Comprehensive Evaluation Of Hippophae Fructus Quality And Study On The Modification Of Its Active Ingredient,Isorhamnoin

Objective:China has a long history of medicinal use of Hippophae Fructus and is also one of the earliest countries in the world to record the medicinal value of Hippophae Fructus.However,the origin of Hippophae Fructus medicinal materials is complex,and the current Chinese Pharmacopoeia only stipulates one type.In reality,many regions use local materials,making it difficult to provide clear evidence and quality control.To clarify the application history of Hippophae Fructus varieties,the application context of Hippophae Fructus recorded throughout history was sorted out.Establish a systematic evaluation method for Hippophae Fructus harvesting time and optimizing steaming time,and determine the optimal harvesting and steaming time.By detecting exogenous pollution and its risk assessment,an evaluation basis is provided for the production of high-quality medicinal materials with controllable quality.Hippophae Fructus has the functions of invigorating the spleen and promoting digestion,relieving cough and phlegm,promoting blood circulation and dispersing stasis.Modern pharmacological research has shown that Hippophae Fructus also has anticancer activity.Hippophae Fructus is closely related to its own active substances in the treatment of diseases.Isorhamnetin is a type of flavonoid compound isolated and purified from medicinal plants such as Hippophae Fructus.Isorhamnetin has strong antioxidant effects and can clear oxygen free radicals to repair cell and tissue damage caused by aging.It has biological activities such as antiviral,anti-inflammatory,immune regulation,anti allergic,anti-tumor,and cardiovascular function protection,and has broad application prospects.However,due to its almost insolubility in water and polar organic solvents,the bioavailability of isorhamnoin is also limited.A novel zwitterionic polymer drug of Isorhamnetin was designed using chemical modification methods.The unique structure and shape of this nanomedicine can effectively kill tumor cells with low toxicity,good solubility,and high bioavailability.Methods:（1）By reviewing historical and modern literature records of Hippophae Fructus throughout history,a systematic review was conducted on the evolution process of Hippophae Fructus recorded applications,and the research progress on the origin of Hippophae Fructus applications and the quality of modern varieties was verified.（2）Establish a systematic dynamic accumulation evaluation method for the active ingredients of Hippophae Fructus based on its harvesting time and drying method,and determine the optimal harvesting time and drying method.Firstly,the main components were screened through a high-resolution mass spectrometry database,and their distribution patterns were studied by classifying and determining flavonoids,fatty oils,water-soluble components,etc.Using liquid chromatography-mass spectrometry to determine flavonoid components in Hippophae Fructus,pre column derivatization gas chromatography to determine fatty acid components in Hippophae Fructus,and HPLC-ELSD fingerprint and quality marker to determine water-soluble components and their distribution patterns in Hippophae Fructus.（3）By examining the different harvesting periods,drying processes,and production areas of Hippophae Fructus,we conducted health risk assessments on exogenous pollutants such as fungal toxins,pesticide residues,and heavy metals.Fungal toxins were detected in Hippophae Fructus using high-performance liquid chromatography tandem mass spectrometry with positive and negative ion detection modes,and evaluated in conjunction with the antibacterial efficacy test of Hippophae Fructus.Pesticide residues were detected by establishing 248 methods for detecting pesticide residues in Hippophae Fructus,using liquid chromatography-mass spectrometry to detect 135 pesticide residues,and gas chromatography-mass spectrometry to detect 113 pesticide residues.Inductively coupled plasma mass spectrometry was used to determine 15 heavy metals,providing an evaluation basis for producing high-quality medicinal materials with controllable quality.