Ethnomedicinal, Phytochemical and Pharmacological Investigations of Tetradenia riparia (Hochst.) Codd (Lamiaceae).

Tetradenia riparia Hochsteter codd. (Lamiaceae) in its native African continent, is considered one of the most popular aromatic medicinal plants. In folk medicine it may be used as an infusion to treat respiratory problems, cough, headache, stomach pain, diarrhea, fever, malaria, and dengue; and in the form of compresses it is applied for the relief of headaches and toothaches. The species T. riparia has been researched for decades to isolate and identify chemical constituents present in extracts or essential oil obtained from the leaves, floral buds, or stems of this plant. The present study reviews the scientific literature on ethnomedicinal, phytochemical, and pharmacological aspects of T. riparia. We discuss issues related to the botanical and geographical description of the species, ethnobotanical uses, phytochemical studies on its essential oil and extracts, and biological activities of T. riparia. Several compounds have already been isolated from leaves, such as ibozol, 7α-hydroxyroileanone, 1',2'-dideacetylboronolide, 8(14),15-sandaracopimaradiene-7α,18-diol; 5,6-dihydro-α-pyrone and α-pyrone. Terpenes predominated in the essential oil, comprising monoterpenes, sesquiterpenes, diterpenes, hydrocarbons, and oxygenates. Most phytocompounds were isolated from the leaves and flower buds, namely fenchone, 14-hydroxy-9-epi (E)-caryophyllene, 9ß, 13ß-epoxy-7-abietene, and 6,7-dehydroroileanone. These compounds provide the species a high pharmacological potential, with antimicrobial, antioxidant, antitumor, analgesic, anti-leishmania, anti-tuberculosis, and anti-parasitic activities. Therefore, this species is a promising herbal medicine.

Bioassay-guided isolation of antibacterial compounds from the leaves of Tetradenia riparia with potential bactericidal effects on food-borne pathogens.

ETHNOPHARMACOLOGICAL RELEVANCE: Tetradenia riparia (commonly known as ginger bush) is frequently used in traditional African medicine to treat foodborne infections including diarrhoea, gastroenteritis, and stomach ache. AIM OF THE STUDY: The present study aims to identify in Tetradenia riparia the compounds active against foodborne pathogens. MATERIALS AND METHODS: Dried Tetradenia riparia leaf powder was consecutively extracted with hexane, ethyl acetate, methanol and water. The hexane extract was counter-extracted with methanol:water (9:1), and after evaporation of the methanol, this phase was extracted with dichloromethane. The water extract was counter-extracted with butanol. All these fractions were tested against a panel of foodborne bacterial pathogens. A bioassay-guided purification was performed to isolate antimicrobial compounds using Staphylococcus aureus as a target organism. Further, antibiofilm activity was evaluated on S. aureus USA 300. RESULTS: The dichloromethane fraction and ethyl acetate extract were the most potent, and therefore subjected to silica gel chromatography. From the dichloromethane fraction, one active compound was crystalized and identified using NMR as 8(14),15-sandaracopimaradiene-7alpha, 18-diol (compound 1). Two active compounds were isolated from the ethyl acetate extract: deacetylumuravumbolide (compound 2) and umuravumbolide (compound 3). Using a microdilution method, their antimicrobial activity was tested against eight foodborne bacterial pathogens: Shigella sonnei, S. flexneri, Salmonella enterica subsp. enterica, Escherichia coli, Micrococcus luteus, S. aureus, Enterococcus faecalis, and Listeria innocua. Compound 1 had the strongest activity (IC50 ranging from 11.2 to 212.5 µg/mL), and compounds 2 and 3 showed moderate activity (IC50 from 212.9 to 637.7 µg/mL and from 176.1 to 521.4 µg/mL, respectively). Interestingly, 8(14),15-sandaracopimaradiene-7alpha, 18-diol is bactericidal, and also showed good antibiofilm activity with BIC50 (8.8 ± 1.5 µg/mL) slightly lower than for planktonic cells (11.4 ± 2.8 µg/mL). CONCLUSIONS: These results support the traditional use of this plant to conserve foodstuffs and to treat gastrointestinal ailments, and open perspectives for its use in the prevention and treatment of foodborne diseases.

