Zootherapeutic uses of animals excreta: the case of elephant dung and urine use in Sayaboury province, Laos.

BACKGROUND: Despite a widespread aversion towards faeces and urine, animal excreta are used in traditional medicine in many countries since centuries, but records are scattered and few therapeutic uses have been accurately documented while in the current context of emerging zoonoses such records may be of major interest. METHODOLOGY: In this study, we investigated the therapeutic uses that mahouts in Xayaboury province, Lao PDR make of elephant urine and faeces as well as of the brood chamber that beetles (Heliocopris dominus) fashion from elephant dung. Semi-structured interviews were conducted with mahouts on elephant diet, health problems and responses to disease, andwhether they use elephant products. Data were supplemented by interviews with traditional healers. RESULTS: Seven respondents reported the use of elephant urine in ethnoveterinary care for elephants and in human medicine in case of diabetes and otitis. 25 respondents reported therapeutic use of elephant faeces (EF) and elephant dung beetle brood chambers. The major indications are gastrointestinal and skin problems. Macerations or decoctions are drunk or used externally as a lotion. The mahouts attribute the therapeutic effectiveness of EFs to their content which includes the remains of many species from the elephant diet which they consider to be medicinal. DISCUSSION: The indications of these uses are consistent with pharmacological and clinical studies highlighting the properties of different animals' urine and faeces and their curative potential tested in vivo. The acknowledgement by the mahouts of medicinal properties of elephant faecal bolus contrasts with the rare justifications of animal material use recorded in zootherapeutic studies, which falls within the symbolic domain. However, numerous studies highlight the preponderant role of the microbiota in physiological processes, raising the hypothesis of a curative action of EF, by rebalancing the user's microbiota. CONCLUSION: The therapeutic uses of EF preparations despite their possible curative properties are a potential source of zoonotic transmission from elephants to humans. In the current context of globalisation of trade which favours the emergence of zoonoses and in relation with the issue of One Health, it becomes crucial to further document the zootherapeutic practices to prevent emerging diseases. As elephants and local related ethnoethological knowledge are threatened, documenting them is urgent to contribute to their preservation.

Forest Fevers: traditional treatment of malaria in the southern lowlands of Laos.

ETHNOPHARMACOLOGICAL RELEVANCE: Malaria is still a highly challenging public health issue in southern Lao PDR, with increasing cases of artemisinin resistance and Plasmodium vivax infections which are more complicated to treat. Traditional medicine has a long history of use in Laos, and is primarily practised by traditional village healers, who possess unique bodies of transmitted knowledge focused on herbal prescriptions, including those for the treatment of malaria. Villagers also use plants for healthcare in the home. The aim of the study is to document local fever concepts and use of herbal remedies, and examine whether they may have potential as complementary treatments against malaria. MATERIALS AND METHODS: The study took place in Champasak province in the far south of Laos, in primarily lowland areas. First, 35 traditional healers across the 10 districts of the province were interviewed to elicit details about knowledge and treatment of fevers. Second, a household survey was conducted in a village in a malaria-endemic area; 97 households were interviewed on fever incidence, differentiation, treatment-seeking behaviour and knowledge of plant-based remedies for fevers. Plants indicated by both healers and villagers were collected and voucher specimens deposited in the herbarium of the National University of Laos for identification. RESULTS: Malaria is a well-known pathology among the healers and villagers of lowland Champasak province; biomedical treatments are preferentially used, but traditional medicine is a popular complementary method, especially in chronic cases with additional symptoms. 30 different fever types were recorded, which were usually named symptomatically, and grouped into 12 categories. Some were described as forms of malaria, which was conceived as a dynamic, changing pathology affecting many body systems. Healers formulate treatments based on symptoms and the person's constitution, and with the intention of creating specific pharmacological actions associated with temperature or flavours. 11 of the healers gave prescriptions for malaria (27 in total), including 47 identified plant species. The most-used plants (4 or more use-reports) were also the most cited in the literature for use against malaria, demonstrating a correspondence between Lao healers and other traditional medical systems. Furthermore, some of these species show promising results for future research, especially Amorphophallus paeniifolius (Dennst.) Nicolson and Alocasia macrorrhizos (L.) G. Don. CONCLUSION: Traditional healers are important actors in the treatment of malaria in southern Laos, and herbal remedies should be evaluated further by the use of reverse treatment outcome trials, especially those which may be of use as complementary remedies in treating P. vivax. Initiatives on knowledge transmission, medicinal plant conservation and healthcare integration are also urgently needed.

Comparison of the antimalarial activity of a Colombian traditional Uitoto remedy with laboratory preparations.

