Separation, Purification And Component Analysis Of Antitumor Active Components In Seabuckthorn Leaf-bound Polyphenols

Colon cancer is one of the most common malignant tumors in the world.According to the latest data in 2018,the incidence of colon cancer ranks third in the world,and the mortality rate ranks second.The traditional method of treating colon cancer is mainly surgery combined with postoperative chemotherapy.Its recurrence,toxicity and side effects are the biggest causes of death for cancer patients.Natural products have significant effects on the treatment of tumors due to their non-toxicity and side effects.Therefore,natural products extracted from plants,including polyphenols,resveratrol,artemisinin,etc.,have become the focus of attention.Hippophae rhamnoides,also known as vinegar,is a perennial deciduous shrub or small tree of the genus Hippophae,which is mainly distributed in a large area of Eurasia,and is mainly distributed in North China,Northwest China,and Southwest China.Hippophae rhamnoides is a very precious medicinal and homologous plant.It was first listed in the Pharmacopoeia of the People’s Republic of China in 1977."Dictionary of Traditional Chinese Medicine" records that sea buckthorn fruit has the effects of promoting blood circulation and dissipating blood stasis,nourishing the spleen and stomach,replenishing and quenching thirst,clearing heat and reducing diarrhea.Studies have shown that fruits,leaves and seeds of sea buckthorn are rich in nutrients and biologically active ingredients,such as multivitamins,amino acids,unsaturated fatty acids,flavonoids,trace elements and serotonin.In particular,the polyphenol compounds rich in sea buckthorn and sea buckthorn leaves have anti-oxidant,anti-inflammatory,antibacterial,and anti-tumor medical functions,and have a good preventive effect on a variety of chronic diseases.Plant-derived polyphenols are substances that are abundant in nature.They are very easy to obtain,and even more rare are non-toxic and harmless.The biological activity of tumors has the potential to be developed into tumor drugs.Hippophae rhamnoides leaves are rich in polyphenols.There have been many studies on the extraction and characterization of flavonoids in Hippophae rhamnoides leaves.However,there is no systematic report on the composition of phenolic acids,especially bound phenolic acids,in seabuckthorn leaves.Therefore,this study Taking sea-buckthorn leaves-bound polyphenols as research objects,the anti-tumor activity and molecular mechanism of sea-buckthorn leaves-bound polyphenols are explained from the perspective of plant polyphenols regulating tumor glucose metabolism,providing a theoretical basis for their use as drugs for treating colon cancer.The research contents and results mainly include the following aspects:(1)Extraction and identification of free-state polyphenols from sea buckthorn leaves.Different solvents were used for high-efficiency extraction of free-state polyphenols.In order to obtain the best solvent for the extraction of free-state polyphenols from sea buckthorn leaves,80 % methanol,80 % ethanol,80 % acetone,and 80 % ethyl acetate were used in the experiment.Extraction of free-state polyphenols from seabuckthorn leaves with four different organic solvents and five different solvents;using Folin-Ciocalteu colorimetric method to determine the content of free-state polyphenols,the results showed that the methanol extraction efficiency was the highest;using MTT method,respectively The effects of the components extracted from the above different solvents on the proliferation of tumor cells HCT-116 were examined,and their antitumor effects were compared.The methanol extracts were screened for the best antitumor effects.(2)Extraction and identification of bound polyphenols from sea buckthorn leaves.The extraction of bound polyphenols from seabuckthorn leaves uses two hydrolysis methods,acid hydrolysis and alkaline hydrolysis,to extract bound polyphenols with high efficiency.The free-state polyphenols were extracted from the residue of seabuckthorn leaves from the residue after extracting free polyphenols from methanol.The Folin-Ciocalteu colorimetric method was used to determine the content of free-state polyphenols.The acid-and alkaline-hydrolysis methods were compared for the combined state Extraction effect of phenol.As a result,it was found that the alkaline hydrolysis method had better effect on the extraction of bound polyphenols from sea buckthorn leaves.The effects of the components extracted by the two hydrolysis methods on the proliferation of HCT-116 tumor cells were tested by MTT method,and their antitumor effect activities were compared.The best antitumor effect of the alkaline hydrolysis extract was screened.(3)Isolation and purification of seabuckthorn leaves bound polyphenols with macroporous resin;separation and identification of the purified seabuckthorn leaves bound polyphenols by high performance liquid chromatography;determination of seabuckthorn leaves bound polyphenols A range of active ingredients that exert antitumor effects among phenol ingredients.The MTT method was used to separately detect the effects of the above purified and isolated components on the proliferation of tumor cell HCT-116,and determine a scheme for obtaining active components with antitumor effects.(4)Elucidate the molecular mechanism of Hippophae rhamnoides leaves bound polyphenols by regulating P alternative splicing to reverse the Warburg effect.Adding different concentrations of Hippophae rhamnoides leaves bound polyphenols to treatment,the kit was used to detect the effects of Hippophae rhamnoides leaves bound polyphenols on the pyruvate consumption,lactic acid production and ATP production of cells.Western blot was used to detect the sea-buckthorn leaf-bound polyphenol-treated cells and regulate the expression of the splicing factors hn RNPA1,hn RNPA2,and PTB in P alternative splicing.And protein P1 and P2 expression levels.In summary,Hippophae rhamnoides leaf-bound polyphenols can play a role by reversing the Warburg effect to inhibit the proliferation of colon cancer cells.Hippophae rhamnoides leaf-bound polyphenols can affect the expression of PKM by affecting the PKM’s shear factor,selective shearing,and To exert its ability to inhibit colon cancer;therefore,the active polyphenols with antitumor effects were isolated from sea buckthorn leaves,making full use of local wild sea buckthorn resources,and clarifying its role in antitumor effects from the perspective of energy metabolism remodeling.The molecular mechanism provides its theoretical basis for its in-depth development and anti-tumor drugs.