Biofilm inhibiting properties of compounds from the leaves of Warburgia ugandensis Sprague subsp ugandensis against Candida and staphylococcal biofilms.

ETHNOPHARMACOLOGICAL RELEVANCE: Warburgia ugandensis Sprague subspecies ugandensis is a plant widely distributed in Eastern, Central and Southern Africa. In humans, it is used to treat respiratory infections, tooth aches, malaria, skin infections, venereal diseases, diarrhea, fevers and aches. AIM OF THE STUDY: This study aims to identify the bioactive compounds against clinically important biofilm-forming strains of Candida and staphylococci that are responsible for tissue and implanted device-related infections. METHODS: Using a bioassay-guided fractionation approach, hexane -, ethanol -, acetone - and water extracts from the leaves of W. ugandensis, their subsequent fractions and isolated compounds were tested against both developing and preformed 24 h-biofilms of Candida albicans SC5314, Candida glabrata BG2, Candida glabrata ATCC 2001, Staphylococcus epidermidis 1457 and Staphylococcus aureus USA 300 using microtiter susceptibility tests. Planktonic cells were also tested in parallel for comparison purposes. Confocal scanning laser microscopy was also used to visualize effects of isolated compounds on biofilm formation. RESULTS: Warburganal, polygodial and alpha-linolenic acid (ALA) were the major bioactive compounds isolated from the acetone extract of W. ugandensis. For both warburganal and polygodial, the biofilm inhibitory concentration that inhibits 50% of C. albicans developing biofilms (BIC50) was 4.5 ±â€¯1 and 10.8 ±â€¯5 µg/mL respectively. Against S. aureus developing biofilms, this value was 37.9 ±â€¯8 µg/mL and 25 µg/mL with warburganal and ALA respectively. Eradication of preformed 24 h biofilms was also observed. Interestingly, synergy between the sesquiterpenoids and azoles against developing C. albicans biofilms resulted in an approximately ten-fold decrease of the effective concentration required to completely inhibit growth of the biofilms by individual compounds. The hydroxyl group in position C-9 in warburganal was identified as essential for activity against staphylococcal biofilms. We also identified additional promising bioactive sesquiterpenoids; drimenol and drimendiol from the structure-activity relationship (SAR) studies. CONCLUSIONS: ALA and four sesquiterpenoids: polygodial, warburganal, drimenol and drimendiol, have shown biofilm-inhibitory activity that has not been reported before and is worth following up. These compounds are potential drug candidates to manage biofilm-based infections, possibly in combination with azoles.

Isolation of Antimicrobial Compounds From Cnestis ferruginea Vahl ex. DC (Connaraceae) Leaves Through Bioassay-Guided Fractionation.

Different parts of Cnestis ferruginea are used in traditional African medicine for treating infectious diseases such as dysentery, bronchitis, eye troubles, conjunctivitis, sinusitis, gonorrhea, and syphilis. Despite its long traditional use in the treatment of infections, this plant is not well studied for its in vitro antimicrobial properties. Therefore, the present study aims to establish the antimicrobial activity profile of extracts from this plant, as well as to isolate and evaluate the antimicrobial activity of the most abundant bioactive compound in C. ferruginea leaves through bioassay-guided purification, using Staphylococcus aureus as a target organism. Although both methanol and water extracts of the plant leaves proved active against S. aureus, a water extract was pursued, and subjected further to liquid-liquid partitioning (ethyl acetate, butanol, and water). The ethyl acetate fraction was found to be the most potent and was subjected to silica gel chromatography. In total, 250 fractions were obtained, and those with similar TLC profiles were clustered into 22 major groups, of which pooled fraction-F6 (83 mg) was the most potent. Additional purification by HPLC resulted in two active peaks, which were identified, using a combination of NMR and mass spectrometry, as hydroquinone and caffeic acid methyl ester. Their antimicrobial activity was confirmed using a microdilution protocol on S. aureus, where hydroquinone had a stronger activity (MIC50 = 63 µg/mL) compared to caffeic acid methyl ester (>200 µg/mL). Traditionally this plant is used as an aqueous preparation to treat many infections, and the present study also demonstrated antimicrobial activity in the aqueous extract, which appears due mainly to two major water-soluble compounds isolated through bioassay-guided purification. This supports the clinical use of the aqueous extract of C. ferruginea leaves as a phytotherapeutic for bacterial infections.