BACKGROUND & OBJECTIVES: In Colombian Amazonia, Uitoto indigenous people use a preparation of Curarea toxicofera (Menispermaceae) to prevent and treat malaria. To open the way for the production of a standardized herbal remedy, we compared the activity of the traditional preparation with laboratory preparations. METHODS: People were interviewed on their mode of use and preparation of what is considered the best remedy against fevers in this area. The herbal remedy was prepared according to the healer's recommendations. The plant was also submitted to continuous distillation and percolation extraction. The preparations were then tested against Plasmodium falciparum, in vitro. Traditional preparation and extract obtained by percolation were tested on Plasmodium berghei infected mice. Chemical profiles were also explored by thin-layer chromatography. RESULTS: Yields of extraction were around 7% in the preparations (percolation was the most efficient). The phytochemical profile showed a mix of steroids, flavonoids and alkaloids qualitatively similar in all preparations. In vitro, the extracts showed inhibitory concentration 50 <10µg/mL: the traditional preparation was almost three times less active than laboratory preparations. In vivo, percolation was also more active than traditional preparation, inhibiting 78% of the parasite growth at 400mg/kg/day by oral route. INTERPRETATION & CONCLUSION: Pharmacological activities suggest that both the original remedy (prepared according to traditional pharmacopeia) and the extracts obtained by percolation extraction exhibit relevant antiparasitic activity. C. toxicofera should therefore be considered for the elaboration of an improved traditional medicine by implementing toxicological studies and carefully following quality control guidelines for its preparation.

From plant selection by elephants to human and veterinary pharmacopeia of mahouts in Laos.

ETHNOPHARMACOLOGICAL RELEVANCE: To what extent has animal observation contributed to the development of human pharmacopeias? We approach this question here through the study of mahouts' knowledge regarding the responses by elephants to their health problems, and the human medicinal uses of plants and the care of domestic animals that result from their observations. MATERIALS AND METHODS: 32 mahouts were interviewed in Thongmyxay district and 28 at the Elephant Conservation Center in the province of Xayabury. Interviews focused on the elephants' diet, health problems, plant items they consume in particular physiological or pathological contexts and the treatments that mahouts provide them. For each plant mentioned, the part of the plant consumed and mode of preparation and administration if used by mahouts were recorded. Species samples were then collected and later identified by specialists. RESULTS: 114 species were recorded as being consumed by elephants during interviews with mahouts and forest outings with them to collect samples. Twenty species were identified as used by elephants in particular pathological conditions or physiological states. According to interviewed mahouts, the consumption of certain plants improves the health of the elephant. We observed clear convergences between the observations interpreted by the mahouts as self-medication behaviour from elephants and their own medicinal practices (for human and veterinary purposes). CONCLUSION: Beyond a mere reproduction of elephant self-medication behaviours observed, the human or veterinary medicinal cares derived from these observations are the result of complex arrangements integrating all available medicinal and conceptual resources into elaborate preparations. We recommend that mahouts' knowledge about traditional medicinal care given to elephants be further compiled, as it could have a beneficial impact on veterinary health care provided in elephant resorts and elephants' well-being.

Hmong herbal medicine and herbalists in Lao PDR: pharmacopeia and knowledge transmission.

BACKGROUND: In Lao PDR, the Hmong ethnic group has extensive knowledge about the use of medicinal plants. However, despite the importance of the Hmong pharmacopeia as a primary health care resource, no study has been undertaken to thoroughly document medicinal plant knowledge and its transmission. Objectives of this study are (i) to describe and characterize Hmong pharmacopeia, and (ii) to understand how medicinal plant knowledge is transmitted and spread among Hmong in Lao PDR, in order to assess whether this knowledge base is under threat. METHODS: In order to describe Hmong pharmacopeia, a total of 14 interlocutors were interviewed in three provinces (Bokeo, Xieng Khouang, and Vientiane), using "walk in the wood" methodology. To gain insight about knowledge transmission, semi-structured interviews were conducted with 28 people. Twenty of them were herbalists. Data analysis was performed using univariate analysis for the description of the pharmacopeia. Medicinal plant knowledge consistency was assessed through use and plant name overlapping. Answers to the semi-structured interview on knowledge transmission were analyzed qualitatively. RESULTS: Three hundred thirty-three different medicinal species were collected. The majority of uses attributed to plants were gastrointestinal conditions (22% of total use reports), gynecological conditions and sexually transmitted disease (12%), skin affections (8%), kidney and bladder problems (5%), physical traumas (5%), and aphrodisiac (or male tonics; 5%). Use convergences are more marked in the gynecological sphere, but there is a strong heterogeneity in practices and knowledge. Medicinal plant knowledge transmission is oral, gained from direct experience since childhood, matrilineal, and kept strictly within the family lineage. Apparent limited consensus on uses might stem from the method of knowledge transmission and to the economic value given to medicinal plants. DISCUSSION: Use pattern of species from the Hmong pharmacopeia does not appear to be strikingly different from the national Lao pharmacopeia. Differences may lie in the methods and reasons for knowledge transmission. It can be proposed that the economic value given to plants helps in keeping the knowledge alive, and encourages its transmission. CONCLUSION: Hmong traditional medicine is constantly evolving in a dynamic process and aims to respond to health problems faced by the local population. Herbalists appear as health fully fledged actors and should be recognized and valued as such.

