. ESB exerted strong antifungal activities against C. albicans ATCC 10231 and SC5314 strains, and C. glabrata ATCC 2001 strain with minimum inhibitory concentration (MIC) values from 3.91 to 31.25 µg/mL and minimum fungicidal concentrations (MFCs) that ranged from 62.5 to 250 µg/mL. It also exhibited potent antifungal activities (MIC = 4-64 µg/mL) against a collection of C. albicans and C. glabrata clinical isolates that were resistant to either nystatin or azole antifungals. The formulated ESB demonstrated higher antifungal potency against the C. albicans and C. glabrata strains with MIC values of 3.91-31.25 µg/mL which was the same as the MFC values. The extract and its microemulsion formulation were active against biofilms of the strains of the Candida species inhibiting their biofilm formations (SMIC50 = 16-64 µg/mL) and their preformed biofilms (SMIC50 = 128 ->512 µg/mL). ESB also exhibited synergistic antifungal action with fluconazole and nystatin against C. albicans ATCC 10231 and C. glabrata ATCC 2001 strains in the checkerboard assay. Chemical characterization of the extract revealed the presence of phenolic compounds such as flavonoids and their prenylated derivatives, anthracene glycosides and alkaloids. UPLC Fingerprints of the extract was also developed and validated for routine identification and authentication of the stem bark of E. senegalensis. The study findings have demonstrated that the stem bark of E. senegalensis is as a potential source of bioactive compounds that could be developed as novel antifungal agents.

Quality assessment of African herbal medicine: A systematic review and the way forward.

BACKGROUND: In Africa, herbalism supplements allopathic medicine's efforts to ensure Universal Health Coverage attainment. This review was conducted to identify and to summarise current literature on methodological approaches used for quality control of herbal medicines in Africa, to evaluate the gaps associated with existing strategies within context of best practices, and make recommendations for future improvements. METHODS: A systematic search was conducted using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Articles were screened and assessed for eligibility. RESULTS: 118 articles were included into the study. There was a high preference for impurity profiling tests (77%) indicating the prioritization for tests that guarantee safety despite the limited analytical resources available. Other classes of tests reported included identification tests (29%), physicochemical tests (18%), and content assays (12%). Although standard methods exist in preparing samples for impurity tests, different techniques were observed in different studies, and this could lead to differences in analytical outcomes. Content assays focused on single marker assessments, which may be inadequate to comprehensively assess the quality of products. CONCLUSION: This review provides knowledge of existing strengths and challenges for herbal medicine quality assessments in Africa. For future it is recommended to implement more studies on contaminants (e.g. mycotoxins) and pharmaceutical adulterants. The use of chemometrics to develop analytical methods should be promoted. Also, stakeholders in the medicine quality industry in Africa need to effectively collaborate to establish a well co-ordinated and harmonized system to provide a sustainable framework for the GACP and GMP guided production and quality assurance of herbal medicines.

Antifungal Activities of Phytochemically Characterized Hydroethanolic Extracts of Sclerocarya birrea Leaves and Stem Bark against Fluconazole-Resistant Candida albicans Strains.

The study evaluated the antifungal activities of the 70% ethanol extracts of Sclerocarya birrea leaves (SBL) and stem bark (SBB) against C. albicans strains and fluconazole-resistant isolates, their antifungal effects in combination with conventional antifungals as well as their effects on the biofilms of the C. albicans strains and isolates. UPLC-QTOF-MS/MS analysis was then carried out to investigate the metabolite profile of the extracts and UPLC fingerprints developed for their routine identification as part of quality control measures. The extracts exhibited considerable antifungal activity with MIC ranging from 12.21 to 97.66 µg/mL and MFC from 12.21 to 390.63 µg/mL against the C. albicans strains and isolates. The antifungal activity of the stem bark extract was higher than the leaf extract. SBL and SBB also significantly inhibited biofilm formation (IC50 = 12.49 to 164.42 µg/mL) and the mature biofilms (IC50 = 91.50 to 685.20 µg/mL) of the strains and isolates of the C. albicans and demonstrated potential for their use in combination therapies with currently used antifungals especially the stem bark extract with nystatin. Metabolite profiling identified the presence of polyphenolic compounds in both leaves and stem bark mostly flavonoids, their derivatives, and proanthocyanidins, which contribute in part to the bioactivity of the plant. Whereas flavonoids like quercetin, myricetin, and their derivatives were abundant in the leaves, epicatechin monomers with their condensed tannins, including procyanidin B2 and procyanidin C, were abundant in the stem bark. Fingerprints of SBL and SBB were developed and validated and could be used as qualitative tools to authenticate the plant. The outcomes of the study show the promise of the leaf and stem bark extracts of S. birrea to be studied further and developed as antifungal agents.

Anthelmintic Agents from African Medicinal Plants: Review and Prospects.

