Pentas longiflora Oliv. (Rubiaceae), a plant used in the treatment of Pityriasis Versicolor in Rwanda: Chemical composition and standardization of leaves and roots

In Rwanda, the roots of Pentas longiflora Oliv. (Rubiaceae) have been used for a long time to treat Pityriasis versicolor. However, many people reported the use of leaves instead of roots. This research was conducted to compare the phytochemical composition and establish chromatographic methods for the standardization of roots and leaves extracts of P. longiflora. During this process, three new pentalongin glycosides (pentalonginoside A, pentalonginoside B, and pentalonginoside C) and two known glycosides of the same type (harounoside and clarinoside), as well as rutin, luteolin-7-rutinoside were isolated from methanol extract of leaves. In addition, pentalongin and psychorubrin, previously isolated from ethylacetate roots extract, were also identified in Pentas longiflora ethylacetate leaves extract. The presence of the antifungal compound pentalongin in leaves may explain the traditional use of leaves in the treatment of Pytiriasis versicolor. Furthermore, harounoside, psychorubrin, and pentalongin were selected as markers for HPLC fingerprints of MeOH extract. The accuracy and risk profile demonstrated the reliability of the validated method. In general, considerable variations of concentration in plant metabolites, including pentalongin, were observed between samples from different sites. The content in pentalongin (expressed as juglone) in collected samples ranged between 1.7 and 70.0 mg/100 g. The highest concentration (70.0 ± 17 mg/100 g) was registered in the cultivated samples from Mukoni. This important variation of pentalongin concentrations according to sampling sites, shows that in order to guarantee equivalent efficacy, finished products with P. longiflora should be standardized based on their pentalongin content

"Contribution to the phytochemical study and evaluation of the antiparasitic and anti-inflammatory potentiality of some Rwandan medicinal plants traditionally used in the treatment of malaria"