Pharmacological activity of Curarea toxicofera in combination with classical antimalarial treatments.

ETHNOPHARMACOLOGICAL RELEVANCE: In the Leticia-Amazonas area, Uitoto indigenous people use a preparation of Curarea toxicofera (Wedd) Barneby & Krukoff (Menispermaceae) alone or combined with prescribed medications to prevent and treat malaria. AIM OF STUDY: To determine the in vitro and in vivo antiplasmodial activity of traditional preparations of Curarea toxicofera alone and in combination with classical antimalarials. MATERIAL AND METHODS: The traditional preparation was evaluated in vitro against P. falciparum FCR3 CQ resistant strain, alone and combined. The preparation was further administered orally alone or combined with chloroquine and artesunate in mice infected with Plasmodium berghei ANKA strain on the four-day antimalarial test model. RESULTS: The herbal remedy used alone was able to significantly decrease the parasitemia both in vitro (IC50 7.3 µg/ml) and in vivo (ED50 328 mg/Kg) but it was less active than chloroquine (IC50 0.29 µg/ml in vitro and ED50 2.3 mg/Kg/day in vivo), and than artesunate (IC50 0.002 µg/ml and ED50 3.7 mg/Kg/day). Interestingly it presented synergism with chloroquine in vitro (Combination Index: 0.39) and in vivo; and was additive with artesunate in vitro (Combination Index: 0.94) and in vivo. CONCLUSION: The traditional preparation showed potential as an antimalarial and, when used in combination, does not negatively affect the efficacy of the drugs evaluated. Pre-clinical studies should be conducted with a standardized preparation to confirm its efficacy and safety alone and in combination with chloroquine and artesunate.

A metabolomic approach to identify anti-hepatocarcinogenic compounds from plants used traditionally in the treatment of liver diseases.

Liver cancer is a major health burden in Southeast Asia, and most patients turn towards the use of medicinal plants to alleviate their symptoms. The aim of this work was to apply to Southeast Asian plants traditionally used to treat liver disorders, a successive ranking strategy based on a comprehensive review of the literature and metabolomic data in order to relate ethnopharmacological relevance to chemical entities of interest. We analyzed 45 publications resulting in a list of 378 plant species, and our point system based on the frequency of citation in the literature allowed the selection of 10 top ranked species for further collection and extraction. Extracts of these plants were tested for their in vitro anti-proliferative activities on HepG2 cells. Ethanolic extracts of Andrographis paniculata, Oroxylum indicum, Orthosiphon aristatus and Willughbeia edulis showed the highest anti-proliferative effects (IC50 = 195.9, 64.1, 71.3 and 66.7 µg/ml, respectively). A metabolomic ranking model was performed to annotate compounds responsible for the anti-proliferative properties of A. paniculata (andrographolactone and dehydroandrographolide), O. indicum (baicalein, chrysin, oroxylin A and scutellarein), O. aristatus (5-desmethylsinensetin) and W. edulis (parabaroside C and procyanidin). Overall, our dereplicative approach combined with a bibliographic scoring system allowed us to rapidly decipher the molecular basis of traditionally used medicinal plants.

Herbal Medicine Practices of Patients With Liver Cancer in Peru: A Comprehensive Study Toward Integrative Cancer Management.