Active principles of Tetradenia riparia. IV. Anthelmintic activity of 8(14),15-sandaracopimaradiene-7α,18-diol.

ETHNOPHARMACOLOGICAL RELEVANCE: Tetradenia (T.) riparia (Hochst.) Codd (Lamiaceae), formerly known as Iboza riparia (Hochst.) N.E.Br., is one of the most frequently used medicinal plants in traditional Rwandese medicine. It was used as a remedy against a wide range of diseases including malaria, angina, yaws, dental abscesses, headache, worm infections and several kinds of fevers and aches. AIM OF THE STUDY: This study aims to identify the compounds active against helminths from Tetradenia riparia. METHODS: A bioassay-guided isolation of anthelmintic compounds from the leaves of Tetradenia riparia was performed using a Caenorhabditis elegans (C. elegans) testing model. RESULTS: The bioassay-guided isolation led to one active compound, i.e. 8(14),15-sandaracopimaradiene-7α,18-diol. Its IC50 value was 5.4 ± 0.9 µg/mL (17.8 ± 2.9 µM). CONCLUSIONS: We identified the bioactive compound from Tetradenia riparia responsible for its anthelmintic activity: 8(14),15-sandaracopimaradiene-7α,18-diol. Although the compound and several of its bioactivities have been described before, this is the first report of its anthelmintic effect.

Investigation of signalling cascades induced by neurotrophic synaptolepis factor K7 reveals a critical role for novel PKCε.

This study elucidates signalling cascades involved in the neurotrophic effects induced by an active compound of Synaptolepis kirkii, a plant that is used against snakebites and for treatment of epilepsy. The active compound of this plant, synaptolepis factor K7 (K7), is suggested to exert anti-tumoral and neurotrophic actions via modulation of PKC. In SH-SY5Y cells synthesis of the neuronal marker growth-associated protein 43 was increased upon 48h treatment with K7. Immunofluorescent staining of neurites revealed an increased neurite formation by synaptolepis factor K7. Short-term signal transduction events were followed at the level of extracellular-regulated kinase phosphorylation. Extracellular-regulated kinase (ERK) phosphorylation was transiently increased upon stimulation with synaptolepis factor K7 (300nM) with a maximal effect at 30min. Use of the general PKC inhibitor bisindolylmaleimide I blocked the K7-induced ERK phosphorylation suggesting involvement of PKC. Conversely, inhibition of conventional PKCs, α, ß and γ by treatment with Go6976 did not inhibit ERK phosphorylation up to 1µM. Use of a specific-PKCε translocation inhibitor peptide or RNAi-mediated knockdown of PKC-epsilon (ε) abolished the K7-induced ERK phosphorylation implicating PKCε in K7 function. This was confirmed by the observed increase in PKCε translocation and autophosphorylation induced by the compound. These data show that synaptolepis factor K7 induces neuronal differentiation of SH-SY5Y cells concomitant with a transient increase in ERK phosphorylation that is mediated by activation of PKCε.

Selective antileishmania activity of 13,28-epoxy-oleanane and related triterpene saponins from the plant families Myrsinaceae, Primulaceae, Aceraceae and Icacinaceae.

Maesa saponins with the 13,28-epoxy-oleanane triterpene core skeleton were described recently to possess strong and selective in vitro and in vivo antileishmania activity. In the absence of direct chemical derivatization possibilities, a structure-based literature search was carried out to explore a structure-activity relationship. Crude alcohol extracts from several plant species of Myrsinaceae, Primulaceae, Aceraceae and Icacinaceae were evaluated for in vitro activity against Leishmania infantum intracellular amastigotes and cytotoxicity on MRC-5(SV2) cells, while the saponin content was evaluated qualitatively by TLC. A clear correlation was found between the presence of close analogue 13,28-epoxy-oleanane triterpene saponins and potent and selective antileishmania activity. This was most striking in Maesa species, except for M. macrosepala. Interesting activities were also found in extracts that did not exactly match the TLC characteristics of the Maesa saponin references, as was the case for Ardisia angusta, A. amherstiana, A. caudata, A. gigantifolia, A. roseiflora, Myrsine affinis, Acer brevipes and A. laurinum var. petelotii. This study indicates that the 13,28-epoxy-oleanane triterpene moiety is essential for selective antileishmania potential and that several other plant species could still be explored for antileishmania drug discovery.