RESUMELes travaux de recherche de la présente thèse sont divisés en deux parties principales:-le screening de plantes médicinales rwandaises pour l’activité antiplasmodiale ;-l’isolement des principes actifs responsables de l’activité antiplasmodiale, l’évaluation de l’activité antiparasitaire et de l’activité anti-inflammatoire des plantes sélectionnées.Dans la première partie, 13 plantes ont été sélectionnées sur base d’une étude ethnobotanique sur les plantes médicinales utilisées pour traiter la malaria au Rwanda. A partir de ces plantes, 19 échantillons ont été collectés et 46 extraits testés contre Plasmodium falciparum. La majorité des extraits testés ont montré une activité in vitro dont 16% avec une activité très élevée (IC50 < 5 µg/ml). Le test in vivo nous a permis de choisir deux plantes, Zanthoxylum chalybeum et Terminalia mollis pour des analyses plus poussées. Dans la deuxième partie, tout d’abord, un fractionnement bio-guidé nous a permis d’identifier des composés responsables de l’activité antiplasmodiale de Z. chalybeum et de T. mollis. Il s’agit respectivement de la nitidine (IC50 ± 77 ng/ml) et de l’acide ellagique (IC50 ± 175 ng/ml). Ensuite, les deux plantes ont été testées in vitro pour l’activité antitrypanosomiale, antileishmaniale, antiamibienne et anti-inflammatoire. Z. chalybeum a montré une activité prometteuse sur la leishmaniose, une activité modérée sur le trypanosome, une activité faible sur l’amibe et une activité intéressante sur l’inflammation. T. mollis a présenté une activité très élevée sur la leishmaniose, une activité insignifiante sur le trypanosome, une activité modérée sur l’amibe et une activité remarquable sur l’inflammation.Il est probable que la nitidine, le composé responsable de l’activité antiplasmodial de Z. chalybeum, joue également un rôle dans l’activité de la plante sur la leishmaniose et sur l’inflammation et que l’acide ellagique intervient dans l’activité de T. mollis sur le trypanosome, l’amibe et l’inflammation. De plus, T. mollis contient de l’acide gallique, des punicalagines et leurs dérivés qui contribuent aux propriétés thérapeutiques de la plante notamment au niveau anti-inflammatoire.En bref, le présent travail a révélé les composés principalement responsables de l’activité antiplasmodiale des plantes sélectionnées, Z. chalybeum et T. mollis et l’effet de ces deux espèces végétales sur d’autres parasites et sur l’inflammation. SUMMARYThe work presented in this thesis is divided into two main parts: -the screening of Rwandan medicinal plants for antiplasmodial activity;-the isolation of the active ingredients responsible for antiplasmodial activity and the evaluation of antiparasitic as well as anti-inflammatory activity of selected plants.In the first part, 13 medicinal plants were selected based on an ethnobotanical survey conducted on medicinal plants used in Rwanda to treat malaria. From the selected plants 19 samples were collected and 46 extracts tested against Plasmodium falciparum. The majority of the plant extracts analysed exhibited in vitro antiplasmodial activity and 16 % presented a high activity (IC50 < 5 µg/ml). The in vivo assay allowed us to select two plants, Zanthoxylum chalybeum and Terminalia mollis for further investigations.In the second part of this work, firstly, a bio-guided fractionation made it possible to identify nitidine (IC50 ± 77 ng/ml) and ellagic acid (IC50 ± 175 ng/ml) as the main compounds responsible for the antiplasmodial activity of Z. chalybeum and T. mollis, respectively. Then, the two plants were evaluated in vitro for antitrypanosomal, antileishmanial, antiamoebic and anti-inflammatory activity. Z. chalybeum showed a very promising antileishmanial activity, a moderate antitrypanosomal activity, a weak antiamoebic and an interesting anti-inflammatory activity. T .mollis presented a very high antitrypanosomal activity, a negligible antileishmanial activity, a moderate antiamoebic and a remarkable anti-inflammatory activity.Nitidine, the most antiplasmodial compound from Z. chalybeum, probably plays a role in the antileishmanial and anti-inflammatory activity of the plant, whereas ellagic acid intervenes in the antitrypanosomal, antiamoebic and anti-inflammatory activity of T. mollis. Additionally, T. mollis contains gallic acid, punicalagins and their derivatives which contribute to the therapeutic properties of the plant, especially in its anti-inflammatory activity. Briefly, this work revealed the most active products responsible for the antiplasmodial activity of selected plants, Z. chalybeum and T. mollis and the effect of these two species on other parasites and on inflammation

In Vitro and In Vivo Antiplasmodial Activity of Three Rwandan Medicinal Plants and Identification of Their Active Compounds

In our previous study, we reported the interesting in vitro antiplasmodial activity of some Rwandan plant extracts. This gave rise to the need for these extracts to also be evaluated in vivo and to identify the compounds responsible for their antiplasmodial activity. The aim of our study was, on the one hand, to evaluate the antiplasmodial activity in vivo and the safety of the selected Rwandan medicinal plants used in the treatment of malaria, with the objective of promoting the development of improved traditional medicines and, on the other hand, to identify the active ingredients in the plants. Plant extracts were selected according to their selectivity index. The in vivo antiplasmodial activity of aqueous, methanolic, and dichloromethane extracts was then evaluated using the classical 4-day suppressive test on Plasmodium berghei infected mice. The activity of the plant extracts was estimated by measuring the percentage of parasitemia reduction, and the survival of the experimental animals was recorded. A bioguided fractionation was performed for the most promising plants, in terms of antiplasmodial activity, in order to isolate active compounds identified by means of spectroscopic and spectrometric methods. The highest level of antiplasmodial activity was observed with the methanolic extract of Fuerstia africana (> 70 %) on days 4 and 7 post-treatment after intraperitoneal injection and on day 7 using oral administration. After oral administration, the level of parasitemia reduction observed on day 4 post-infection was 44 % and 37 % with the aqueous extract of Terminalia mollis and Zanthoxylum chalybeum, respectively. However, the Z. chalybeum extract presented a high level of toxicity after intraperitoneal injection, with no animals surviving on day 1 post-treatment. F. africana, on the other hand, was safer with 40 % mouse survival on day 20 post-treatment. Ferruginol is already known as the active ingredient in F. Africana, and ellagic acid (IC50 = 175 ng/mL) and nitidine (IC50 = 77.5 ng/mL) were identified as the main active constituents of T. mollis and Z. chalybeum, respectively. F. africana presented very promising antiplasmodial activity in vivo. Although most of the plants tested showed some level of antiplasmodial activity, some of these plants may be toxic. This study revealed for the first time the role of ellagic acid and nitidine as the main antimalarial compounds in T. mollis and Z. chalybeum, respectively

