Phytochemistry and bioactivity studies of cassia singueana del. and c. sieberiana dc. (fabaceae)

Cassia singueana Del. and C. sieberiana DC. are medicinal plants used for treating diabetes, ulcer, malaria, and wound healing and also used as poison by hunters in Africa. This study discuss the phytochemicals and bioactivity of the root and leaf of C. singueana Del. and C. sieberiana DC. Extraction of the plant samples by maceration in n-hexane, ethyl acetate, and methanol sequentially, followed by fractionation and purification using various chromatographic techniques led to the isolation of twenty compounds. Structural elucidation of these compounds using spectroscopic methods enabled the identification of seven anthraquinones, four flavonoids, three stilbenes, three terpenes, one bianthrone, one xanthone, and a benzoic acid derivative. C. singueana root extract afforded islandicin, xanthorin, monodictyxanthone, 3-hydroxy-5-methoxystilbene, and 4-hydroxybenzoic acid, which were reported for the first time from Cassia genus, while C. sieberiana root extract gave a new compound, cassiberianol A. The efficiency of ultrasonic assisted extraction (UAE) was optimised using response surface methodology (RSM) for high extraction yield from the root of C. singueana. The Box-Behnken design (BBD) was employed to propose optimised UAE conditions of time (25 min), temperature (50ºC), and solvent-sample ratio (10 mL/g) for high extraction yield. The ability of the BBD model equation for predicting the optimum yield was verified and the predicted yield (1.64%) was in good agreement with the experimental yield (1.65 ± 0.07%). This agreement indicated the suitability of the model and also the success of using RSM in optimising UAE conditions for root of C. singueana. The extraction yield (1.65 ± 0.07%) obtained under the optimised UAE conditions resulted in two-fold improvement when compared to yield obtained by maceration (0.71 ± 0.60%) or Soxhlet extraction (0.79 ± 0.40%) techniques. The RSM was successfully used to optimise UAE conditions for improved efficiency of UAE over maceration and Soxhlet extraction techniques. Bioactivity screenings which include antioxidant, a-glucosidase, acetylcholinesterase, tyrosinase, and 15-lipoxygenase inhibitory activities were conducted on the crude extracts and pure compounds. The results revealed that the ethyl acetate extract from the root of C. sieberiana was the most potent DPPH radical scavenger (1.88 µg/mL), and also gave the highest total phenolic (927 mg gallic acid equivalent/g) and total flavonoid content (346 mg quercetin equivalent/g). The ethyl acetate extract from the root of C. singueana also displayed the strongest ferric reducing power (2298 µmol Fe2+/g dry weight). Meanwhile, the methanol extract from the leaf of C. singueana demonstrated the most effective ß-carotene bleaching activity (87.05%). Quercetin and piceatannol showed broad spectrum of inhibitory activities against a-glucosidase (IC50 5.73 and 7.37 µM, respectively), acetylcholinesterase (IC50 2.89 and 10.57 µM, respectively), tyrosinase (IC50 92.40 and 95.14%, respectively), and 15-lipoxygenase (IC50 0.98 and 1.27 µM, respectively) compared to the positive controls used in these assays. However, physcion highly suppressed the activity of tyrosinase enzyme (79.66%), while cassiberianol A showed significant inhibition (IC50 2.63 µM) towards 15-lipoxygenase enzyme. The significant bioactivities demonstrated by the polar extracts from C. singueana and C. sieberiana can be attributed to the presence of phytochemicals such as flavonoids and stilbenes isolated from these polar extracts

Phytochemistry and bioactivity studies of Cassia singueana Del. and C. sieberlana DC. (FABACEAE)

