Chemical Fractionation Joint to In-Mixture NMR Analysis for Avoiding the Hepatotoxicity of Teucrium chamaedrys L. subsp. chamaedrys.

Dietary supplements based on Teucrium chamaedrys L. subsp. chamaedrys aerial parts were banned, due to the hepatotoxicity of furan-containing neo-clerodane constituents. Indeed, the plant leaf content in phenolic compounds could be further exploited for their antioxidant capability. Accordingly, bio-guided fractionation strategies have been applied, obtaining seven partially purified extracts. These latter were chemically investigated through 1D and 2D NMR techniques and tested for their antiradical, reducing and cytotoxic capability. Data acquired highlighted that, through a simple phytochemical approach, a progressive neo-clerodane depletion occurred, while maximizing phenylethanoid glycosides in alcoholic fractions. Thus, although the plant cannot be used as a botanical remedy as such, it is suggested as a source of healthy compounds, pure or in mixture, to be handled in pharmaceutical, nutraceutical and/or cosmeceutical sectors.

Hepatotoxicity in Cattle Associated with Salvia reflexa Diterpenes, including 7-Hydroxyrhyacophiline, a New Seco-Clerodane Diterpene.

A case of baled alfalfa hay contaminated with multiple weeds induced hepatotoxicity and death in cattle. The hepatotoxic compounds were isolated by bioassay-guided fractionation using a mouse model and identified as salviarin, salvianduline D, rhyacophiline, and 7-hydroxyrhyacophiline. The structure of 7-hydroxyrhyacophiline has not been previously reported. All compounds were found to induce severe acute hepatic necrosis within 24-48 h after a single oral dosage (260-280 mg/kg). The identified diterpenes are known to be found among different Salvia species which led to finding dried plant parts of Salvia reflexa within bales of weedy hay and subsequently a population of S. reflexa was found along the field edges and irrigation ditch banks of the alfalfa hay field. It was thus determined that S. reflexa was responsible for the hepatotoxicity observed in cattle fed the contaminated hay.

Toxicological findings about an anticancer fraction with casearins described by traditional and alternative techniques as support to the Brazilian Unified Health System (SUS).

ETHNOPHARMACOLOGICAL RELEVANCE: Extracts, essential oils and molecules from Casearia sylvestris have popularly shown pharmacological actions against chronic diseases, as anxiety, inflammation, cancer and dyslipidemia. In the context of antitumoral therapy, we investigated in vitro, ex vivo and in vivo toxicological changes induced by a Fraction with Casearins (FC) and its component Casearin X isolated from C. sylvestris on animal and vegetal cells, and upon invertebrates and mammals. MATERIAL AND METHODS: Cytotoxicity was carried out using normal lines and absorbance and flow cytometry techniques, Artemia salina nauplii, Danio rerio embryos and meristematic cells from Allium cepa roots. Acute and 30 days-mice analysis were done by behavioral, hematological and histological investigations and DNA/chromosomal damages detected by alkaline Cometa and micronucleus assays. RESULTS: FC was cytotoxic against lung and fibroblasts cells and caused DNA breaks, loss of integrity and mitochondrial depolarization on ex vivo human leukocytes. It revealed 24 h-LC50 values of 48.8 and 36.7 µg/mL on A. salina nauplii and D. rerio embryos, reduced mitotic index of A. cepa roots, leading to cell cycle arrest at metaphase and anaphase and micronuclei. FC showed i.p. and oral LD50 values of 80.9 and 267.1 mg/kg body weight. Subacute i.p. injections induced loss of weight, swelling of hepatocytes and tubules, tubular and glomerular hemorrhage, microvesicular steatosis, lung inflammatory infiltration, augment of GPT, decrease of albumin, alkaline phosphatase, glucose, erythrocytes, and lymphocytes, and neutrophilia (p > 0.05). FC-treated animals at 10 mg/kg/day i.p. caused micronuclei in bone marrow and DNA strand breaks in peripheral leukocytes. CONCLUSIONS: This research postulated suggestive side effects after use of FC-related drugs, demonstrating FC as antiproliferative and genotoxic on mammal and meristematic cells, including human leukocytes, teratogenicity upon zebrafish embryos, myelosuppression, clastogenicity, and morphological and biochemical markers indicating liver as main target for FC-induced systemic toxicity.

