Study On Isolation, Purification Of Anti-tumor Constituents Derived From Dendrobium Officinale

The article adopted two processes to explore the anti-tumor constituents fromDendrobium officinale：1. The components extracted with three solvents (ethyl acetate,95%ethanol, and water) were separated and purified by silica gel column chromatography, TLCand Sephadex LH-20, and the whole process was guided by MTT method. The Structure ofthe component with strongest anti-tumor activity was identified by spectroscopic data.2.The crude protein prepared by ammonium sulfateprecipitation method was hydrolyzed bydifferent enzymes (Trypsin, Alcalase2.4L, and alkaline protease37017) under their givenconditions respectively. Then these hydrolysates obtained by preciouss steps were purifiedby Sephedax G-25and the whole process was guided by MTT method. Finally wesynthesized three potential active peptides whose sequence was identified byMS techniques.1) Results showed that the strongest active components (TD1and TD2) were achievedby the ethyl acetate extracting, the proliferation of Human hapetocelluar carcinoma cell line(HepG-2), human gastric carcinoma cell line (SGC7901) and human breast cancer cell line(MCF-7) were significantly inhibited by TD1and its hemi–inhibitory concentrations (IC50)were0.45μg/mL、0.36μg/mL、0.34μg/mL, respectively. TD1was identified as denbinobinwhich was first isolated from Dendrobium nobile Lindl and of which anti-tumor effect hasbeen certified. The results have great importance to the development of novel medicine andhealth food using Dendrobium officinale. TD2is a broad-spectrum antitumor compoundwhich can not only inhibited the tumor cells directly but also enhance other medicines’anti-tumor activity, thus worth further studying. However, the proliferation of Huamn livercell line (L-O2) was inhibited by TD1and TD2too, so before the medicine’s developed,their cytotoxicity need to be studied first.2) The A3component which was obained from Alcalase2.4L hydrolyzates has threeadvantages：low-molecular-weight，high peptide level(99.2%) and high inhibition ratio tohuman gastric carcinoma cell line (SGC7901), so it was chose to do the further studys.Studys show that the proliferation of human hapetocelluar carcinoma cell line (HepG-2),human gastric carcinoma cell line (SGC7901) and human breast cancer cell line (MCF-7) were inhibited by A3and its hemi–inhibitory concentrations (IC50) were169.62μg/mL、155.20μg/mL、107.53μg/mL, respectively and A3had no cytotoxicity to Huamn liver cellline (L-O2) at the concentration of1000μg/mL.3) Accroding to the pepides fingerprint, three potential active peptides were chose besynthesized, their sequences were identified as RHPFDGPLLPPGD(C→N),P2KPEEVGGAGDRWTC(C→N) and RCGVNAFLPKSYLVHFGWKLLFHFD(C→N),respectively. The synthetic peptides were identified by HPLC and MS method, results showthat their purity were over98%and their molecular weights are less than0.1%as compared with their theoretical molecular weights.4) The synthetic peptides’ anti-tumor activity were analyzed by MTT assay, resultsshow that they don’t have obvious antitumor activity, which is very different from A3. Thedifference may be caused by a variety of factors, including the change of spatial structure，poor stability and so on, specific situation has yet to be studied further.