Optimized extraction and purification technology of Gekko sulfated glycopeptide based on its anticancer activities

Objective:To optimize the extraction and purification technologies of Gekko sulfated glycopeptide based on the content of glycopeptide,removal ratio of proteins,and anticancer activities.Methods:Different extraction methods,namely,water extraction,ultrasonic extraction,enzymatic hydrolysis-water extraction,and enzymatic hydrolysis-ultrasonic extraction were considered to determine the best extraction method.Single factor and orthogonal experiments were performed to determine the optimum extracting conditions.Sevage,enzymatic hydrolysis-Sevage,trichloroacetic acid(TCA),TCA-Sevage and enzymatic hydrolysis-TCA methods were tested to determine the best deproteinization method.The glycopeptide content and protein removal ratio were analyzed by the phenol-sulfuric acid and Coomassie Brilliant Blue methods.Results:Gekko sulfated glycopeptide obtained by water extraction could inhibit the proliferation of HepG2 and SKBR3,as well as promote that of lymphocytes.The glycopeptide content was 4.049%in the optimal extracting condition of a triple decoction extraction for 1 hour each time with a material to solvent ratio of 1:15.The enzymatic hydrolysis-TCA method was found to be the optimal deproteinization method,with a protein removal ratio of 50.46%,glycopeptide content of 14.27%,and inhibitory ratio on human HepG2 cells of 49.06%.Conclusion:This extraction and purification technique for Gekko sulfated glycopeptide is reasonable,feasible,and provides a scientific basis for industrial production.

Synergetic effects of aqueous extracts of Fuzi(Radix Aconiti Lateralis Preparata) and Tubeimu(Rhizoma Bolbostemmatis) on MDA-MB-231 and SKBR3 cells

OBJECTIVE： To test the synergistic effects of the aqueous extract of Tubeimu （Rhizoma Bolbosternmatis） and Fuzi （Radix Aconiti Lateralis Preparata） on MDA-MB-231 and SKBR3 breast cancer cells.METHODS： A combined index was created for the effects of Tubeimu （Rhizoma Bolbostemmatis） and Fuzi （Radix Aconiti Lateralis Preparata） extracts. Cell proliferation was performed by trypan blue exclusion and 3-（4,5-dimethylthiazol-2-yl）-5-（3- carboxy- methoxyphenyl）-2-（4-sulfophenyl）-2H-tetrazolium （MTS） assays. Flow cytometry was used to assess cell cycle distribution and apoptosis. Cell migration was determined by wound-healing and transwell assays. Confocal microscopy was used to detect E-cadherin and actin filaments. RESULTS： The aqueous extract from Tubeimu （Rhizoma Bolbostemmatis）and Fuzi （Radix Aconiti lateralis Preparata） exerted synergetic effects on the growth of MDA-MB-231 cells and G1 phase arrest. When exposed to extracts at concentrations of 62.5 ：62.5 and 62.5：31.3 μg/mL, the combination index was 0.83 and 0.74, respectively. Interestingly, 62.5： 31.3 μg/mL of combined drugs enhanced the inhibitory effect of Tubeimu （Rhizoma Bolbostemmatis） on the migration of SKBR3 cells and reduced the stimulative effect of Fuzi （Radix Aconiti Lateralis Preparata） （P 〈 0.01）, in which cells showed an increased expression of E-cadherin and reorganization of actin filaments （P 〈 0.001）. 62.5：62.5 μg/mL extract also synergistically induced apoptosis of MDA-MB-231 cells （P 〈 0.05 and P 〈 0.001）. Acting as the main active ingredients in the extract, tubeimoside I and acetylbenzoylaconine at 10：10μg/ mL and 5：2.5 μg/mL also produced inhibitory effects on the proliferation and migration of cells （P 〈 0.01）. CONCLUSION： Tubeimu （Rhizoma Bolbostemmatis） and Fuzi （Radix Aconiti Lateralis Preparata） extracts had synergic effects on MDA-MB-231 and SKBR3 cells.