Soil-transmitted helminthiasis affects more than 1.5 billion people globally and largely remains a sanitary problem in Africa. These infections place a huge economic burden on poor countries and affect livestock production, causing substantial economic losses and poor animal health. The emergence of anthelmintic resistance, especially in livestock, and the potential for its widespread in humans create a need for the development of alternative therapies. Medicinal plants play a significant role in the management of parasitic diseases in humans and livestock, especially in Africa. This report reviews anthelmintic studies that have been conducted on medicinal plants growing in Africa and published within the past two decades. A search was made in various electronic databases, and only full articles in English were included in the review. Reports show that aqueous and hydroalcoholic extracts and polar fractions obtained from these crude extracts form the predominant (80%) form of the extracts studied. Medicinal plants, extracts, and compounds with different chemical groups have been studied for their anthelmintic potential. Polyphenols and terpenoids are the most reported groups. More than 64% of the studies employed in vitro assays against parasitic and nonparasitic nematode models. Egg hatch inhibition, larval migration inhibition, and paralysis are the common parameters assessed in vitro. About 72% of in vivo models involved small ruminants, 15% rodents, and 5% chicken. Egg and worm burden are the main factors assessed in vivo. There were no reports on interventions in humans cited within the period under consideration. Also, few reports have investigated the potential of combining plant extracts with common anthelmintic drugs. This review reveals the huge potential of African medicinal plants as sources of anthelmintic agents and the dire need for in-depth clinical studies of extracts, fractions, and compounds from African plants as anthelmintic agents in livestock, companion animals, and humans.

Maerua angolensis DC. (Capparaceae) Stem Bark Extract Protects against Pentylenetetrazole-Induced Oxidative Stress and Seizures in Rats.

INTRODUCTION: The stem bark of Maerua angolensis DC. (Capparaceae) is traditionally used for management of epilepsy. Our aim was to evaluate the antiseizure potential and identify possible mechanisms by which the effects are registered. METHODS: The petroleum ether/ethyl acetate extract (100-1000 mg kg-1) was administered per os to male Sprague-Dawley rats after pretreatment with flumazenil (0.3 mg kg-1) or L-arginine (150 mg kg-1) or sildenafil (5 mg kg-1) and they subsequently received a subcutaneous injection of pentylenetetrazole (65 mg kg-1). Rats were observed for latency to and duration of myoclonic seizures and additionally the level of protection against oxidant markers and products was assessed in vitro and in vivo. RESULTS: The extract (300 and 1000 mg kg-1, p.o.) significantly delayed the onset and decreased the duration and frequency of PTZ-induced convulsions. The anticonvulsant effect of MAE (300 mg kg-1, p.o.) was reversed by pretreatment with flumazenil, L-arginine, or sildenafil. Also, MAE (300 mg kg-1) treatment reversed significantly PTZ-induced oxidative stress in rat brain tissue. CONCLUSION: The petroleum ether/ethyl acetate fraction exhibits antiseizure activity by affecting GABAergic and nitric oxide-cGMP pathways. In addition, the extract protects against the generation of free radicals and the oxidative products of the PTZ-induced seizures.

Antiinflammatory properties of betulinic acid and xylopic acid in the carrageenan-induced pleurisy model of lung inflammation in mice.

This study investigated the antiinflammatory properties of betulinic acid (BA) and xylopic acid (XA) extracted from Margaritaria discoidea and Xylopia aethiopica, respectively. M. discoidea and X. aethiopica are plants native in Ghana and the West-African region and used traditionally to treat different pathologies including inflammatory conditions. The antiinflammatory effect of BA and XA was established by an in vivo assay using the carrageenan-induced pleural inflammation model in mice. Also, the ability of BA and XA to increase catalase, superoxide dismutase, glutathione levels and decrease lipid peroxidation level in reactive oxidative assays was assessed. In addition, the ability of XA and BA to prevent potential lung tissue damage was quantified. Pretreatment with BA and XA reduced significantly, signs of inflammation: neutrophil infiltration, oedema, and alveoli septal thickening in carrageenan-treated lung tissue. Additionally, BA or XA pretreatment lowered the degree of lipid peroxidation in the lung tissue while increasing the levels of catalase, superoxide dismutase, and glutathione in vivo. Comparatively, XA was more efficacious than BA in the prevention of lung tissue damage. BA and XA derived from X. aethiopica and M. discoidea possess antiinflammatory and in vivo antioxidant activities in mice pleurisy model. The effect of these compounds gives credence to the traditional use in the management of inflammatory conditions of the airway.

Pharmacognostic standardisation of Hilleria latifolia (Lam.) H. Walt. (Phytolaccaceae)

Objective: To establish the pharmacognostic characters for the correct identification and quality control of Hilleria latifolia (H. latifolia), an important herb in Ghanaian folklore medicine, for the treatment of infections, pain and inflammation. Methods: The macro-morphological, qualitative and quantitative microscopic features, physicochemical and phytochemical features of the medicinally used parts of H. latifolia were evaluated using standard methods. Results: The plant has simple, alternate leaves with entire margin. The lamina is ovate to broadly lanceolate with an acuminate apex. It is hypostomatic with anomocytic stomata. The plant contains abundant prismatic crystals in all parts. Starch grains abound in the roots. The quantitative indices of the leaf and physicochemical parameters have also been established. Conclusions: The pharmacognostic features established in this study may be used as part of the pharmacopoeial standard for the correct identification and quality control of H. latifolia.