LC-MS analysis of 13,28-epoxy-oleanane saponins in Maesa spp. extracts with antileishmanial activity.

INTRODUCTION: Saponins are natural products that are well known for a wide range of biological activities. For saponins of Maesa balansae, selective antileishmanial activity has been described. OBJECTIVE: In view of their pharmacological interest, several Maesa species from the National Botanical Garden of Meise (Belgium) and wild-grown plants from Vietnam were screened for their antileishmanial potential and saponin content. METHODOLOGY: Different parts of the plants (mainly leaves and twigs) were collected, dried and extracted. Plant extracts were evaluated by liquid chromatography/mass spectrometry (LC-MS) using electrospray ionisation in the negative ion mode and their saponin content was compared with those of Maesa balansae (maesabalides) and Maesa lanceolata (maesasaponins). RESULTS: Several Maesa species (M. ambigua, M. argentea, M. brevipaniculata, M. japonica and M. perlarius) showed potent antileishmanial activity (<0.1 microg/mL) and indeed contained known maesasaponins and maesabalides. However the leaves of M. argentea also revealed two new compounds. Two saponins with [M - H]- ions at m/z 1465 and 1477 were characterised. Their mass spectrometric fragmentation pattern revealed a structure that was the same or closely related to maesasaponin V.3 and VI.2, respectively, but had a glycan part with one additional hexose residue. CONCLUSION: Several known as well as new saponins from Maesa species active against leishmaniasis were characterised using LC-MS.

Coumarins from Cnidium monnieri and their antiosteoporotic activity.

Bioactivity-guided fractionation has led to the successful isolation of antiosteoporotic components, i. e., osthole, imperatorin and bergapten from an ethanolic extract of the fruits of Cnidium monnieri (L.) Cusson. Among them, osthole was determined as the major compound possessing antiosteoporotic activity. Further study showed that osthole not only promoted the proliferation and activity of alkaline phosphatase of osteoblasts in neonatal calvaria cultures, but also inhibited the bone resorption by decreasing the formation, differentiation and TRAP activity of osteoclasts derived from rat marrow cells.

In vitro and in vivo anti-leishmanial activity of triterpenoid saponins isolated from Maesa balansae and some chemical derivatives.

The methanolic extract from the leaves of the Vietnamese medicinal plant Maesa balansae showed potent in vitro and in vivo activity against the tropical protozoal parasite Leishmania infantum. Bioassay-guided purification of the extract led to the identification of six triterpenoid saponins, maesabalides I-VI (1-6), each having a strong and specific anti-leishmanial activity. Maesabalide III (3) and IV (4) were the most potent with IC(50) values against intracellular amastigotes of about 7 and 14 ng/mL. In comparison, the IC(50) value of sodium stibogluconate, the reference drug for treatment of leishmaniasis, is only 5.6 microg/mL. No cytotoxicity was present on a human fibroblast (MRC-5) cell line (CC(50) > 32 microg/mL). In vivo evaluation in the BALB/C mouse model demonstrated that >90% reduction of liver amastigote burdens was obtained 1 week after a single subcutaneous dose at 0.2-0.4 mg/kg was administered. Several chemical derivatives of maesabalides I-VI were prepared in order to study the structure-activity relationship.

Use of medicinal plants and pharmaceuticals by indigenous communities in the Bolivian Andes and Amazon.