（4）Constructing a cancer treatment association network with Hippophae Fructus through network pharmacology,collecting effective ingredients and their targets in Hippophae Fructus through TCMSP and Unipro databases,finding intersection targets through Venny2.1,collecting and screening disease targets through Gene Cards and OMIM databases,conducting PPI network analysis through STRING v11.0 database,and confirming protein and small molecule structures through PDB and Pub Chem,By constructing a network diagram using Cytoscape（Version 3.7.1）software,we identified the potential mechanism of Hippophae Fructus in treating cancer,which is the action of multiple components such as resveratrol on key targets for cancer treatment.Due to the poor solubility and almost insolubility in water and polar organic solvents,the excellent anti-tumor activity of resveratrol compounds is greatly limited.Response surface optimization method was used to extract and separate resveratrol from Hippophae Fructus fruit.Amphoteric polymers were synthesized by self-assembly of isothiocyanate and polymer compound poly（carboxybetaine metacrylate）.Different strong acids HCl,H₂SO₄,H₃PO₄,CH₃SO₃H,and CF₃SO₃H were selected to adjust the p H value.The spectral properties of the zwitterionic polymer in organic solvents were measured,and the dynamic self-assembly changes of the polymer drug were determined by scanning electron microscopy.The morphology of different acid binding was detected one by one using transmission electron microscopy.Activity evaluation was conducted on in vitro cells,and in terms of biological activity,the synthesized compound was used as a positive control.The cytotoxicity of the compound on human colon cancer cell line（HCT-116）,human cervical cancer cell line（He La）,and normal liver cell line（L-02）was determined using MTT method.Through experiments,a highly targeted nano drug of Isorhamnetin was screened.Results:（1）The research mainly found that before the Ming Dynasty,although there were widespread records of the use of Hippophae Fructus,it was difficult to determine which species of Hippophae Fructus was used through description.It was not until the Uyghur"Yihetilati Baidiyi"first recorded the plant morphology of Hippophae Fructus and determined that its origin was Chinese Hippophae Fructus.In the Qing Dynasty,the Tibetan medical literature"Golden Classic Annotation"divided the Hippophae Fructus into two kinds for the first time,and the"Jingzhu Materia Medica"of the same period was divided into three kinds in more detail,and detailed morphology.The basic judgment basis was Gyantse Hippophae Fructus,costal fruit Hippophae Fructus,and Xizang Hippophae Fructus.In Mongolian medical literature,it is recorded as Chinese Hippophae Fructus.At the same time,it is corrected that in the"Compendium of Materia Medica",the concept of vinegar forest is Hippophae Fructus,and the character of vinegar forest in the"Compendium of Materia Medica"is described as large folic acid vine.The application of Hippophae Fructus in this herb is mainly concentrated in the Tibetan,Mongolian,and Uyghur ethnic groups,and the basis used includes various plants of the Hippophae Fructus genus.However,the2020 edition of the Chinese Pharmacopoeia stipulates that the original Hippophae Fructus medicinal material is only one species of Chinese Hippophae Fructus,which contradicts the varieties used in the Tibetan medical system.Through combing the current drug standards of Hippophae Fructus,it is found that the application of varieties is chaotic.The local standards are based on Wolong Hippophae Fructus,Gyantse Hippophae Fructus,Xizang Hippophae Fructus,Yunnan Hippophae Fructus,costal Hippophae Fructus,and Chinese Hippophae Fructus.The existing control items in the current standards have weak specificity,and some standards lack some necessary control items,most of which are general inspection items such as leachables,moisture ash content,acid insoluble ash content,etc.Identification using thin-layer chromatography with Isorhamnetin,quercetin,and kaempferol as reference substances,with total flavonoids,Isorhamnetin,quercetin,and kaempferol content control indicators.There were no control substances with strong specificity as identification items,and common flavonoid reference substances were used for comparative identification,resulting in ineffective differentiation from the original.There are 50 kinds of traditional Chinese patent medicines and simple preparations with Hippophae Fructus ingredients as medicine,among which 8 kinds of Hippophae Fructus are identified by flavonoids,accounting for 16%.There are 4 varieties,accounting for 8%,with vitamins as the control indicator for content determination;There are three varieties,accounting for 6%,with total flavonoids as the control indicator for content determination;There are 4 varieties,accounting for 8%,whose content is determined using Isorhamnetin as the control indicator.There are few quality control items for Hippophae Fructus in the current traditional Chinese patent medicines and simple preparations standard.Further research is needed to determine whether the material basis,safety,and clinical efficacy of different species of Hippophae Fructus are equivalent,and to conduct comparative studies on closely related species to determine whether their pharmacological effects are equivalent.