Cassia singueana Del. and C. sieberiana DC. are medicinal plants used for treating diabetes, ulcer, malaria, and wound healing and also used as poison by hunters in Africa. This study discuss the phytochemicals and bioactivity of the root and leaf of C. singueana Del. and C. sieberiana DC. Extraction of the plant samples by maceration in n-hexane, ethyl acetate, and methanol sequentially, followed by fractionation and purification using various chromatographic techniques led to the isolation of twenty compounds. Structural elucidation of these compounds using spectroscopic methods enabled the identification of seven anthraquinones, four flavonoids, three stilbenes, three terpenes, one bianthrone, one xanthone, and a benzoic acid derivative. C. singueana root extract afforded islandicin, xanthorin, monodictyxanthone, 3-hydroxy-5-methoxystilbene, and 4-hydroxybenzoic acid, which were reported for the first time from Cassia genus, while C. sieberiana root extract gave a new compound, cassiberianol A. The efficiency of ultrasonic assisted extraction (UAE) was optimised using response surface methodology (RSM) for high extraction yield from the root of C. singueana. The Box-Behnken design (BBD) was employed to propose optimised UAE conditions of time (25 min), temperature (50ºC), and solvent-sample ratio (10 mL/g) for high extraction yield. The ability of the BBD model equation for predicting the optimum yield was verified and the predicted yield (1.64%) was in good agreement with the experimental yield (1.65 ± 0.07%). This agreement indicated the suitability of the model and also the success of using RSM in optimising UAE conditions for root of C. singueana. The extraction yield (1.65 ± 0.07%) obtained under the optimised UAE conditions resulted in two-fold improvement when compared to yield obtained by maceration (0.71 ± 0.60%) or Soxhlet extraction (0.79 ± 0.40%) techniques. The RSM was successfully used to optimise UAE conditions for improved efficiency of UAE over maceration and Soxhlet extraction techniques. Bioactivity screenings which include antioxidant, α-glucosidase, acetylcholinesterase, tyrosinase, and 15-lipoxygenase inhibitory activities were conducted on the crude extracts and pure compounds. The results revealed that the ethyl acetate extract from the root of C. sieberiana was the most potent DPPH radical scavenger (1.88 μg/mL), and also gave the highest total phenolic (927 mg gallic acid equivalent/g) and total flavonoid content (346 mg quercetin equivalent/g). The ethyl acetate extract from the root of C. singueana also displayed the strongest ferric reducing power (2298 μmol Fe2+/g dry weight). Meanwhile, the methanol extract from the leaf of C. singueana demonstrated the most effective β-carotene bleaching activity (87.05%). Quercetin and piceatannol showed broad spectrum of inhibitory activities against α-glucosidase (IC50 5.73 and 7.37 μM, respectively), acetylcholinesterase (IC50 2.89 and 10.57 μM, respectively), tyrosinase (IC50 92.40 and 95.14%, respectively), and 15-lipoxygenase (IC50 0.98 and 1.27 μM, respectively) compared to the positive controls used in these assays. However, physcion highly suppressed the activity of tyrosinase enzyme (79.66%), while cassiberianol A showed significant inhibition (IC50 2.63 μM) towards 15-lipoxygenase enzyme. The significant bioactivities demonstrated by the polar extracts from C. singueana and C. sieberiana can be attributed to the presence of phytochemicals such as flavonoids and stilbenes isolated from these polar extracts

Back to plants for drug discovery: from ethnomedicine to more conventional approaches

