Two new sesquiterpenes from Chloranthus japonicus Sieb

Abstract Two new sesquiterpenes, namely, 1β,10β-dihydroxy-eremophil-7(11), 8-dien-12,8-olide (1) and 8,12-epoxy-1β-hydroxyeudesm-3,7,11-trien-9-one (2), together with three known sesquiterpenoids, shizukolidol (3), 4α-hydroxy-5α(H)-8β-methoxy-eudesm-7(11)-en-12,8-olide (4), and neolitacumone B (5), and two known monoterpenes, (3R,4S,6R)-p-menth-1-en-3,6-diol (6) and (R)-p-menth-1-en-4,7-diol (7), were isolated from the whole plant of Chloranthus japonicus Sieb. Their structures were elucidated on the basis of spectroscopic data analysis and comparison with those of related known compounds. Compounds 4–7 were isolated from this plant for the first time.

In view of compounds isolated from title species showed various bioactivities, all compounds 1-7 were evaluated in vitro for Brine shrimp (Artemia salina) larvae toxicities. unfortunately, they did not exhibit toxic activities towards brine shrimps (LC 50 > 200 ppm).

Plant material
The whole plant of C. japonicus was collected from Taibai mountain of Qinling area, Shaanxi Province, P.r. China, in July 2013 and authenticated by dr Zhou, Y. F. of Institute of Botany of Shaanxi. A voucher specimen (No. 03054) has been deposited in the Plants Herbarium of Institute of Botany of Shaanxi in China.

Brine shrimp lethality assay
The brine shrimp (Artemia salina) lethality assay was considered as useful tool for preliminary assessment of toxicities of compounds or extracts (Li et al. 2012;Tanamatayarat et al. 2012). In this work, compounds 1-7 were tested for the toxic activity towards brine shrimp larvae. Brine shrimp eggs (ocean Star International, Inc., uSA) were hatched in a large beaker containing artificial seawater (2.5%, pH 8.0-8.5) and they were cultured at 28 °C for 48 h. Compounds 1-7 were dissolved in dimethyl sulfoxide (dMSo) at the concentration of 2 mg/ mL, and then transferred 10, 8, 6, 4, 2, 1, 0.5 μL to 96 well plate, respectively, diluted with artificial seawater (15-20 nuclei larvae) to final volume of 200 μL, dMSo as a blank control and podophyllotoxin as a positive control, each concentration had three repetitions. After 24 h incubation, the number of dead shrimp in each well was counted under microscope.
The mortality rate was calculated using the formula: where M = per cent of the dead larvae after 24 h; A = number of the dead larvae after 24 h; B = average number of the dead larvae in the blank control after 24 h; N = number of the dead larvae before starting the test; G = number of selected larvae for test. In addition, the LC 50 values were calculated.

Supplementary material
Supplementary material relating to this article is available online, alongside Table S1 and Figures S1-S17.