Clerodane furanoditerpenoids as the probable cause of toxic hepatitis induced by Tinospora crispa.

Tinospora crispa is a popular traditional herbal plant commonly used throughout the world for treatment of various diseases, in particular type 2 diabetes mellitus. We report here a new case of toxic hepatitis in a 57-year old male patient in the French West Indies following the consumption of two aqueous extracts of fresh Tinospora crispa stems. It thus differs from two previously reported cases that concerned the chronic intake of powdered dry stems delivered in solid oral dosage forms (i.e. pellets and tablets). Liquid Chromatography-Diode Array Detection-Mass Spectrometry (LC/DAD/MS) analyses were performed on an aqueous extract of the offending sample that mimics the swallowed preparation. They revealed the presence of species-specific molecular marker borapetoside C (1) and thus enabled an unambiguous phytochemical identification. The exploration of tandem MS/MS data obtained by ultra-high performance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-QTOF-HRMS) allowed the identification of 17 additional cis-clerodane-type furanoditerpenoid lactones, analogues of 1. These results support the hypothesis that the mechanisms underlying hepatotoxicity of Tinospora crispa are the same as those encountered with furanoditerpenoids-containing plants such as Teucrium chamaedrys or Dioscorea bulbifera. In the context of type 2 diabetes treatment, we recommend that Tinospora crispa intake should be more closely monitored for signs of hepatotoxicity.

Bioassay-guided isolation of active principles from Nigerian medicinal plants identifies new trypanocides with low toxicity and no cross-resistance to diamidines and arsenicals.

ETHNOPHARMACOLOGICAL RELEVANCE: Leaves from the plant species studied herein are traditionally used in northern Nigeria against various protozoan infections. However, none of these herbal preparations have been standardized, nor have their toxicity to mammalian cells been investigated. In search of improved and non-toxic active antiprotozoal principles that are not cross-resistant with current anti-parasitics, we here report the results of the in vitro screening of extracts from seven selected medicinal plant species (Centrosema pubescens, Moringa oleifera, Tridax procumbens, Polyalthia longifolia, Newbouldia laevis, Eucalyptus maculate, Jathropha tanjorensis), used traditionally to treat kinetoplastid infections in Nigeria, and the isolation of their bioactive principles. AIM OF THE STUDY: To investigate the efficacies of medicinal plant extracts, and of compounds isolated therefrom, against kinetoplastid parasites, assess cross-resistance to existing chemotherapy, and assay their toxicity against mammalian cells in vitro. MATERIAL AND METHODS: Plants were extracted with hexane, ethyl acetate and methanol. Active principles were isolated by bioassay-led fractionation, testing for trypanocidal activity, and identified using NMR and mass spectrometry. EC50 values for their activity against wild-type and multi-drug resistant Trypanosoma brucei were obtained using the viability indicator dye resazurin. RESULTS: Seven medicinal plants were evaluated for activity against selected kinetoplastid parasites. The result shows that crude extracts and isolated active compounds from Polyalthia longifolia and Eucalyptus maculata, in particular, display promising activity against drug-sensitive and multi-drug resistant Trypanosoma brucei. The EC50 value of a clerodane (16α-hydroxy-cleroda-3,13(14)-Z-dien-15,16-olide) isolated from Polyalthia longifolia was as low as 0.38µg/mL, while a triterpenoid (3ß,13ß-dihydroxy-urs-11-en-28-oic acid) isolated from Eucalyptus maculata displayed an EC50 of 1.58µg/mL. None of the isolated compounds displayed toxicity towards Human Embryonic Kidney cells at concentrations up to 400µg/mL. In addition, the isolated compounds were active against Leishmania mexicana, as well as against T. congolense. CONCLUSION: We have isolated a clerodane compound from Polyalthia longifolia that shows low toxicity, no cross-resistance with current treatments, and promising activity against both human-infective and veterinary Trypanosoma species.

Salvia divinorum: toxicological aspects and analysis in human biological specimens.