OBJECTIVE: To investigate, by means of household surveys, the use of medicinal plants and pharmaceuticals in Apillapampa, a large Andean community of Quechua peasants, and in six small communities of Yuracaré-Trinitario "slash-and-burn" cultivators of the National Park Isiboro-Secure (the NPIS) in the Bolivian Amazon. METHODS: A total of 12% of households in Apillapampa and nearly all households in the NPIS were interviewed about their use of medicinal plants and pharmaceuticals for treating illnesses. Informants were also asked to name any medicinal plants they knew. FINDINGS: In spite of the presence of a primary health care service (PHC) with medical doctor in Apillapampa, an equal number of informants used medicinal plants and pharmaceuticals. In the NPIS, the prevalent use of medicinal plants or pharmaceuticals in any community depended on the distance of the community from the nearest village and from a PHC with medical doctor (r = 0.85 and r = -0.96; both P = 0.05. The NPIS communities' knowledge of plants expressed as the average number of medicinal plants mentioned correlated positively and negatively with distance from the nearest village and use of pharmaceuticals, respectively (r= 0.95, P < 0.005 and r = -0.90, P < 0.05, respectively). CONCLUSION: The cultural importance of traditional medicine and the physical isolation of communities, both in general and from PHCs, are factors that influence the use of and knowledge about medicinal plants.

A comparison of traditional healers' medicinal plant knowledge in the Bolivian Andes and Amazon.

Medicinal plant knowledge of two groups of traditional healers was thoroughly studied during a 2-year ethnobotanical survey in the Bolivian Andes (Quechua farmers from Apillapampa) and Amazon rainforest (Yuracaré-Trinitario slash-and-burn cultivators from Isiboro-Sécure National Park), respectively. Both areas represent ecologically and culturally diverse zones, differing in floristic diversity, physical accessibility to health care and degree of modernization, the latter evidenced by presence or intensity in use of modern services such as electricity, water distribution, and materials for house construction. It is generally believed that indigenous people have an impressive knowledge of useful plant species and that this knowledge reflects the plant wealth of their living environment. However, the present study shows that healers' knowledge of collected medicinal plants (expressed as percentage of plants known by name and use by the majority of healers) is higher in the Andean area characterised by a long history of anthropogenic activity, than in the biodiversity-rich rainforest (protected since 1965). Therefore, medicinal plant knowledge does not seem to depend on the level of plant diversity, degree of modernization or absence of Western health care infrastructure. Indeed, although Andean healers live in a floristically poorer environment, have adopted more modern services and have easier access to primary health care facilities, they are more knowledgeable about medicinal plants than rainforest healers who live isolated in an environment with considerable floristic/ecological variation and lack of Western health care. It is hypothesised that social factors underlying traditional medical practices (background of extensive family in traditional medicine) play an important role in transmission--and hence survival of knowledge on medicinal plants.

Comparative activities of the triterpene saponin maesabalide III and liposomal amphotericin B (AmBisome) against Leishmania donovani in hamsters.

Maesabalide III (MB-III), an oleane triterpene saponin isolated from the Vietnamese plant Maesa balansae, is a new antileishmanial lead compound whose activity against Leishmania donovani (MHOM/ET/67/L82) in groups of five golden hamsters was evaluated after administration of a single subcutaneous dose on either day 1 (prophylactic treatment) or day 28 (curative treatment) after infection. Liposomal amphotericin B (AmBisome), administered intravenously at 5 mg/kg of body weight, was used as the reference drug. Amastigote burdens in liver, spleen, and bone marrow were determined either 7 days (early effects) or 56 days (late effects) after treatment. Prophylactic administration of MB-III at 0.2 mg/kg reduced liver amastigote burdens by 99.8 and 83% within 7 and 56 days after treatment, respectively. In the latter group, however, all animals became ill and some died. Both MB-III at 0.8 mg/kg and liposomal amphotericin B were 100% effective against liver stages, but clearance from the spleen and bone marrow was not achieved. Curative administration of MB-III at 0.2 and 0.4 mg/kg was not protective, as no survivors were left at the termination of the experiment on day 84. Despite the high level of reduction of the liver amastigote burden after treatment with MB-III at 0.8 mg/kg (94.2%) or liposomal amphotericin B (99.4%), clinical protection could not be obtained in either group, with two deaths occurring and the residual liver burdens persisting. It is concluded that administration of a single dose of MB-III at 0.8 mg/kg has efficacy potential comparable to that of a single dose of liposomal amphotericin B at 5 mg/kg and is therefore considered a promising new antileishmanial lead compound. However, multiple-dose pharmacological, toxicological, and pharmacokinetic studies are still needed before it can become a valid drug candidate for development.