RATIONALE: The highest burden of liver cancer occurs in developing countries, where the use of herbal medicine (HM) is still widespread. Despite this trend, few studies have been conducted to report HM practices of patients with a hepatic tumor in the developing world. Hence, this study aimed to document the use of HM among patients with liver cancer in Peru. STUDY DESIGN AND METHODS: A comparative behavioral epidemiological survey was conducted among liver cancer patients attending the National Cancer Institute of Peru. Information was obtained by direct interviews based on a semistructured questionnaire. The use of HM in Peruvian liver cancer patients was reported, first, regarding general consumption prior to the onset of disease, and second, after the appearance of symptoms that patients would relate to their tumor. In parallel, general consumption of HM in noncancerous people was assessed as a comparative figure. A correspondence analysis was performed to reveal potential associations between the symptoms of cancer and the specific use of HM. RESULTS: Eighty-eight patients and 117 noncancerous individuals participated in the survey. Overall, 68.3% of the people interviewed claimed to use HM on a regular basis for general health preservation. Furthermore, 56.8% of the patients turned to plants first to treat the disorders for which they later came to the cancer care center. When compared with the number of plant species used routinely (n = 78), a selection of plants was made by patients in response to the symptoms of cancer (n = 46). At least 2 plant species, Aloe vera and Morinda citrifolia, were significantly associated with the treatment of liver cancer-related symptoms in the patient group. CONCLUSIONS: The present study is the first survey on the HM practices of patients with liver cancer in Latin America and, more broadly, in the developing world. Our findings confirm that HM remains one of the principal primary health care resources in Peru, even for a severe disease like liver cancer. These traditional, complementary and alternative medicine practices should be taken into consideration in Peruvian health programs aiming to educate the population in cancer prevention and treatment, as well as integrative cancer management.

Quassia "biopiracy" case and the Nagoya Protocol: A researcher's perspective.

Biopiracy accusations are common in the world of biodiversity research. At the end of 2015, a French NGO accused researchers from the Institut de Recherche pour le Développement (IRD) of biopiracy. These researchers had applied for a patent for a natural bioactive molecule against malaria and cancer, the Simalikalactone E, isolated from Quassia amara L. (Simaroubaceae) leaves. This biopiracy allegation triggered a huge wave of attacks from the media and social networks, and vehement recrimination from political officials in French Guiana against researchers who have been accused of ethical misconduct, by stealing the traditional knowledge of indigenous people. These accusations were made in the contentious context of the ratification of the Nagoya Protocol in the frame of implementing the French law on biodiversity, nature and landscapes. So, in an atmosphere of heightened emotions it is crucial to understand the issues behind these accusations. We describe herein the genesis of our discovery, present the detractors' arguments, and discuss the consequences of such biopiracy denunciations for scientific research. We also address our concerns about the gap between rhetoric and reality and the real impact of the Nagoya Protocol on biodiversity conservation.

Adaptation and optimization of a fluorescence-based assay for in vivo antimalarial drug screening.

The in vivo efficacy of potential antimalarials is usually evaluated by direct microscopic determination of the parasitaemia of Plasmodium-infected mice on Giemsa-stained blood smears. This process is time-consuming, requires experienced technicians and is not automatable. Therefore, we optimized a SYBR Green I (SYBRG I) fluorescence-based assay to fluorometers commonly available in many research laboratories. This technique was originally developed to assess parasitaemia in humans by cytometry. We defined optimal conditions with Plasmodium berghei-infected mice, standard lysis buffer (Tris, EDTA, saponin and Triton), whole blood cells and 2 h staining incubation with SYBRG I 2X. The fluorescence background generated by uninfected whole blood cells was low (around 4.6%), and the linearity high (r 2 = 0.96), with parasitaemia ranging from 1.4 to 60%. The Bland-Altman plot showed a strong correlation between SYBRG I and Giemsa gold standard method; Z'-factor was >0.5. These findings suggest that our fluorescence-based assay is suitable for in vivo antimalarial drug assessment in a malaria murine model. It can help to overcome the human bias found with microscopic techniques.

Treatment and management of liver diseases by Khmer traditional healers practicing in Phnom Penh area, Cambodia.

ETHNOPHARMACOLOGICAL RELEVANCE: Liver disorders are a major health problem in Cambodia, where some patients prefer to seek treatment from traditional healers. The aim of the study was to document the knowledge and practices of these healers in four Southern Cambodian provinces. MATERIALS AND METHODS: An ethnopharmacological survey was carried out from September 2015 to January 2016 in Cambodian urban and rural areas. Thirty-three Khmer traditional healers (KTH) were interviewed using a semi-structured questionnaire including socio-demographic data, healer's formation and their professional practice conditions, perception of liver diseases (types and causes of liver disorders, diagnostic methods and symptoms of liver problems), dietary recommendations given to patients, and herbal remedies used to treat them. For each medicinal plant mentioned in herbal remedies, the local name, part of the plant, mode of preparation and administration, and their properties, according to the healers, were recorded. The plants mentioned by the traditional therapists were collected and later identified by specialists. RESULTS: Different types of liver disease are identified by the healers, and diagnosis was mostly based on reading medical records, and by observing the yellow discoloration of the skin and eyes. A total of 42 herbal remedies including 83 medicinal plants belonging to 40 families were mentioned for treating liver disorders. The most predominant families were Leguminosae and Poaceae. Among the plants reported, Cananga latifolia, Andrographis paniculata, Smilax aff. glabra, Gomphrena celosioides, Passiflora foetida and Physalis minima were the most cited species. A large part of the herbal remedies used were multi-ingredient recipes, and were prepared mainly by a decoction administered orally. Plants are combined in multi-ingredient recipes, and selected on the basis of their properties (trocheak, psah, somrap mé rok, ktchol) which originate from Khmer medical concepts. Most of the plants used by healers have a wide ethnobotanical use for liver disorders, and have been studied for their hepatoprotective activity and related activities on the liver. CONCLUSION: In the diagnosis and treatment of liver diseases, KTH have incorporated biomedical concepts and new practices, which suggest that they could be defined as neotraditional healers. Medicinal plants constitute the core of traditional medicine practice by these healers, and these plants play a very important role in the health care of people with liver problems in Cambodia. Therefore, more attention should be paid to the integration of healers in national health care programs for the development of combined therapies. Furthermore, two plant species (i.e. Cananga latifolia and Willughbeia edulis) were found to be widely used for treating liver disorders in our survey, and should be studied for their pharmacological potential for liver problems.