The identification and quantitation of the main psychoactive component of Salvia divinorum (salvinorin A) in biological specimens are crucial in forensic and clinical toxicology. Despite all the efforts made, its uncontrolled abuse has increased quickly, exposing its users' health to serious risks both in the short and long term. The use of alternative biological matrices in toxicological analyzes can be advantageous as complementary postmortem samples, or in situations when neither blood nor urine can be collected; they may be useful tools in those determinations, providing important information about prior exposure. The aim of this article is to present a brief summary of legal aspects of Salvia divinorum and salvinorin A, including the methods used for the determination of the latter in biological matrices.

Cytotoxic Effects of Salvinorin A, A Major Constituent of Salvia divinorum.

S. divinorum is a psychoactive plant that has been consumed as a recreational drug of abuse in the last years. Salvinorin A is its main constituent, and is responsible for the observed psychoactive effects. Both S. divinorum and salvinorin A have become controlled drugs in several countries, but they are not listed in the Schedules of the United Nations Drug Conventions. Regarding the effects of S. divinorum consumption, almost all studies are based on in vivo or on surveys, and there are no studies in vitro on its toxicity. Furthermore, all studies are focused on the acute toxicological effects of the plant. So, it is of utmost importance to further investigate the effects of S. divinorum and salvinorin A, particularly using in vitro models, after prolonged exposures. In this context, the present work evaluated the in vitro toxicity induced by S. divinorum or salvinorin A in six cell lines, through MTT assays and LC50 determination. Overall, results showed that both S. divinorum and salvinorin A are cytotoxic, dose- and time-dependent. Also, Hep G2 and Caco 2 (to a lesser extent) cells showed lower sensitivity to S. divinorum and salvinorin A when compared to the other studied cell lines. To our knowledge, this is the first work focused on the in vitro toxicity of S. divinorum and salvinorin A using a variety of cell lines, which are extensively described in literature and have been widely used in several in vitro studies.

Gastroprotective effect of epoxy clerodane diterpene isolated from Tinospora cordifolia Miers (Guduchi) on indomethacin-induced gastric ulcer in rats.

The present study evaluated the gastroprotective effect of epoxy clerodane diterpene (ECD), isolated from Tinospora cordifolia on indomethacin-induced gastric ulcer in rats. Administration of indomethacin exhibits extreme levels of ulcer index (UI) and myeloperoxidase (MPO) activity. Indomethacin down regulated PGE2, anti-inflammatory cytokines (IL-4, IL-10) and pro-angiogenic factors (VEGF and EGF). The ECD pretreatment considerably increased the levels of PGE2, anti-inflammatory cytokines and pro-angiogenic factors. The ulcer-healing activity of ECD was inhibited by pre-administration of the specific COX-1 inhibitor (SC560) and nonspecific NOS inhibitor (L-NAME), which indicates the involvement of PGE2 and NOS in ECD induced ulcer healing activity. These findings suggest that ECD exerts its antiulcer activity by reinforcement of defensive elements and diminishing the offensive elements.

cis-clerodane diterpenes from the liverwort Scapania ciliata.

Chemical investigation of the Chinese liverwort Scapania ciliata led to the isolation of four new cis-clerodane lactones, named ciliatolides A-D (1-4, resp.), among which compound 1 was found to be a tetranorclerodanoid. Their structures were determined by extensive analysis of spectroscopic data, and, in the case of compound 1, together with a single-crystal X-ray diffraction analysis. The absolute configurations were established by analysis of the CD spectra and by quantum-chemical CD calculations. The cytotoxicities of compounds 1-4 were preliminarily tested against the PC3 and MCF-7 cell lines.

Learning and memory impairment induced by salvinorin A, the principal ingredient of Salvia divinorum, in wistar rats.

The effects of salvinorin A (Salvia divinorum principal ingredient), a potent κ-opioid natural hallucinogen, on learning and memory were investigated. Wistar rats were tested in the 8-arm radial maze, for object recognition and passive avoidance tasks for spatial, episodic, and aversive memory. Attention was assessed using a latent inhibition task. Salvinorin A (80-640 µg/kg subcutaneous [sc]) did not affect short-term memory, but it impaired spatial long-term memory. Episodic and aversive memories were impaired by salvinorin A (160-640 µg/kg). Memory impairment was blocked by the selective κ-opioid receptor antagonist, nor-binaltorphimine ([nor-B]; 0.5-1 mg/kg, intraperitoneal [ip]). Salvinorin A (160 µg/kg) disrupted latent inhibition, after LiCl treatment, such as reduced sucrose intake, suggesting an attention would result in an impairment of cognitive behavior. These findings demonstrate for the first time that salvinorin A has deleterious effects on learning and memory, through a κ-opioid receptor mechanism.