Over the last years, there is rekindling of interest in drug discovery from botanical resources. This thesis proposed two different approaches (from ethnomedicine to scholarly strategies) for drug discovery from medicinal plants, identifying the areas of knowledge involved and addressing the challenges encountered, with the aim of enhancing the chance of success of the overarching process. The first approach consists to review the literature to increase understanding of a plant of interest and generate strong hypotheses for future drug development research on this plant. Illustrating such an approach, we focused on Tithonia diversifolia (Hemsl.) A. Gray (TD). Knowledge about TD was collected from both online databases and non-electronic resources. Overall, a total of 1,804 reports have been collected. After subsequent duplicates removal and screening for relevant titles and abstracts, a total of 119 text articles were obtained and assessed for eligibility. Finally, 168 articles were selected, of which forty-nine were added after analyses of the reference lists of the included papers. We found that all parts of the plant are valued in several cultures for a wide scope of ailments ranging from topical issues \u2014wounds, skeleto-muscular disorders, abscesses, dermatological conditions, and stomach pains\u2014 to systemic disorders such as diabetes, malaria, fever, hepatitis and infectious diseases. Importantly, most of the ethnomedical claims of TD have been substantiated in several studies conducted in vitro and in vivo in animals. Sometimes, findings were conflicting and thanks to this review, we were able to assess the weight of evidence for each pharmacological effect of TD. The anti-inflammatory, antimalarial, antidiabetic, antioxidant and anticancer effects do stand out but there is also a stunning array of other relevant pharmacological effects. Chemically, a hundred of chemicals, mainly terpenoids and phenols, have been isolated from various TD extracts so far. Of these, some compounds including Tagitinin C have been linked to the pharmacology of TD. About the toxicological profile, we were able to conclude based on evidence that short-term oral administration of TD is relatively well-tolerated in animals when taken at doses less than 100 mg/kg. The second approach consists to screen the ethnomedical knowledge of indigenous people to select the best plant candidate to launch a drug discovery campaign. So, we carried out a 6-month cross-sectional questionnaire-based survey to explore the use of medicinal plants (MP) in People living with HIV (PLHIV) in the city of Dschang (West Region, Cameroon). Of the 247 HIV-infected respondents, 54.9% reported to use plants. MP users were then kindly invited to provide photographs and/or specimens of plants for botanical identification. A total of 70 MP, chiefly the herbs, were mentioned by informants (82.2% of total MP users, mean\ub1SEM: 2.2\ub10.2 MP/subject, min 1, max 11), of which forty-nine have been botanically identified. Commonly reported pathological conditions or symptoms treated with MP included malaria (n = 27, 18.4% of total citations), cough (n = 20, 13.6%) and abdominal pain (n = 16, 10.9%). The benefits of using MP reportedly ranged from moderate (n = 60, 57.7%) to complete (n = 35, 33.7%) relief, while only 8 subjects (7.7% of MP users) reported no change in their terms. Interestingly, 2 subjects (33.3 % of respondents) denounced fatigue and weight loss. We also observed that THPs were the main advisors of PLHIV on the use of MP. Thus, in the rest of our study, their knowledge and attitudes towards HIV/AIDS were surveyed aiming at understanding whether they may be an appropriate resource to assist in the scaling up of HIV prevention and treatment delivery services in Cameroon. 16 THPs were recruited by the chairperson of the Cooperative Society of Producers of Medicinal Plants of West Region based on their good reputation in traditional healing practice. Three of them acknowledged the use of MP to manage HIV diseases in their clients. All THPs who agreed to participate in the survey were also evaluated for their knowledge of HIV transmission, prevention and diagnosis. We found that their knowledge related to HIV was relatively low raising concern about their aptitude to effectively assist conventional health practitioners in fighting against HIV/AIDS. Resulting from literature mining and ethnomedical claims is the adoption of a relevant pharmacological testing system. In any case, the testing systems should represent the biological activities that best match the ethnomedical uses of the selected plant species. In addition, it is important to bear in mind that plant extracts are complex mixtures containing various components and, therefore, their overall activity results from interactions between their naturally occurring ingredients. It is with this background that we conducted a study of comparison of the effects of a whole extract of a particular strain of Cannabis sativa L. to that of cannabidiol (CBD). We knew cannabis is endowed with a potent anti-inflammatory effect attributable mainly to CBD, but also to its entourage. This mechanism by which other compounds occurring in cannabis may contribute to its clinical effects has been espoused as an \u201centourage effect\u201d. The concept of entourage effect was first introduced in 1998 by Ben-Shabat and Raphael Mechoulam but still, there was no hard evidence that the entourage effect is real. So, thanks to a collaboration with a pharmaceutical company, we grew a particular strain of cannabis deprived of THC and standardized in 5% CBD (CM5). Then, we tested the effects of an extract of CM5 in parallel to that of pure CBD at equimolar concentrations on neutrophil functions including oxidative metabolism, migration and production of proinflammatory cytokines. Results show that CM5 0.05-50 \u3bcg/mL and CBD 10-8-10-5 M inhibit the neutrophil functions including ROS production, cell migration, mRNA levels of proinflammatory cytokines (but at the protein level, only TNF-a was inhibited) to a comparable extent, indicating that CBD may be the main responsible of the anti-inflammatory effects of Cannabis. The effects of CBD and CM5 show however remarkable differences in terms of potency and efficacy, suggesting that beyond CBD, other components of cannabis may contribute to its biological effects. As a whole, such results support the use of cannabis and CBD to stem inflammation, however also warrant in-depth investigation of the underlying cellular and molecular mechanisms to better exploit their therapeutic potential

Pharmacological and phytochemical evaluation of seven plants used for microbial-related ailments in South African traditional medicine.

