Studies On The Chemical Constituents And Content Determination Of Pteris Semipinnata L.

The Pteris semipinnata L.,belonged to the Pteris L.,is a very ordinary fern plant and broadly distributed in south regions of China.It is pungent and bitter in flavour,warm in nature,and the whole plant has been used in Traditional Chinese Medicine for clearing away heat and toxic material,cooling blood for hemostasis,and rlieving pain.Some of the recent advances in the phytochemical investigations have showed that it’s a prolific source of sesquiterpenoids,diterpenoids,flavonoids,lignans,and organic acids.With the development of modern pharmacology research,some of the ent-kaurane-type diterpenoids exhibited significant antitumor activities in vitro.In order to replenish the chemical constituents data,search new bioactive antitumor compounds,and improve the quality standards,we carried out a phytochemical investigation on the whole plant of it and established a method for the content determination.Furthermore,this study was performed to evaluate the cytotoxicity of the ent-kaurane-type diterpenoids.The MeOH extract of the whole plant of P.semipinnata was chromatographed on silica gel,sephadex LH-20,ODS,macroporous resin,MCI GEL resin,and semi-preparation HPLC to yield 27 compounds.Their strctures were determined on the basis of IR,NMR,HR-MS,and ORD analysis,and the absolute configuration of 1 and 2 were determined by the results of single crystal X-ray diffraction experiment.All compounds were elucidated as(16R)-ent-11β-hydroxy-15-oxokaurane-19-oic acid(1),(16R)-ent-11α-hydroxy-15－oxokaurane-19-oic acid(2),(16R)-nt-7β,11α-dihydroxy-15-oxokaurane-19-oic acid(3),ent-l la-hydroxy-15-oxokaur-16-en-19-oic acid(4),paniculoside Ⅲ(5),ent-7α,11α-dihydroxy-15-oxokaur-16-en-19,6β-olide(6),ent-7α,9-dihydroxy-15-oxokaur-16-en-19,6β-olid(7),apigenin(8),apigenin-7-O-β-D-glucopyranoside(9),apigenin-7-O-β-D-gentiobioside(10),apigenin-7-O-β-D-glucopyranosyl-4’-O-α-L-rhamnopyranoside(11),isoviolanthin(12),luteolin(13),luteolin-7-O-β-D-glucopyranoside(14),luteolin-7-O-β-D-gentiobioside(15),kaempferol-3-O-β-D-glucopyranoside(16),quercetin-3-O-β-D－glucopyranoside(17),rutin hydrate(18),epigallocatechin(19),pinoresinol-4-O-β-D-glucopyranoside(20),phillyrin(21),bergeninum(22),protocatechuate(23),caffeic acid(24),gallic acid(25),β-sitosterol(26),β-daucosterol(27).Compounds 1 and 3 were new hydroxylated ent-kauranoic acids.Compound 2 was a previously known diterpenoid,but only partially NMR data were described.Therefore,the complete structural assignment of the NMR signals and stereochemical structure of 2 were also discussed in this paper.Compounds 10,12,19,20,and 22-25 were isolated from the Pteris L.,and 9,11,15-18,26,and 27 were isolated from this plant for the first time.Ent-kaurane-type diterpenoids 1-6 were evaluated the in vitro cytotoxicity against A549,CT26.WT,and Hep G2 cells via the MTT method with cis-DDP as a positive control.Compound 6 exhibited significant cytotoxicity to CT26.WT and Hep G2 cells with the IC50 values of 13.80 ± 2.85 and 15.6 ± 1.17μM,respectively.Meanwhile,compound 4 only possessed strong cytotoxicity to CT26.WT cells,and the IC50 values was 17.60 ± 3.00 μM.In contrast,no cytotoxicity of the compounds 1-3 and 5 to these cancer cells were observed herein.The IC50 values of the positive control,cis-DDP,to A549,CT26.WT,and Hep G2 cells were 20.00 ±1.60,10.70 ± 0.78,and 21.30 ± 0.66μM..An UPLC-Q-TOF/MS method was developed for the content determination of apigenin-7-O-β-D-gentiobioside,paniculoside III,apigenin-7-O-β-D-glucopyranoside,luteolin,5F,apigenin,and 4F in P.semipinnata.This method was carried out on an ACQUITY UPLC BEH C18 column(1.7μm,100 x 2.1 mm)using a mixture of MeCN-H2O(the H2O containing 0.1%formic acid)and under the following conditions:flow rate 0.4 mL/min and temperature 50 ℃.The mass spectrometer experiments were subjected to analysis by the ESI source,in the negative ion mode,with the following optimized parameters:mass range(m/z)100-1000,declustering potential-130 V,and other parameters were at their default value.The regression equations revealed good linear relationship(r ≥0.998 9)between the peak areas of the compounds and their concentrations.The average recovery values of seven compounds ranged from 98.72 to 101.01%,and the relative standard deviation(RSD)of precision,stability,repeatability,and average recoveries was within 2.01%.In conclusion,the described method revealed to be simple,repaid,reproducible accurate,and sensitive,which suggested that it could be used to the quality control for the P.semipinnata and its preparations.