mezerein and simplexin

The leaves of Phaleria nisidai Kaneh. (Thymelaeaceae) are brewed into a tea commonly used as a tonic, strengthening beverage and immune enhancer in Palau, Micronesia. Recently, the leaves of P. nisidai have been shown to contain toxic daphnane diterpene esters which may pose a public health threat to Palauans.. This project documents the use frequency, preparation and side effects of P. nisidai. The content of daphnane diterpene esters in aqueous and methanol extracts and infusions prepared by healers in Palau is compared to assess the risk of daphnane ingestion associated with traditional consumption. Quantitative results are correlated with an in vitro assessment of the immunomodulating activity of the extracts.. Research participants, comprising traditional healers and laypeople, were interviewed concerning use patterns and side effects of P. nisidai. Several traditional healers prepared and provided boiled tea samples for chemical analysis. Leaves were collected and methanolic and aqueous extractions were prepared in the laboratory. Peripheral blood mononuclear cells (PBMCs) were cultured with various concentrations of methanol and aqueous leaf extracts and their output of IFNγ was measured using ELISA. Cell proliferation was also assessed using the MTT assay. The concentration of selected daphnane diterpene esters in healer-prepared infusions, lab methanol and lab aqueous extracts was quantified using ultraperformance liquid chromatography-mass spectrometry-triple quadrupole detection (UPLC-MS-TQD).. Through structured interviews it was determined that P. nisidai tea was used frequently, with many participants drinking it daily. The reported side effects were mild, and with the exception of diarrhea (n=2), no side effect was mentioned more than once. Methanol extracts contained 4.0μg simplexin, 17.6μg acetoxyhuratoxin and 2.3μg huratoxin per g dry leaf material. In traditional water infusions provided by healers and in standardized lab-prepared aqueous extracts all three compounds were below the limit of detection (16.3ng/mL) using our UPLC-MS-TQD method. Methanol and aqueous extracts increased the release of IFNγ by PBMCs (p<0.05); however, methanol extracts were significantly more active than aqueous extracts (p<0.05). Methanol and aqueous extracts significantly increased proliferation of PBMCs, causing at least 60% more cell proliferation than negative control (p<0.05).. The presence of daphnane diterpene esters in a frequently consumed traditional beverage was initially viewed as a public health concern, though interview data reveal that Palauans do not observe toxicity or side effects associated with their use of P. nisidai tea. Concurrently, daphnanes are present in methanolic extracts but not detected in aqueous preparations indicating that the traditional method of preparation avoids the extraction of these potentially toxic compounds, while still maintaining immunostimulant activity.

Topics: Cell Survival; Cells, Cultured; Diterpenes; Esters; Humans; Immunologic Factors; Interferon-gamma; Leukocytes, Mononuclear; Methanol; Palau; Plant Leaves; Tea; Terpenes; Thymelaeaceae; Water

This study presents a strategy based on repeatable reversed-phase LC-TOF-MS methods and chemometric statistical tools, including untargeted PCA and supervised OPLS-DA models, to identify low-yielding compounds with potent immunostimulant activity in Phaleria nisidai (Thymelaeaceae), a plant with a history of use as an adaptogen on the islands of Palau in Micronesia. IFNγ ELISA assays were used to classify chromatographic fractions according to imunomodulatory activity prior to LC-TOF-MS chemometric analysis to target and identify compounds likely to contribute to observed activity. Simplexin, a daphnane diterpene ester, was identified for the first time from this genus and caused an increase in the production of cytokines (IFNγ, IL1β, IL6, and IL13) by peripheral blood mononuclear cells. Five other daphnane diterpene esters were tentatively identified for the first time from this plant based on mass spectral data and are marker metabolites distinguishing active from inactive fractions. This analytical approach increased the efficiency of bioactivity-guided fractionation and has the potential to minimize redundant isolation and identify minor constituents with potent activity from a complex matrix.

Topics: Chromatography, Reverse-Phase; Cytokines; Diterpenes; Esters; Humans; Immunologic Factors; Leukocytes, Mononuclear; Mass Spectrometry; Plant Extracts; Terpenes; Thymelaeaceae

Cattle grazing in arid rangelands of Australia suffer periodic extensive and serious poisoning by the plant species Pimelea trichostachya, P. simplex, and P. elongata. Pimelea poisoning (also known as St. George disease and Marree disease) has been attributed to the presence of the diterpenoid orthoester simplexin in these species. However, literature relating to previous studies is complicated by taxonomic revisions, and the presence of simplexin has not previously been verified in all currently recognized taxa capable of inducing pimelea poisoning syndrome, with no previous chemical studies of P. trichostachya (as currently classified) or P. simplex subsp. continua. We report here the isolation of simplexin from P. trichostachya and the development of a liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method to measure simplexin concentrations in pimelea plant material. Simplexin was quantified by positive-ion atmospheric pressure chemical ionization (APCI) LC-MS/MS with selected reaction monitoring (SRM) of the m/z 533.3 > 253.3 transition. LC-MS/MS analysis of the four poisonous taxa P. trichostachya, P. elongata, P. simplex subsp. continua, and P. simplex subsp. simplex showed similar profiles with simplexin as the major diterpenoid ester component in all four taxa accompanied by varying amounts of related orthoesters. Similar analyses of P. decora, P. haematostachya, and P. microcephala also demonstrated the presence of simplexin in these species but at far lower concentrations, consistent with the limited reports of stock poisoning associated with these species. The less common, shrubby species P. penicillaris contained simplexin at up to 55 mg/kg dry weight and would be expected to cause poisoning if animals consumed sufficient plant material.

Topics: Animals; Australia; Cattle; Cattle Diseases; Chromatography, Liquid; Diterpenes; Plant Extracts; Plant Poisoning; Tandem Mass Spectrometry; Terpenes; Thymelaeaceae