Medicinal plants, malaria and biotechnology

The first part of the talk will be dedicated to the investigation of medicinal plants with the objective to identify new antimalarial treatments. According to the last World Malaria Report [1], there were 584 000 deaths for 198 millions malaria cases worldwide in 2013. Particularly, the disease caused an estimated 437 000 African children died before their fifth birthday, still in 2013. Malaria is caused by a parasite, Plasmodium sp. and transmitted by Anopheles mosquitoes. The problem of parasite resistance towards common available medicines such as chloroquine, mefloquine, quinine, is increasing. In this context, the vegetal kingdom remains the main source of pharmacologically active compounds against this parasitic infection as attested by the famous quinine, isolated from Cinchona sp., artemisinin extracted from Artemisia annua and also atovaquone derived from lapachol found in several Bignoniaceae. All these substances are related to plants with traditional use against fever and malaria. Beside these well-known examples, various new antiplasmodial compounds are frequently discovered from Nature, particularly following an ethnopharmacological approach, as reviewed by several authors in recent years [2-6]. Then, the pharmacological and phytochemical study of plants from traditional pharmacopoeias can be of first interest not only to discover new antimalarial “lead compounds”, but also to valorize local vegetal species whose efficacy and safety would have been demonstrated in laboratory and by clinical investigations [7,8]. Some results obtained with Dicoma tomentosa from Burkina-Faso [9] and Terminalia mollis from Rwanda [10] will be presented. In the second part of the talk, two applications of biotechnology for the production of artemisinin and paclitaxel and then some works developed at the ‘Université de la Réunion’ will be presented. In the framework of this collaboration, Psiadia arguta, an endemic plant from Reunion Island, which is known to have cytotoxic, anti-plasmodial and anti-inflammatory properties, was subjected to micropropagation. The objective of the work was to compare the biological properties and the phytochemical composition of callus, vitroplants and acclimatized plants of Psiadia arguta [11]. 1. WHO, World Malaria Report 2014, December 2014, Geneva (http://www.who.int/malaria/publications/world_malaria_report_2014/en/). 2. Batista R, Silva Ade J Jr, de Oliveira AB: Plant-derived antimalarial agents: new leads and efficient phytomedicines. Part II. Non-alkaloidal natural products. Molecules 2009, 14:3037-72. 3. Bero J, Frédérich M, Quetin-Leclercq J : Antimalarial compounds isolated from plants used in traditional medicine. Journal of Pharmacy and Pharmacology 2009, 61:1401–1433. 4. Bero J and Quetin-Leclercq J: Natural products published in 2009 from plants traditionally used to treat malaria. Planta Medica 2011, 77:631-40. 5. Kaur K, Jain M, Kaur T, Jain R: Antimalarials from nature. Bioorganic & Medicinal Chemistry 2009, 17:3229–3256. 6. Nogueira CR and Lopes LMX: Antiplasmodial Natural Products. Molecules 2011, 16:2146-2190 7. Ginsburg H and Deharo E: A call for using natural compounds in the development of new antimalarial treatments – an introduction. Malaria Journal 2011, 10 (suppl. 1):S1 8. Willcox M, Graz B, Falquet J, Diakite C, Giani S, Diallo D: A “reverse pharmacology” approach for developing an antimalarial phytomedicine. Malaria journal 2011, 10(suppl1):S8 9. Jansen, O., Tits, M., Angenot, L., Nicolas, J.-P., De Mol, P., Nikiema, J.-B., & Frédérich, M : Anti-plasmodial activity of Dicoma tomentosa (Asteraceae) and identification of urospermal A-15-O-acetate as the main active compound. Malaria Journal 2012, 11, 289. 10. Muganga, R., Angenot, L., Tits, M., & Frédérich, M : In vitro and in vivo antiplasmodial activity of three Rwandan medicinal plants and identification of their active compounds. Planta Medica 2013, 80(6), 482-489. 11. Mahy Justine, Comparative study of biological activities and analysis of volatile compounds of Psiadia arguta in various cultures: vitroplants and acclimatized plants. Mémoire de M2, 2013, Université de Liège/Université de la Réunion