Structure elucidation and hepatotoxicity evaluation against HepG2 human cells of neo-clerodane diterpenes from Teucrium polium L.

Seven neo-clerodanes (teupolins VI-XII) and eleven known compounds were isolated and characterized from leaf extracts of Teucrium polium L., a medicinal plant used in traditional and herbal medicine for its hypolipidemic, hypoglycemic, antioxidant and antiproliferative properties. The structures of these compounds were elucidated by 1D (1H, 13C and DEPT) and 2D (COSY, TOCSY, HSQC, HMBC) NMR experiments and by mass spectrometry analysis. The complete stereostructure of each compound was defined with a NOESY experiment. Because the overexploitation of herbal remedies containing T. polium extracts has resulted in several cases of hepatitis, the hepatotoxic activity of pure metabolites against the human hepatoblastoma cancer cell line HepG2 was assessed by the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) test. All of the compounds showed low toxicity values at the highest concentration tested (200 µM).

Ultrapotent effects of salvinorin A, a hallucinogenic compound from Salvia divinorum, on LPS-stimulated murine macrophages and its anti-inflammatory action in vivo.

The hallucinogenic compound, salvinorin A, is a potent κ-opioid receptor (KOR) agonist. However, other target(s) than the KOR, such as the cannabinoid CB1 receptor, have been proposed to explain its multiple pharmacological actions. Here, we have evaluated the effect of salvinorin A in lipopolysaccharide (LPS)-stimulated macrophages as well as in models of inflammation in vivo. Salvinorin A (0.1-10 pM) reduced LPS-stimulated nitrite, TNF-α and IL-10 (but not IL-1ß) levels as well as iNOS (but not COX-2) LPS-induced hyperexpression. The effect of salvinorin A on nitrite levels was reverted by the opioid antagonist naloxone, the KOR antagonist nor-binaltorphimine and by the CB1 antagonist rimonabant Salvinorin A also prevented KOR and CB1 hyperexpression induced by LPS. In vivo, salvinorin A reduced the LPS- and the carrageenan-induced paw oedema and formalin-induced inflammatory pain, in a nor-binaltorphimine and rimonabant-sensitive manner. It is concluded that salvinorin A-via KORs and CB1 receptors-exerts ultrapotent actions on macrophages and also shows moderate antinflammatory effects in vivo.

Vitamin E ameliorates high dose trans-dehydrocrotonin-associated hepatic damage in mice.

trans-Dehydrocrotonin (t-DCTN), the diterpenoid from Croton cajucara Bentham, exhibits hypoglycemic and hypolipidemic activities, but in high doses is associated with a discrete hepatotoxicity. In the search for measures to mitigate this, pretreatment with the antioxidants N-acetylcysteine and vitamin E has been examined. Mice that received a high dose t-DCTN (100 mg/kg) manifested hepatic damage, as evidenced by significant elevations in serum ALT and AST, and hepatic GSH, and histological alterations, which could be obliterated by pretreatment with vitamin E, but not with N-acetylcysteine, possibly by creating an effective antioxidant balance.

Casearin X, its degradation product and other clerodane diterpenes from leaves of Casearia sylvestris: evaluation of cytotoxicity against normal and tumor human cells.

An EtOH extract of the leaves of Casearia sylvestris afforded new clerodane diterpene, casearin X, together with the known compounds casearins B, D, L, and O, and caseargrewiin F. Casearin X degraded to the corresponding dialdehyde when stored in CDCl(3). The diterpenes isolated were cytotoxic to human cancer cell lines, with caseargrewiin F being the most active and the new clerodane, casearin X, the second active compound with IC(50) values comparable to the positive control doxorubicin. All isolated diterpenes showed lower activities against normal human cells than against cancer cell lines, which might indicate a possible selective action on cancer cells. Casearin X dialdehyde was not cytotoxic to cancer cells indicating that the occurrence of these CO groups at C(18) and C(19) is incompatible with the cytotoxic activity.

