Gynostemma Pentaphyllum Seed Oil Anti-tumor Active Components Analysis And Biological Function Research

Gynostemma Pentaphyllum(Thunb.)Makino is the traditional Chinese herbal medicine which is the only plant contains ginseng saponin except Panax.It is named as"Southern Ginseng" and a large number of clinical and pharmacological studies have shown that G.Pentaphyllum has treatment or prevention effects on various diseases,at the same time,there are no toxic and side effects in long-term use.In recent years,G.Pentaphyllum has been developed and utilized in food,health products,medicine and other fields as a medicine edible plant.Pingli County is the origin and production region of G.Pentaphyllum in Shaanxi,China.The products of G.Pentaphyllum from Pingli County are very popular around the world.Rhizome transplanting is the main form of G.pentaphyllum cultivation,and the production of seeds was low,therefore,the seeds become by-products in the production process and have not yet been fully utilized or commercially explored.We first found that there are plenty of a-eleostearic acid in G.pentaphyllum seeds in 2012 and it has drawn greater attention in recent years.In order to explore the products of G.Pentaphyllum seed,we have taken some measures including selecting good tetraploid cultivated varieties and changing the ratio of male and female,to enhance the seeds' yield.As a necessary theoretical basis,the main components and biological activity of G.Pentaphyllum seed are the key technical problems to be solved in this developing process.We have found that the vegetable oil content in G.Pentaphyllum seeds is more than 40%and almost comparable to those of rapeseed.Based on this,in this study,we choose the cultivated tetraploid G.pentaphyllum seeds planting in our base as experiment materials.The research contents mainly included the main components,oil extraction technology,food safety toxicology evaluation,antioxidant activities and anti-tumor effects in vivo and in vitro of G.pentaphyllum seed oil(GPSO).The results of the study are as follows:1.The contents of moisture,ash,crude protein,crude fat,crude fiber,reducing sugar,carbohydrate,total saponin and total flavonoid in seed powder were 4.43%,2.83%,20.33%,48.55%,4.98%,0.22%,0.32%,4.55%and 0.36%,respectively.G.pentaphyllum seed is a good source of natural vegetable oil and protein.The seed oil is yellowish green which is a drying oil with clear and bright apprance.The peroxide value of GPSO is a little high so that the stability is not good and prone to get lipid oxidation.The content of unsaturated fatty acid(UFA)is 95.69%including 88.17%conjugated linolenic acid(CLnA).GPSO can be a potential source of conjugated linolenic acids represented by a-eleostearic acid.In addition,there are various useful compounds including squalene,tocopherol and phytosterols in the unsaponifiable matter of GPSO.2.Response surface methodology(RSM)was applied to extraction of GPSO through Supercritical CO2 extraction(SCE)method.The significant parameters including extraction temperature,pressure and time were investigated and the optimal conditions identified from RSM analysis comprised 43?,32 MPa and 160 min,respectively.The experimental extraction yields under these conditions were 35.96%,which showed a good correlation with the value predicted by the RSM model.Compared with conventional screw pressing and solvent extraction,SCE could improve oil quality and tend to extract high content of UFA(95.69%)than conventional methods,while the CLnA contents is about 88.17%.SCE is a recommended method which could maintain the bioactive compounds and has a higher yield than traditional method to extract GPSO.3.According to Procedures and Methods for Toxicolohical Assessment on Food Safety issued by the Ministry of Health in China,the toxicity of GPSO was evaluated through the acute oral toxicity test and two genetic toxicity tests.The results show that GPSO belongs to actually non-toxic grade,so it can be used for the further research of functional food or health care products.4.The antioxidant potential of GPSO in vitro was evaluated according to radical-scavenging activity and ferric-reducing antioxidant power assay.In addition,the antioxidant activity of GPSO in vivo was examined by using an aged mice model.As the results,the overall antioxidant capacity of GPSO in vitro was shown to be comparable to that of Camellia seed oil as a positive control.GPSO could antagonize the aged-related increases in serum alanine aminotransferase,aspartate aminotransferase and triglyceride,respectively.It also elevated the activity of antioxidant enzymes and inhibited the formation of malondialdehyde in mice livers.GPSO has direct and potent antioxidant activities;it could be utilised as a functional food to supplement or replace some conventional oils.5.The cytotoxicity of triglycerides,fatty acids and unsaponification matter from GPSO were investigated by cell viability assay.The results show that triglycerides and unsaponification matter have much weaker cytotoxic effects than fatty acids from GPSO(GPFA),which can significantly inhibite cell viability in a concentration-dependent manner against various cancer cells especially leukemia cells,suggesting its potential benefits as an anticancer agent.6.The cytotoxicity of GPFA was much higher and more selective against some cancer cells such as K562 and U937 than normal cells(PBMCs).GPFA treatment could cause DNA damage and DNA fragment which were detected by nuclear DAPI staining and comet assay.The cell membrane would be injured and physicochemical properties may be changed,including membrane fluidity decline,membrane potential changes,intracellular Ca2+ level variation and loss of mitochondrial membrane:potential.GPFA could promote the yield of radical oxygen species(ROS),which drive cells toward apoptotic or necrotic death.The possible mechanism of apoptosis induced by GPFA might be involved with the increasement of cleaved caspase-3,caspase-9 and PARP,the ratio of Bcl-2/Bax decreased,and Cytochrome C release from mitochondria to cytosol,suggesting GPFA cause cell death through the mitochondria dependent apoptosis pathway.Meanwhile,the release of apoptosis-inducing factor(AIF)is enhanced,implying that GPFA could induce apoptosis through caspase-independent pathway.7.S180 cells and S180 tumor-bearing mice were selected as the research model to investgate the anti-tumor activity of GPSO in vitro and in vivo.The results show that GPFA treatment in vitro could increase intracellular ROS generation and induce lipid peroxidation,then cause cell membrane damage,nuclear condensation and a decline in mitochondrial membrane potential,and initiate the apoptotic response in S180 cells.GPSO administration on S180 tumor-bearing mice could decrease the activity of SOD and promote the formation of MDA in tumor tissue.Meanwhile,cell apoptosis was detected in tumor tissue by TUNEL assay.GPSO has a good anti-tumor activity in S180 tumor-bearing mice and the inhibitory rate for tumor growth reached 69.11%.GPSO represented a great potential in anti-tumor treatments and certainly deserve further investigation.