HPLC-UV method for standardization of Neorautanenia mitis, an African plant used in an anti-scabies ointment

An HPLC-UV method for standardization of the methanol-soluble extracts from tubers of Neorautanenia mitis (A.Rich.) Verdc., Leguminosae, harvested during different periods and from different sites, is described. The chemical fingerprint was established with six identified markers using LC-ESI (+)-MS/MS, including rotenone; the total error was used as validation criterion, the accuracy and risk profiles demonstrated the reliability of the method. The study verified that the major degradation product of rotenone in methanol is dehydrorotenone. The detection range of rotenone was between 40 and 400 µg/ml. The collected samples contained 868-5732 µg/g of rotenone. The concentrations of rotenone in the wild samples from the Ngoma site (5167 ± 565 µg/g) were higher than those registered in the samples from the other sites. No significant differences were observed among the remaining sampling sites, and most of the rotenone was located in the inner part of the tubers (2165 ± 1051 µg/g) when compared to that in their peels (961 ± 320 µg/g)

ESTABLISHMENT AND VALIDATION OF CHROMATOGRAPHIC METHODS FOR STANDARDIZATION OF NEORAUTANENIA MITIS, A MEDICINAL PLANT USED IN AN ANTI-SCABIES OINTMENT IN RWANDA.

An HPLC-UV method for standardization of the methanol-soluble extracts from tubers of Neorautanenia mitis (A.Rich.) Verdc., Leguminosae, harvested during different periods and from different sites, is described. The chemical fingerprint was established with six identified markers using LC-ESI (+)-MS/MS, including rotenone; the total error was used as validation criterion, the accuracy and risk profiles demonstrated the reliability of the method. The study verified that the major degradation product of rotenone in methanol is dehydrorotenone. The detection range of rotenone was between 40 and 400 µg/ml. The collected samples contained 868-5732 µg/g of rotenone. The concentrations of rotenone in the wild samples from the Ngoma site (5167 ± 565 µg/g) were higher than those registered in the samples from the other sites. No significant differences were observed among the remaining sampling sites, and most of the rotenone was located in the inner part of the tubers (2165 ± 1051 µg/g) when compared to that in their peels (961 ± 320 µg/g)