Natural remedies used by Bunong people in Mondulkiri province (Northeast Cambodia) with special reference to the treatment of 11 most common ailments.

ETHNOPHARMACOLOGICAL RELEVANCE: In this paper we present a comprehensive ethnomedicinal study conducted in Mondulkiri province. Traditional knowledge about natural medicine (plants, animals, mushrooms) was investigated in Cambodia's largest indigenous community: the Bunong people. The survey aims to document the medicinal plant use of this ethnic, by focusing on the eleven most frequent diseases encountered in the area, in order to highlight species that could be recommended in public health programs. MATERIALS AND METHODS: During the years 2013 and 2014, 202 villagers were interviewed in 28 villages from the five districts in Mondulkiri. Two types of methodology were employed: (1) an ethnobotanical field survey (walk-in-the-wood interviews) and (2) semi-structured household interviews with a special emphasis on the treatment of 11 most common ailments encountered in the area. Medicinal plants and mushrooms were collected and identified together with medicinal animals. The factor informant consensus (FIC) and fidelity level (FL) were calculated. RESULTS: Bunong people use a total of 214 plants belonging to 72 families, 1 mushroom and 22 animal species in their traditional healthcare practices in order to treat 51 different ailments. Among the medicinal plants, Fabaceae was the most predominant family; Chromolaena odorata (L.) R.M. King and H.Rob. (Asteraceae), Zingiber montanum (J.Koenig) Link ex A.Dietr. (Zingiberaceae) and Kalanchoe pinnata (Lam.) Pers. (Crassulaceae) were the most cited medicinal plants; and four ailments (cold/fever, diarrhea, postpartum disorders and stomachache) were described as major ailments in the community. The root was the most important part of plants used, and decoction was the most cited method of preparation. During our survey, we also discovered a "new to science" plant species called Ardisia mondulkiriensis Hul and Chassagne, and we recorded for the second time the plant species recently described, Solanum sakhanii Hul. CONCLUSION: Most of the species reported for the treatment of the 11 most frequent ailments have already been proven to be efficient and safe. Furthermore, 10 plant species are reported for the first time as medicinal and some of them are widely used in the community. Further pharmacological and phytochemical investigations should be undergone to assess the pharmaceutical potential of these species. While undergoing considerable changes, Bunong people have maintained extensive traditional medicine knowledge. As this indigenous hill tribe depend mainly on natural remedies for their daily healthcare, environmental preservation is of high importance for the community.

Wayanin and guaijaverin, two active metabolites found in a Psidium acutangulum Mart. ex DC (syn. P. persoonii McVaugh) (Myrtaceae) antimalarial decoction from the Wayana Amerindians.

