title: Traditional preparation of Phaleria nisidai, a Palauan tea, reduces exposure to toxic daphnane-type diterpene esters while maintaining immunomodulatory activity.
abstract: BACKGROUND
  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.
  
  
  OBJECTIVE
  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.
  
  
  METHODS
  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).
  
  
  RESULTS
  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).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Biological Sciences, Lehman College and The Graduate Center, City University, United States.
authors.forename: Daniel
authors.initials: D
authors.lastname: Kulakowski
authors.affiliation: Department of Biological Sciences, Lehman College and The Graduate Center, City University, United States; Institute of Economic Botany, The New York Botanical Garden, Bronx, NY 10458, USA.
authors.forename: Christopher
authors.initials: C
authors.lastname: Kitalong
authors.affiliation: Department of Biological Sciences, Lehman College and The Graduate Center, City University, United States.
authors.forename: Adam
authors.initials: A
authors.lastname: Negrin
authors.affiliation: Belau National Museum, Natural History Section, P.O. Box 666, Koror 96940, Palau.
authors.forename: Van-Ray
authors.initials: VR
authors.lastname: Tadao
authors.affiliation: Institute of Economic Botany, The New York Botanical Garden, Bronx, NY 10458, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Balick
authors.affiliation: Department of Biological Sciences, Lehman College and The Graduate Center, City University, United States. Electronic address: edward.kennelly@lehman.cuny.edu.
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Kennelly
mesh_terms: D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004224:Diterpenes; D004952:Esters; D006801:Humans; D007155:Immunologic Factors / Q000009:adverse effects / Q000494:pharmacology; D007371:Interferon-gamma / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism; D000432:Methanol / Q000737:chemistry; D019029:Palau; D018515:Plant Leaves / Q000737:chemistry; D013662:Tea / Q000009:adverse effects; D013729:Terpenes / Q000032:analysis; D029645:Thymelaeaceae; D014867:Water / Q000737:chemistry
chemical_list: D004224:Diterpenes; D004952:Esters; D007155:Immunologic Factors; D013662:Tea; D013729:Terpenes; D014867:Water; C011110:simplexin; C044222:huratoxin; C011309:mezerein; D007371:Interferon-gamma; D000432:Methanol
keywords: Acetoxyhuratoxin (PubChem CID: 44559853); Adaptogen; Daphnane diterpene esters; Huratoxin (PubChem CID: 6375646); Immunomodulation; Micronesia; Panacea; Simplexin (PubChem CID: 119045); Thymelaeaceae; Tonic; UPLC-MS-TQD