Doctor of Philosophy in Biological Sciences. University of KwaZulu-Natal, Pietermaritzburg 2018.Since antiquity, man has always believed in the healing properties of plants. The age-old practice of phytotherapy is now justified by numerous phytochemical and pharmacological studies which substantiate the presence of biologically active compound(s) in some medicinal plants. Quite often, ethnopharmacological studies provide important leads for the development of different types of plant-based therapeutic drugs. New effective antibiotics are urgently needed to combat multiple and extensively drug-resistant bacterial strains that are currently threatening public health globally. The rich floral diversity in southern Africa and the resultant extensive chemical diversity provide encouraging prospects for discovering novel pharmacologically important phytocompounds within this region. Against this background, the current study was designed to evaluate the in vitro antibacterial properties, phenolic profiles and mutagenic potentials of extracts obtained from seven South African plants used traditionally to manage bacterial infections and where the active principles were unknown. The study also aimed at identifying the principal antibacterial compound(s) in selected plants that demonstrated potent and broad-spectrum antibacterial properties. Aqueous and organic solvent extracts of the seven selected plants namely Bolusanthus speciosus, Cucumis myriocarpus, Ekebergia capensis, Protea caffra, Prunus africana, Searsia lancea and Solanum panduriforme were screened for antibacterial (minimum inhibitory concentration, MIC) properties against a panel of seven bacterial strains (Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, multiple drug-resistant (MDR) E. coli, MDR K. pneumoniae, drug-sensitive Staphylococcus aureus and penicillin-resistant S. aureus) using the microdilution technique. The extracts were also screened for antigonococcal properties using microdilution and agar disk-diffusion techniques. In addition, combinations of the different plant extracts, as well as plant extracts with four antibiotics (ampicillin, cefotaxime, chloramphenicol and penicillin) were evaluated for antibacterial synergistic interactions against MDR Gram-negative bacterial strains (E. coli and K. pneumoniae) using the checkerboard titration and time-kill bio-assays. Preliminary phytochemical analyses for phenolics in aqueous methanol (50% MeOH) plant extracts were conducted using spectrophotometric methods. In addition, specific phenolic acids in 80% MeOH extracts of the plants were quantified with the use of ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The mutagenic properties of plant extracts that demonstrated noteworthy antibacterial activities (MIC<1 mg/ml) were assessed using the Ames Salmonella/Microsome assay involving two tester strains, TA98 and TA102 (without S9 metabolic activation). The most potent antibacterial activities were exhibited by the MeOH bark extracts of B. speciosus which yielded MIC values of 0.039 and 0.078 mg/ml against S. aureus (penicillin-resistant and drug-sensitive strains, respectively). Dichloromethane (DCM) leaf extracts of S. lancea yielded an MIC value of 0.63 mg/ml against five of the seven test bacterial strains including MDR E. coli and MDR K. pneumoniae. B. speciosus bark (MeOH and DCM), P. caffra seeds (DCM) and twigs (MeOH) also demonstrated broad-spectrum antibacterial activities. B. speciosus MeOH bark extracts, as well as the DCM leaf extracts of P. africana and S. lancea demonstrated moderate antigonococcal properties (MIC range: 0.31-0.63 mg/ml). The checkerboard assay detected antibacterial synergistic interactions in combinations of chloramphenicol with each of B. specious leaf MeOH extracts, P. africana leaf MeOH and DCM extracts against MDR E. coli (fractional inhibitory concentration index ≤ 0.5). However, the time-kill assay did not detect any significant synergistic interactions in any of these three aforementioned combinations. Total phenolic content in the plant extracts investigated ranged from 2.38 to 62.73 mg gallic acid equivalents (GAE)/g dry matter. Variations in the quantities of flavonoids, condensed tannins, hydroxybenzoic and hydroxycinnamic acids among these plant extracts were also observed. In generally, higher levels of phenolic compounds were detected in S. lancea (leaves), P. africana (leaves) and P. caffra (leaves, twigs), while lower levels were detected in B. speciosus (barks), C. myriocarpus (fruits) and S. panduriforme (fruits, leaves, roots, stem). Based on the Ames test, all test plant extracts were non-mutagenic against Salmonella typhimurium tester strain TA98 and TA102 (without S9 metabolic activation). S. lancea leaves and P. caffra twigs demonstrated broad-spectrum antibacterial properties and as a result, a detailed analysis of their phytochemical constituencies was conducted. Antibacterial activity directed fractionation led to the isolation of two compounds from the aqueous (80%) MeOH leaf extracts of S. lancea. The isolated compounds were characterised using NMR data and both of them demonstrated noteworthy antibacterial activities (MIC range: 0.016-0.25 mg/ml) against E. faecalis and S. aureus. 1-Tetracosanol (43.98%), 1-nonadecanol (37.5%), eicosane (7.67%), 1,7-di-iso-propylnaphthalene (4.23%), 1-pentadecanol (3.04%), 2,6-di-iso-propylnaphthalene (1.96%) 1,3-iso-propylnaphthalene (1.62 %) were identified by GC-MS in an active S. lancea leaf ethyl acetate sub-fraction. The sub-fraction inhibited the growth of both E. faecalis and S. aureus at an MIC value of 0.25 mg/ml. 1-Tetracosanol and 1-nonadecanol, which accounted for more than 80% of the total phytochemical constituents of the fraction, are known antibacterial compounds which most likely contributed to the antibacterial properties of S. lancea leaves. Gas chromatography-mass spectrometry (GC-MS) analysis also revealed the presence of 15 compounds in three fractions obtained from a methanolic P. caffra twig extract. Two of the identified compounds, 1-adamantanecarboxylic acid and levoglucosan, are often incorporated into antimicrobial moieties to improve the efficacy of the therapeutic molecule, or as carbon skeletons. Two known antibacterial compounds, namely 1-heptacosanol and 1-nonadecanol were also among the compounds detected in the methanolic P. caffra twig extract. Overall, the present study reaffirmed that botanical medicines can potentially be used to manage drug-sensitive and drug-resistant bacterial infections often prevalent in humans. The study also provided the scientific rationale for the use of P. caffra, C. myriocarpus and S. panduriforme in South African folk medicine