（2）Through the measurement results and cluster analysis of different parts of Hippophae Fructus,it can be seen that the seeds,fruit peels,and flesh of Hippophae Fructus are clustered into one group,and the content of flavonoids,water-soluble components,and fatty acid components can be significantly different,which can be used as important indicators for identifying different parts of Hippophae Fructus.Through the measurement results and cluster analysis of different harvest periods of Hippophae Fructus,it can be seen that the optimal harvest period for Hippophae Fructus fruit is from mid August to the end of September when the fruit is mature.At this stage,enzymatic hydrolysis and environmental factors are relatively small,and the measured content of free flavonoids is the lowest.The content of water-soluble components such as uridine,adenine,fructose,mannose,and glucose is the highest.The measured content of fatty acids is the lowest during this period,and the ratio of palmitic acid to palmitic oleic acid is the lowest.It also conforms to the commonly used Hippophae Fructus harvesting method.After the Hippophae Fructus ripens,the fruit is pruned with fruit,and after freezing,the fruit branches are knocked.The causal stem is easily detached after being frozen,and the fruit is collected by shaking.At the same time,it also has the function of pruning,increasing plant ventilation and light transmission,promoting the growth of new branches,and increasing the fruit set rate for the next year.The fresh Hippophae Fructus fruit harvested after maturity has a moisture content of about80%.It is easy to ferment when placed at room temperature and can easily spoil after prolonged storage.The skin of Hippophae Fructus fresh fruit has a dense waxy oil film layer that affects the drying effect and reduces the diffusion channel of water.The natural drying method takes a long time and is prone to spoilage.By examining the steaming time and drying method of Hippophae Fructus,the evaluation criteria are flavonoids,water-soluble components,and fatty acids.After steaming for 20 minutes,Hippophae Fructus fruit increases the water diffusion channel of the skin,shortens the drying time,and has the best content of flavonoids,water-soluble components,and fatty acids.The highest content of flavonoid glycosides plays a role in killing enzymes and protecting glycosides.In addition,steaming plays a role in killing microorganisms to prevent spoilage and facilitate storage.Through cluster analysis,flavonoids can be used as indicator components to distinguish the best indicators for Hippophae Fructus samples from different regions.Based on a ratio of0.60 between palmitic acid and palmitoleic acid,the ratio of fresh Hippophae Fructus fruit is less than 0.60.The longer the storage year,the greater the ratio.The storage time of Hippophae Fructus can be determined by the ratio of palmitoleic acid to palmitoleic acid.The antibacterial test on fresh Hippophae Fructus showed that it has no inhibitory effect on bacteria and fungi,and there is a risk of fungal toxin contamination during the harvesting season,drying process,and storage conditions.Using high-performance liquid chromatography tandem mass spectrometry with positive and negative ion modes,11 fungal toxins including aflatoxin B₁,aflatoxin B₂,aflatoxin G₁,aflatoxin G₂,zearalenone,ampicillin,fumatoxin B₁,fumatoxin B₂,ochratoxin A,T-2 toxin,emetomycin,etc.were determined.Zearalenone,aflatoxin,and ampicillin were detected in Hippophae Fructus fruit.Through the established pesticide detection method,248 types of pesticide residues were screened,and pesticide residues such as carbendazim,dimethoate,and atrazine were detected.According to the test results,a health risk assessment is conducted,and the risk prompts that Fe,Al,Ag,Pb,Cr,and As elements need to be paid attention to,with Al element requiring special attention.The elements measured in the carcinogenic risk assessment do not pose a carcinogenic risk.The intake risk through health risk assessment is within an acceptable range,but there is an intake risk that should be rando mly monitored dynamically to ensure the safety of Hippophae Fructus use.