In vitro and in vivo activities of a triterpenoid saponin extract (PX-6518) from the plant Maesa balansae against visceral leishmania species.

The in vitro and in vivo activities of a mixture of six oleane triterpene saponins, recovered from the methanolic extract of the leaves of the Vietnamese plant Maesa balansae (PX-6518), were evaluated against drug-sensitive visceral Leishmania strains. The in vitro 50% inhibitory concentration (IC(50)) against intracellular Leishmania infantum amastigotes was 0.04 micro g/ml. The cytotoxic concentrations causing 50% cell death (CC(50)s) were about 1 micro g/ml in murine macrophage host cells and >32 micro g/ml in human fibroblasts (MRC-5 cell line). Evaluation in the Leishmania donovani BALB/c mouse model indicated that a single subcutaneous administration of 0.4 mg/kg at 1 day after infection reduced liver amastigote burdens by about 95% in all treated animals. If treatment was delayed until 14 days after infection, a dose of 1.6 mg/kg of body weight was required to maintain the same level of activity. Single 250-mg/kg doses of sodium stibogluconate (Pentostam) 1 and 14 days after infection produced comparable efficacies. A single dose of PX-6518 at 2.5 mg/kg administered 5 days before infection was still 100% effective in preventing liver infection, suggesting a particularly long residual action. Spleen and bone marrow could not be cleared by PX-6518 nor sodium stibogluconate. PX-6518 did not show activity after oral dosing at up to 200 mg/kg for 5 days. This study concludes that triterpenoid saponins from M. balansae show promising in vitro and in vivo antileishmanial potential and can be considered as new lead structures in the search for novel antileishmanial drugs.

Liquid chromatographic method for analysis of saponins in Maesa balansae extract active against leishmaniasis.

A liquid chromatographic method was developed for the separation of six related triterpenoid saponins in Maesa balansae extracts with different purity, active against leishmaniasis. As stationary phase a Hypersil BDS C18 column (3 microm), 100 x 4.6 mm was used. The mobile phase was a mixture of methanol, acetonitrile, 5% (m/v) ammonium acetate, pH 6.5 and water. A linear gradient was developed for the analysis of crude extracts. An isocratic method was developed to analyze purified samples that mainly contained saponins 3 and 4, the most active saponins. The isocratic LC method was optimized and the robustness was evaluated with an experimental design. The method showed good selectivity, repeatability, linearity and sensitivity.

Antitrichomonas in vitro activity of Cussonia holstii Engl.

Cussonia holstii is a traditional medicinal plant used in Kenya, which has not been studied chemically up-to-date. Upon biological screening, a substantial antitrichomonas activity was found in the dichloromethane extract of the bark of Cussonia holstii. A detailed chromatographic study using centrifugal partition chromatography revealed that a pentacyclic triterpenoid, i.e., hederagenin, is the antitrichomonas constituent of the plant with a IC50 of 2.8 microM.

New pyranonaphthoquinone and pyranonaphthohydroquinone from the roots of Pentas longiflora.

Several quinone type compounds were isolated from the hexane, dichloromethane, and ethyl acetate extracts of the roots of Pentas longiflora. The hexane extract afforded two new compounds, [(3alpha,3'alpha,4beta,4'beta)-3,3']-dimethoxy-cis-[4,4'-bis(3,4,5,10-tetrahydro-1H-naphtho[2,3-c]pyran)]-5,5',10,10'-tetraone (1) and cis-3,4-dihydroxy-3,4-dihydromollugin (2), together with six known compounds, namely, pentalongin, mollugin, trans-3,4-dihydroxy-3,4-dihydromollugin, methyl-2,3-epoxy-3-prenyl-1,4-naphthoquinone-2-carboxylate, tectoquinone, and 3-hydroxymollugin. From the dichloromethane extract were isolated the three known compounds 3-methoxymollugin, methyl-3-prenyl-1,4-naphthoquinone-2-carboxylate, and scopoletin, while the ethyl acetate extract afforded the known 2-methoxy-3-methylanthraquinone.