Secondary metabolites from Rubiaceae species

This study describes some characteristics of the Rubiaceae family pertaining to the occurrence and distribution of secondary metabolites in the main genera of this family. It reports the review of phytochemical studies addressing all species of Rubiaceae, published between 1990 and 2014. Iridoids, anthraquinones, triterpenes, indole alkaloids as well as other varying alkaloid subclasses, have shown to be the most common. These compounds have been mostly isolated from the genera Uncaria, Psychotria, Hedyotis, Ophiorrhiza and Morinda. The occurrence and distribution of iridoids, alkaloids and anthraquinones point out their chemotaxonomic correlation among tribes and subfamilies. From an evolutionary point of view, Rubioideae is the most ancient subfamily, followed by Ixoroideae and finally Cinchonoideae. The chemical biosynthetic pathway, which is not so specific in Rubioideae, can explain this and large amounts of both iridoids and indole alkaloids are produced. In Ixoroideae, the most active biosysthetic pathway is the one that produces iridoids; while in Cinchonoideae, it produces indole alkaloids together with other alkaloids. The chemical biosynthetic pathway now supports this botanical conclusion. © 2015 by the authors; licensee MDPI

Aflatoxins

Aflatoxins are a group of highly toxic and carcinogenic substances that occur naturally and can be found in food substances. Aflatoxins are secondary metabolites of certain strains of fungi Aspergillus flavus and Aspergillus parasiticus as well as the less common Aspergillus nomius. Aflatoxins B1, B2, G1, and G2 are the most important members, which can be categorized into two groups according to chemical structure. As a result of the adverse health effects of mycotoxins, their levels have been strictly regulated, especially in food and feed samples. Therefore, their accurate identification and determination remain a herculean task due to their presence in the complex food matrix. The great public concern and the strict legislation incited the development of sensitive analytical methods that are discussed in this book

Forests for a Better Future Sustainability, Innovation and Interdisciplinarity

This book highlights the role of research in innovation and sustainability in the forest sector. The contributions included fall within the broad thematic areas of forest science and cover crucial topics such as biocontrol, forest fire risk, harvesting and logging practices, quantitative and qualitative assessments of forest products, urban forests, and wood treatments—topics that have also been addressed from an interdisciplinary perspective. The contributions also have practical applications, as they deal with the ecological and economic importance of forests and new technologies for the conservation, monitoring, and improvement of services and forest value