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Lignans isolated from the ethyl acetate extract of Knema pachycarpa fruit

Knema is a genus of tropical evergreen trees of the family Myristicaceae found in South East Asian countries such as Vietnam, Thailand, and Malaysia. In this paper, four lignans, (+)-pinoresinol (1),(+) epi-pinoresinol (2), piperitol (3), and pluviatilol (4), were isolated from the ethyl acetate extract of the fruit of Knema pachycarpa, an indigenus tree in Vietnam. The chemical structures were determined by spectroscopic data and comparison with the reported literature. These compounds were isolated from Knema genus for the first time.


INTRODUCTION
Knema is a genus of tropical evergreen trees of the family Myristicaceae found in South East Asian countries such as Vietnam, Thailand, and Malaysia. At least 13 species are found in Vietnam, where they are commonly known as "mau cho" referring to the red resin secreted in the bark [1]. Traditionally, Knema species have been used to treat sore, pimples, cancers and skin diseases. The genus Knema contains variety of natural compounds including cardanols, flavonoids, acetophenones, lignans, acylphloroglucinols, acylresorcinols, and anacardic acids [2][3][4].
Knema pachycarpa de Wilde "Mau cho trai day" is an indigenus tree in Vietnam and the chemical study of this Knema species has not been reported. In this paper, we report the isolation of 4 lignan compounds from the ethyl acetate extract of K. pachycarpa fruit including (+)-pinoresinol (1), epipinoresinol (2), piperitol (3), and pluviatilol (4). Their chemical structures were determined by spectroscopic data and comparison with the reported literature.

General Experimental Procedures
The 1 H-NMR (500 MHz) and 13 C-NMR (125 MHz) spectra were recorded by a Bruker AM500 FT-NMR spectrometer using TMS as an internal standard. The electrospray ionization mass spectra (ESI-MS) were obtained on an Agilent 1260 series single quadrupole LC/MS system. Column chromatography (CC) was performed on silica gel (Merck, 230-400 mesh) or Sephadex LH-20. Thin layer chromatography used precoated silica gel plates (Merck 60 F 254 ). Compounds were visualized by spraying with Ce-Mo stain.

Plant material
The fruit of Knema pachycarpa de Wilde was collected at A-Luoi, Hue city, Viet Nam, in 2015 and identified by Dr. Nguyen The Cuong, Institute of Ecology and Biological Resources, VAST. A voucher specimen (VN-1527) was deposited at the Institute of Marine Biochemistry, VAST.

Extraction and Isolation
The fruits of K. pachycarpa were sliced into small pieces and dried. The material (380 g) was extracted with MeOH at room temperature (3 times, 1 day/time). The extracts were combined and evaporated in vacuo and the residue was suspended in H 2 O. The suspension was successively partitioned with n-hexane and ethyl acetate to give n-hexane residue (105 g) and ethyl acetate residue (2.3 g).

CONCLUSION
A phytochemical investigation of the ethyl acetate extract of the fruit of K. pachycarpa led to the isolation of four lignans including (+)-pinoresinol (1), (+) epi-pinoresinol (2), piperitol (3), and pluviatilol (4). Their chemical structures were elucidated by spectroscopic NMR and MS data. These lignans were isolated from Knema genus for the first time.