Two novel neo-clerodane diterpenoids from Scutellaria barbata.

Two novel neo-clerodane diterpenoids, barbatellarines A (1) and B (2), were isolated from the whole plants of Scutellaria barbata, along with the known compound scutebarbatine F (3). The chemical structures and relative stereochemistry of the isolated compounds were established by NMR (1D and 2D) and mass spectroscopic analyses. Compounds 2 and 3 were evaluated for in vitro cytotoxic activity against the HL-60 (human leukemia), MCF7 (human breast cancer), and LLC (Lewis lung carcinoma) cancer cell lines. Compound 2 exhibited weak cytotoxic activity against HL-60 cells, with an IC(50) value of 41.4microM.

Antimicrobial evaluation of clerodane diterpenes from Polyalthia longifolia var. pendula.

Phytochemical investigation of the ethanolic extract of leaves of Polyalthia longifolia var. pendula has led to the isolation of seven clerodane diterpenoids and five alkaloids. (-)-14, 15-bisnor-3, 11E-kolavadien-13-one (1), (-)-16-oxocleroda-3,13(14)E-dien-15-oic acid (2), (-)-16alpha-hydroxycleroda-3,13 (14)Z-dien-15,16-olide (3), (+)-(4-->2)-abeo-16(R/S)-2, 13Z-kolavadien-15, 16-olide-3-al (4), (-)-3beta, 16beta-dihydroxycleroda-4(18), 13(14)Z-dien-15,16-olide (5), (-)-3, 12E-kolavadien-15-oic acid-16-al (6), (-)-labd-13E-en-8-ol-15-oic acid (7), liriodenine (8), (-)-anonaine (9), (+)-isoboldine (10), (-)-asimilobine (11) and hordenine (12) have been isolated. This is the first report of 1, 6 and 10 from this plant species while 12 is reported for first time from this genus. Clerodane derivatives 1-7 were evaluated for their antimicrobial activity. Diterpene 3 was found to be most potent agent with MIC value of 6.25 microg/mL against Staphylococcus aureus and Sporothrix schenckii.

neo-Clerodane diterpenoids from Scutellaria barbata with cytotoxic activities.

Three neo-clerodane diterpenoids, named barbatins A-C (1-3), and the neo-clerodane diterpenoid nicotinyl ester, named scutebarbatine B (4), were isolated from the whole plant of Scutellaria barbata D. Don. Their structures were elucidated by spectroscopic analyses (UV, IR, HRFAB-MS, 1D NMR and 2D NMR). In vitro, compounds 1-4 showed significant cytotoxic activities against three human cancer lines, namely, HONE-1 nasopharyngeal, KB oral epidermoid carcinoma, and HT29 colorectal carcinoma cells, with IC50 values in the range 3.5-8.1 microM.

Antiulcer activity and subacute toxicity of trans-dehydrocrotonin from Croton cajucara.

The antiulcerogenic activity of trans-dehydrocrotonin (DHC), a nor-clerodane diterpene isolated from Croton cajucara Benth. (Euphorbiaceae), and its subacute (35 days) toxicity were studied in mice and rats, respectively. For the antiulcerogenic tests, models of gastric ulcers induced in mice by ethanol/HCl or stress were used. In both models, an oral dose of DHC (100 mg/kg) significantly reduced (P < 0.01) the formation of gastric lesions. DHC was also tested for its ability to scavenge free radicals, but no such action was observed in rat liver mitochondria. To assess the subacute toxicity, rats were treated orally with DHC (25, 50 and 100 mg/kg) for 5 weeks. A significant increase in liver weight was observed in male and female rats at highest doses, whereas a significant reduction in plasma alkaline phosphatase and cholesterol levels and an increase in gamma glutamyl transpeptidase were observed only at the highest dose (100 mg/kg) in female rats. DHC caused histopathological alterations in the liver that included a turbid tumefaction, microvacuolar degeneration and nuclear alterations. Despite the beneficial antiulcerogenic activity of DHC, our results suggest that the long-term use of this compound may induce liver damage.