ETHNOPHARMACOLOGICAL RELEVANCE: Psidium acutangulum Mart. ex DC is a small tree used by the Wayana Amerindians from the Upper-Maroni in French Guiana for the treatment of malaria. AIM OF THE STUDY: In a previous study, we highlighted the in vitro antiplasmodial, antioxidant and anti-inflammatory potential of the traditional decoction of P. acutangulum aerial parts. Our goal was then to investigate on the origin of the biological activity of the traditional remedy, and eventually characterize active constituents. MATERIALS AND METHODS: Liquid-liquid extractions were performed on the decoction, and the antiplasmodial activity evaluated against chloroquine-resistant FcB1 ([(3)H]-hypoxanthine bioassay) and 7G8 (pLDH bioassay) P. falciparum strains, and on a chloroquine sensitive NF54 ([(3)H]-hypoxanthine bioassay) P. falciparum strain. The ethyl acetate fraction (D) was active and underwent bioguided fractionation. All the isolated compounds were tested on P. falciparum FcB1 strain. In vitro anti-inflammatory activity (IL-1ß, IL-6, IL-8, TNFα) of the ethyl acetate fraction and of an anti-Plasmodium active compound, was concurrently assessed on LPS-stimulated human PBMC and NO secretion inhibition was measured on LPS stimulated RAW murine macrophages. Cytotoxicity of the fractions and pure compounds was measured on VERO cells, L6 mammalian cells, PBMCs, and RAW cells. RESULTS: Fractionation of the ethyl acetate soluble fraction (IC50 ranging from 3.4 to <1µg/mL depending on the parasite strain) led to the isolation of six pure compounds: catechin and five glycosylated quercetin derivatives. These compounds have never been isolated from this plant species. Two of these compounds (wayanin and guaijaverin) were found to be moderately active against P. falciparum FcB1 in vitro (IC50 5.5 and 6.9µM respectively). We proposed the name wayanin during public meetings organized in June 2015 in the Upper-Maroni villages, in homage to the medicinal knowledge of the Wayana population. At 50µg/mL, the ethyl acetate fraction (D) significantly inhibited IL-1ß secretion (-46%) and NO production (-21%), as previously observed for the decoction. The effects of D and guiajaverin (4) on the secretion of other cytokines or NO production were not significant. CONCLUSIONS: The confirmed antiplasmodial activity of the ethyl acetate soluble fraction of the decoction and of the isolated compounds support the previous results obtained on the P. acutangulum decoction. The antiplasmodial activity might be due to a mixture of moderately active non-toxic flavonoids. The anti-inflammatory activities were less marked for ethyl acetate fraction (D) than for the decoction.

Sacha Inchi Oil (Plukenetia volubilis L.), effect on adherence of Staphylococus aureus to human skin explant and keratinocytes in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Plukenetia volubilis L. (Euphorbiaceae) is a domesticated vine distributed from the high-altitude Andean rain forest to the lowlands of the Peruvian Amazon. Oil from the cold-pressed seeds, sold under the commercial name of Sacha Inchi Oil (SIO) is actually much in favour because it contains a high percentage of omega 3 and omega 6, and is hence used as a dietary supplement. SIO is also used traditionally for skin care, in order to maintain skin softness, and for the treatment of wounds, insect bites and skin infections, in a tropical context where the skin is frequently damaged. AIMS OF THE STUDY: This study was designed in order to verify whether the traditional use of SIO for skin care would have any impact on Staphylococcus aureus growth and skin adherence, as S. aureus is involved in many skin pathologies (impetigo, folliculitis, furuncles and subcutaneous abscesses) being one if the main pathogens that can be found on the skin. Therefore, our objective was to assess SIO bactericidal activity and interference with adherence to human skin explants and the keratinocyte cell line. Cytotoxicity on that cells was also determined. The activity of SIO was compared to coconut oil (CocO), which is widely used for skin care but has different unsaturated fatty acids contents. MATERIALS AND METHODS: Laboratory testing with certified oil, determined antibacterial activity against radio labelled S. aureus. Cytotoxic effects were measured with XTT on keratinocyte cells and with neutral red on human skin explants; phenol was used as cytotoxic control. Adherence assays were carried out by mixing H3-labelled S. aureus bacteria with keratinocyte cells and human skin explants, incubated with oils 2h before (to determine the inhibition of adherence, assimilated to a preventive effect) or 2h after the contact of the biological material with S. aureus (to assess the detachment of the bacteria, assimilated to a curative effect). Residual radioactivity measured after washings made it possible to determine the adherence intensity. Bactericidal effect was determined by colony counting on trypticase soy agar. RESULTS: Laboratory assays showed that SIO and CocO, tested undiluted, were not cytotoxic on keratinocytes nor human explants and were not bactericidal neither. SIO was more active as antiadherent (preventive) than CocO on keratinocytes. There was no significant difference between detachment effects (curative) of both oils on keratinocytes but SIO was almost 5 times more active on the detachment of S. aureus from human skin explants. CONCLUSION: From that study it can be concluded that the use of SIO on dermal cells is safe and efficient in the inhibition of S. aureus adherence. Our results tend to support the traditional use of undiluted SIO in skin care.

Antiplasmodial and anti-inflammatory effects of an antimalarial remedy from the Wayana Amerindians, French Guiana: takamalaimë (Psidium acutangulum Mart. ex DC., Myrtaceae).