Ethnopharmacology and malaria in Africa

According to the last World Malaria Report [1], there were 584 000 deaths for 198 millions malaria cases worldwide in 2013. Particularly, the disease caused an estimated 437 000 African children died before their fifth birthday, still in 2013. Malaria is caused by a parasite, Plasmodium sp. and transmitted by Anopheles mosquitoes. The problem of parasite resistance towards common available medicines such as chloroquine, mefloquine, quinine, is increasing. In this context, the vegetal kingdom remains the main source of pharmacologically active compounds against this parasitic infection as attested by the famous quinine, isolated from Cinchona sp., artemisinin extracted from Artemisia annua and also atovaquone derived from lapachol found in several Bignoniaceae. All these substances are related to plants with traditional use against fever and malaria. Beside these well-known examples, various new antiplasmodial compounds are frequently discovered from Nature, particularly following an ethnopharmacological approach, as reviewed by several authors in recent years [2-6]. Then, the pharmacological and phytochemical study of plants from traditional pharmacopoeias can be of first interest not only to discover new antimalarial “lead compounds”, but also to valorize local vegetal species whose efficacy and safety would have been demonstrated in laboratory and clinical investigations [7]. As demonstrated in several works from Willcox [8], better knowledge of plants from traditional pharmacopoeias and local valorization of validated traditional remedies in Improved Traditional Medicine (ITM) could allow the access to effective, standardized, available and affordable therapeutics for management of malaria by local populations. After this introductive section, the second part of the talk will be dedicated to the presentation of some results obtained in Liège with Dicoma tomentosa from Burkina-Faso [9], Strychnos icaja from Cameroun [10] and Terminalia mollis from Rwanda [11]. 1. WHO, World Malaria Report 2014, December 2014, Geneva (http://www.who.int/malaria/publications/world_malaria_report_2014/en/). 2. Batista R, Silva Ade J Jr, de Oliveira AB: Plant-derived antimalarial agents: new leads and efficient phytomedicines. Part II. Non-alkaloidal natural products. Molecules 2009, 14:3037-72. 3. Bero J, Frédérich M, Quetin-Leclercq J : Antimalarial compounds isolated from plants used in traditional medicine. Journal of Pharmacy and Pharmacology 2009, 61:1401–1433. 4. Bero J and Quetin-Leclercq J: Natural products published in 2009 from plants traditionally used to treat malaria. Planta Medica 2011, 77:631-40. 5. Kaur K, Jain M, Kaur T, Jain R: Antimalarials from nature. Bioorganic & Medicinal Chemistry 2009, 17:3229–3256. 6. Nogueira CR and Lopes LMX: Antiplasmodial Natural Products. Molecules 2011, 16:2146-2190 7. Ginsburg H and Deharo E: A call for using natural compounds in the development of new antimalarial treatments – an introduction. Malaria Journal 2011, 10 (suppl. 1):S1 8. Willcox M, Graz B, Falquet J, Diakite C, Giani S, Diallo D: A “reverse pharmacology” approach for developing an antimalarial phytomedicine. Malaria journal 2011, 10(suppl1):S8 9. Jansen, O., Tits, M., Angenot, L., Nicolas, J.-P., De Mol, P., Nikiema, J.-B., & Frédérich, M : Anti-plasmodial activity of Dicoma tomentosa (Asteraceae) and identification of urospermal A-15-O-acetate as the main active compound. Malaria Journal 2012, 11, 289. 10. Tchinda, A. T., Jansen, O., Nyemb, J.-N., Tits, M., Dive, G., Angenot, L., & Frédérich, M. Strychnobaillonine, an unsymmetrical bisindole alkaloid with an unprecedented skeleton from Strychnos icaja roots. Journal of Natural Products 2014, 77(4), 1078–82. 11. Muganga, R., Angenot, L., Tits, M., & Frédérich, M : In vitro and in vivo antiplasmodial activity of three Rwandan medicinal plants and identification of their active compounds. Planta Medica 2013, 80(6), 482-489

17-O-acetyl, 10-hydroxycorynantheol, a selective antiplasmodial alkaloid isolated from Strychnos usambarensis leaves

In the course of our investigations on Strychnos usambarensis leaves in order to isolate isostrychnopentamine, the main alkaloid responsible for the antiplasmodial activity of the plant, a new tertiary indolic alkaloid has been isolated: 17-O-acetyl, 10-hydroxycorynantheol 1. Its structure was determined by means of spectroscopic and spectrometric methods such as UV, IR, CD, NMR and ESI-MS. 17-O-acetyl, 10-hydroxycorynantheol 1 is one of the most active monomeric indole alkaloid known to date showing an in vitro activity against Plasmodium falciparum close to 5 µM and a high selectivity

LC-SPE-NMR-MS analysis of Strychnos usambarensis fruits from Rwanda

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