（3）Through network pharmacology screening,it was found that the active substances associated with the treatment of cancer by Hippophae Fructus are those that act on key targets such as isorhamnoin to treat cancer.Design Expert 8.0.6 and Box Behnken software were used to optimize the extraction and purification of isorhamnoin from fresh Hippophae Fructus fruit.Isorhamnoin was used as the lead compound and the polymer compound poly（carboxybetaine methylate）was used to synthesize a zwitterionic polymer（ISPCM-1）through self-assembly mode,ISPCM-1 was further synthesized with hydrochloric acid,phosphoric acid,sulfuric acid,methanesulfonic acid,and trifluoromethanesulfonic acid（ISPCM-6）.Using transmission electron microscopy to simulate acidic gastrointestinal environments at p H 4,5,and 6,we observed（ISPCM-1 to ISPCM-6）amphoteric polymer drugs and found that they are fir mly encapsulated in water in oil form.This structure has the characteristics of high dispersion and high aggregation,which is conducive to the dynamic self-assembly of drugs into cells and can significantly improve drug solubility.The cytotoxicity of compounds ISPCM1-6 on human colon cancer cell lines（HCT-116）,human cervical cancer cell lines（He La）,and normal liver cell lines（L-02）was determined using MTT method with Isorhamnetin as the positive control.The cytotoxicity data of ISPCM-1and ISPCM-2 are similar to the positive control,and the anti-tumor activity of ISPCM-1 and ISPCM-2 is slightly lower than the positive control,but the difference is not significant;For ISPCM-3 to ISPCM-6,there is almost no activity or it exceeds the range of effective evaluation.Samples（ISPCM1-6）were tested using confocal microscopy,and significant fluorescence characteristics were observed in both ISPCM-1 and ISPCM-2,indicating good biocompatibility between ISPCM-1 and ISPCM-2 with the target cells and producing significant fluorescence characteristics.The fluorescence generated within the cells is mainly distributed around the cytoplasm and rarely dispersed around the nucleus,indicating that ISPCM-1 and ISPCM-2 have targeting properties towards the cytoplasm and nucleus,Especially for cytoplasm,it has good targeting ability.ISPCM-1～2 has a better inhibitory effect on tumor cells in vitro than ISPCM-3～6.The results indicate that ISPCM-1 and ISPCM-2 can be used as novel anti-tumor amphoteric polymer drugs,providing new design ideas and directions for further research on Isorhamnetin.Conclusion:This study is based on literature research and clarifies the historical evolution of Hippophae Fructus.It corrects the recognition of vinegar forest in the"Compendium of Materia Medica"as Hippophae Fructus,and the description of vinegar forest in the"Compendium of Materia Medica"as large folic acid vine.Establish a comprehensive quality evaluation plan for flavonoids,fatty acids,and water-soluble components in Hippophae Fructus,detect different parts of Hippophae Fructus fruit,different harvesting periods,steaming times,and drying methods,and determine the optimal harvesting period from mid August to the end of September when the fruit is ripe.The optimal steaming time is 20 minutes before drying treatment.The storage time of Hippophae Fructus can be preliminarily determined for quality evaluation by the ratio of palmitic acid to palmitoleic acid.The health risk assessment of exogenous pollutants in Hippophae Fructus requires attention to elements such as Fe,Al,Ag,Pb,Cr,and As,with a focus on Al.The elements measured in the carcinogenic risk assessment do not pose a carcinogenic risk.Detected that Hippophae Fructus contains zearalenone,aflatoxin G2,penicillin,carbendazim,dimethoate,atrazine exogenous pesticide residues,and fungal toxins.The intake risk through health risk assessment is within an acceptable range,but there is an intake risk that should be rando mly monitored dynamically to ensure the safety of Hippophae Fructus use.The ISPCM series of zwitterionic polymers（ISPCM-1～ISPCM-6）were prepared by self-assembly of isoproterenol as the lead compound and polymer compounds.The prepared ISPCM series of zwitterionic polymers significantly increased the solubility of isoproterenol.MTT assay was used to determine the cytotoxicity of zwitterionic polymers（ISPCM-1～ISPCM-6）on human colon cancer cell lines（HCT-116）,human cervical cancer cell lines（He La）,and normal liver cell lines（L-02）.Transmission electron microscopy,scanning electron microscopy,and confocal microscopy were used to observe that ISPCM-1and ISPCM-2 have good biocompatibility with target cells and produce significant fluorescence characteristics.The fluorescence generated inside the cells is mainly distributed around the cytoplasm,It is rarely dispersed around the nucleus,indicating that ISPCM-1 and ISPCM-2 have targeting properties towards the cytoplasm and nucleus,especially towards the cytoplasm.ISPCM-1～2 has a better inhibitory effect on tumor cells in vitro than ISPCM-3～6.The experimental results indicate that ISPCM-1 and ISPCM-2 can serve as novel anti-tumor amphiphilic polymers,providing new design ideas and directions for further research on Isorhamnetin.