ETHNOPHARMACOLOGICAL RELEVANCE: Field investigations highlighted the use of Psidium acutangulum Mart. ex DC (syn. P. persoonii McVaugh), a small tree used by the Wayana Amerindians in Twenke-Taluhwen and Antecume-Pata, French Guiana, for the treatment of malaria, and administered either orally in the form of a decoction or applied externally over the whole body. This use appears limited to the Wayana cultural group in French Guiana and has never been reported anywhere else. Our goal was to evaluate the antimalarial and anti-inflammatory activities of a P. acutangulum decoction to explain the good reputation of this remedy. MATERIALS AND METHODS: Interviews with the Wayana inhabitants of Twenke-Taluhwen and Antecume-Pata were conducted within the TRAMAZ project according to the TRAMIL methodology, which is based on a quantitative and qualitative analysis of medicinal plant uses. A decoction of dried aerial parts of P. acutangulum was prepared in consistency with the Wayana recipe. In vitro antiplasmodial assays were performed on chloroquine-resistant FcB1 ([(3)H]-hypoxanthine bioassay) and 7G8 (pLDH bioassay) P. falciparum strains and on chloroquine sensitive NF54 ([(3)H]-hypoxanthine bioassay) P. falciparum strain. In vitro anti-inflammatory activity (IL-1ß, IL-6, IL-8, TNFα) was evaluated on LPS-stimulated human PBMC and NO secretion inhibition was measured on LPS stimulated RAW murine macrophages. Cytotoxicity of the decoction was measured on L6 mammalian cells, PBMCs, and RAW cells. A preliminary evaluation of the in vivo antimalarial activity of the decoction, administered orally twice daily, was assessed by the classical four-day suppressive test against P. berghei NK65 in mice. RESULTS: The decoction displayed a good antiplasmodial activity in vitro against the three tested strains, regardless to the bioassay used, with IC50 values of 3.3µg/mL and 10.3µg/mL against P. falciparum FcB1 and NF54, respectively and 19.0µg/mL against P. falciparum 7G8. It also exhibited significant anti-inflammatory activity in vitro in a dose dependent manner. At a concentration of 50µg/mL, the decoction inhibited the secretion of the following pro-inflammatory cytokines: TNFα (-18%), IL-1ß (-58%), IL-6 (-32%), IL-8 (-21%). It also exhibited a mild NO secretion inhibition (-13%) at the same concentration. The decoction was non-cytotoxic against L6 cells (IC50>100µg/mL), RAW cells and PBMC. In vivo, 150µL of the decoction given orally twice a day (equivalent to 350mg/kg/day of dried extract) inhibited 39.7% average parasite growth, with more than 50% of inhibition in three mice over five. The absence of response for the two remaining mice, however, induced a strong standard deviation. CONCLUSIONS: This study highlighted the in vitro antiplasmodial activity of the decoction of P. acutangulum aerial parts, used by Wayana Amerindians from the Upper-Maroni in French Guiana in case of malaria. Its antioxidant and anti-inflammatory potential, which may help to explain its use against this disease, was demonstrated using models of artificially stimulated cells.

Therapeutic switching: from antidermatophytic essential oils to new leishmanicidal products.

This study examined whether the antidermatophytic activity of essential oils (EOs) can be used as an indicator for the discovery of active natural products against Leishmania amazonensis. The aerial parts of seven plants were hydrodistilled. Using broth microdilution techniques, the obtained EOs were tested against three strains of dermatophytes (Trichophyton mentagrophytes, Microsporum gypseum and Microsporum canis). To compare the EOs antifungal and antiparasitic effects, the EOs activities against axenic amastigotes of L. amazonensis were concurrently evaluated. For the most promising EOs, their antileishmanial activities against parasites infecting peritoneal macrophages of BALB/c mice were measured. The most interesting antifungal candidates were the EOs from Cymbopogon citratus, Otacanthus azureus and Protium heptaphyllum, whereas O. azureus, Piper hispidum and P. heptaphyllum EOs exhibited the lowest 50% inhibitory concentration (IC50) values against axenic amastigotes, thus revealing a certain correspondence between both activities. The P. hispidum EO was identified as the most promising product in the results from the infected macrophages model (IC50: 4.7 µg/mL, safety index: 8). The most abundant compounds found in this EO were sesquiterpenes, notably curzerene and furanodiene. Eventually, the evaluation of the antidermatophytic activity of EOs appears to be an efficient method for identifying new potential drugs for the treatment of L. amazonensis.

Therapeutic switching: from antidermatophytic essential oils to new leishmanicidal products

This study examined whether the antidermatophytic activity of essential oils (EOs) can be used as an indicator for the discovery of active natural products against Leishmania amazonensis. The aerial parts of seven plants were hydrodistilled. Using broth microdilution techniques, the obtained EOs were tested against three strains of dermatophytes (Trichophyton mentagrophytes, Microsporum gypseum and Microsporum canis). To compare the EOs antifungal and antiparasitic effects, the EOs activities against axenic amastigotes of L. amazonensis were concurrently evaluated. For the most promising EOs, their antileishmanial activities against parasites infecting peritoneal macrophages of BALB/c mice were measured. The most interesting antifungal candidates were the EOs from Cymbopogon citratus, Otacanthus azureus and Protium heptaphyllum, whereas O. azureus, Piper hispidum and P. heptaphyllum EOs exhibited the lowest 50% inhibitory concentration (IC50) values against axenic amastigotes, thus revealing a certain correspondence between both activities. The P. hispidum EO was identified as the most promising product in the results from the infected macrophages model (IC50: 4.7 µg/mL, safety index: 8). The most abundant compounds found in this EO were sesquiterpenes, notably curzerene and furanodiene. Eventually, the evaluation of the antidermatophytic activity of EOs appears to be an efficient method for identifying new potential drugs for the treatment of L. amazonensis.

Development and validation of liquid chromatography combined with tandem mass spectrometry methods for the quantitation of simalikalactone E in extracts of Quassia amara L. and in mouse blood.

INTRODUCTION: Simalikalactone E (SkE) from Quassia amara, has been proved to be a valuable anti-malarial and anti-cancer compound. As SkE is very scarce, methods of quantitation are needed in order to optimise its isolation process and to determine pharmacokinetic data. OBJECTIVE: To validate methods using liquid chromatography coupled to mass spectrometry for the quantitation of SkE in plant extracts and in biological fluids. METHODS: High- and ultrahigh-performance liquid chromatography (UHPLC) coupled to ion trap mass spectrometry (MS) with single ion monitoring detection and to triple quadrupole-linear ion trap tandem mass spectrometry with multiple reaction monitoring detection methods were developed. Validation procedure was realised according to the International Conference on Harmonisation guideline. Methanol extracts of dried Quassia amara leaves, and mouse-blood samples obtained after various routes of administration, were analysed for SkE. RESULTS: Methods were validated and gave similar results regarding the content of SkE expressed per kilogram of dry leaves in the traditional decoction (160 ± 12 mg/kg) and in the methanol extract (93 ± 2 mg/kg). The recovery of the analyte from mouse blood ranged from 80.7 to 119.8%. Simalikalactone E was only detected using UHPLC-MS/MS (0.2 ± 0.03 mg/L) in mouse blood after intravenous injection: none was detected following intraperitoneal or oral gavage administration of SkE. CONCLUSION: The LC-MS methods were used for the quantitation of SkE in plant extracts and in mouse blood. These methods open the way for further protocol optimisation of SkE extraction and the determination of its pharmacokinetic data.

Antiplasmodial and leishmanicidal activities of 2-cyano-3-(4-phenylpiperazine-1-carboxamido) quinoxaline 1,4-dioxide derivatives.

Malaria and leishmaniasis are two of the World's most important tropical parasitic diseases. Thirteen new 2-cyano-3-(4-phenylpiperazine-1-carboxamido) quinoxaline 1,4-dioxide derivatives (CPCQs) were synthesized and evaluated for their in vitro antimalarial and antileishmanial activity against erythrocytic forms of Plasmodium falciparum and axenic forms of Leishmania infantum. Their toxicity against VERO cells (normal monkey kidney cells) was also assessed. None of the tested compounds was efficient against Plasmodium, but two of them showed good activity against Leishmania. Toxicity on VERO was correlated with leishmanicidal properties.

New findings on Simalikalactone D, an antimalarial compound from Quassia amara L. (Simaroubaceae).

Quassia amara L. (Simaroubaceae) is a species widely used as tonic and is claimed to be an efficient antimalarial all over the Northern part of the Amazon basin. Quassinoid compound Simalikalactone D (SkD) has been shown to be one of the molecules responsible for the antiplasmodial activity of a watery preparation made out of juvenile fresh leaves of this plant. Because of its strong antimalarial activity, we decided to have a further insight of SkD pharmacological properties, alone or in association with classical antimalarials. At concentrations of up to 200µM, we showed herein that SkD did not exert any apoptotic or necrotic activities in vitro on lymphoblastic cells. However, an antiproliferative effect was evident at concentrations higher than 45nM. SkD was inefficient at inhibiting heme biomineralization and the new permeability pathways induced by the parasite in the host erythrocyte membrane. With respect to Plasmodium falciparum erythrocytic stages, SkD was almost inactive on earlier and later parasite stages, but potently active at the 30th h of parasite cycle when DNA replicates in mature trophozoites. In vitro combination studies with conventional antimalarial drugs showed that SkD synergizes with atovaquone (ATO). The activity of ATO on the Plasmodium mitochondrial membrane potential was enhanced by SkD, which on its own had a poor effect on this cellular parameter.