Chemical Composition Analysis And Safety Evaluation Of Youguiyin That A Classic Prescription Of "bu Shen Yi Jing"

BackgroundYouguiyin is derived from "Jingyue Quanshu" that a famous classic recipe,which is composed of 8 traditional Chinese medicines of Rehmannia glutinosa,wolfberry,cornus,yam,aconite,cinnamon,eucommia,and licorice.It has the effect of "Yin" and"Yang" and filling essence and blood.It is mainly used for the treatment of inadequate treatment of life and deficiency of kidney essence.Youguiyin has a wide range of clinical applications and many studies on pharmacological effects.However,it has not yet been systematically reported which chemical compositions are contained in Youguiyin and how safe they are.Youguiyin is also the main field of our research for "Kidney essence" biological substance basis and the mechanism of action,and is a tool drug interpretation for"Bushen Yijing" prescription drug to interpretate the "Kidney essence deficiency syndrome" pharmacological mechanism.The drug composition of Youguiyin covers different polarities and multiple types of compounds.It analyzes and identifies these chemical components,which is the key link to control the quality of the experimental drugs and clarify the substance basis of its pharmacological effects.Therefore,it is of great significance to study the chemical composition and safety of You Guiyin.This research was supported by the Chongqing Science and Technology Commission-Family Planning Commission Key Chinese Medicine Research Project(zy201801001)"The core functional substance group of Youguiyin in the treatment of deficiency syndrome of kidney essence and its proteomics interpretation".Purpose1.It qualitatively analyzed the chemical components of Youguiyin,and quantitatively detected the index and main active components;2.It evaluated the acute toxicity and long-term toxicity of Youguiyin.Methods1.Preparation of two test samples?Preparation of Youguiyin water decoction:According to the prescription composition,weigh 8 kinds of traditional Chinese medicine with water 8 times,boil 2 times,0.5 h each time,combine the liquid medicine,concentrate to the mass concentration of the crude drug for 2 g·mL-1 that store at 4? for use.?Preparation of Youguiyin formula granule liquid:According to the dosage conversion,weigh 8 kinds of traditional Chinese medicine formula particles,add 43 mL of water and heat(75?)to prepare the crude drug mass concentration for 1.9 g·mL-1.?Preparation of samples:we weighed 0.1ml of the above-mentioned,diluted 100 times(positive ion mode detection)or 1000 times(negative ion mode detection),ultrasound,centrifugation and sucked the supernatant.2.Qualitative analysis method for the chemical compositions of Youguiyin?Chromatographic conditions:waters,ACQUITY UPLC BEH C18 column(2.1×100 mm,1.7?m)for separation,mobile phase:0.1%formic acid acetonitrile(A)-0.1%formic acid aqueous solution(B),gradient elution,positive and negative Collect data in mode.?Qualitative detection method:It uses UPLC-QTOF-MS/MS to characterize the chemical constituents in Youguiyin decoction and its formula granules.The first-order mass spectrometry was used to determine the precise relative molecular mass,the second-order mass spectrometry was used to obtain the cleavage information and the chemical structure was determined by comparing with the standard or literature data.3.Determining content of analytical method for the chemical compositions of Youguiyin ?Selection of quantitative ingredients:It is used as quantitatively determined components as follows:the common components obtained by the qualitative analysis of the decoction and the granule liquid are used to predict the network pharmacological effects,and the components corresponding to the main pharmacological effects in the prediction results and the index components of the 2015 Chinese Pharmacopoeia are taken.?Chromatographic conditions:waters,ACQUITY UPLC BEH C18 column(2.1×100 mm,1.7 ?m)was used for separation.The mobile phase was 0.1%formic acid in acetonitrile(A)-0.1%formic acid in water(B).It was used an electrospray ion source to collect data in positive and negative ion modes.?Qualitative detection method:It used UPLC-QqQ-MS/MS to determine the contents of 18 chemical components in Youguiyin decoction and formula granules4.The acute toxicity test of Youguiyin 1)Grouping and administration:KM mice are randomly divided into a blank group and a drug administration group,10 mice in each group.The drug administration group is given 240 g·kg-1 Youguiyin formula granule liquid for 0.04 ml-10 g-1/piece/time,3 times a day that the total numbers of 720 g·kg-1 which is equivalent to 533 times the clinical dosage for humans.2)Observation indicators:?Appearance:observe general signs,activity status and weekly weight.?Organs:The appearance of the brain,thymus,heart,spleen,stomach,lungs and kidneys and calculate the organ index.?Routine blood indexes:use blood cell analyzer to detect the content of WBC,HGB,MCH,MCHC,HCT,MCV and PLT in whole blood.?Liver function indexes:Determine the content of ALT,AST,ALP,TP,GLU,T-CHO and TG in the serum by automatic blood biochemical analyzer.?Kidney function index:Determine the content of CREA,UREA and CK in serum with automatic blood biochemical analyzer.5.Long-term toxicity test of Youguiyin 1)Grouping and administration:SD rats were randomly divided into blank group and administration group,60 rats in each group.And the administration group was given 60 g·kg-1 of Youguiyin formula granule liquid by intragastric administration that equivalent to human clinical dosage 45 times.Its administration time is once a day for 90 days,1 mL·100 g-1/piece/time.After the administration,the recovery period is observed for 30 days.2)Observation indicators:?Appearance:Observe general signs,activity status,and weekly weight.?Organs:Observating the appearance and calculation of organ index of brain,thymus,heart,liver,spleen,lung,kidney,adrenal gland,stomach,ovary,uterus,epididymis and testis that the HE staining of paraffin sections to observe histomorphology.?Routine blood index:It employed the blood cell analyzer to detect the content of HGB,RBC,HCT,MCH,MCHC and PLT in whole blood.?Liver function indexes:The contents of AST,ALP,ALB,TP,GLU,TG and T-CHO in serum was measured by automatic blood biochemical analyzer.?Kidney function index:Determine the content of CREA,UREA and CK in serum with automatic blood biochemical analyzer.Result1.Qualitative analysis of the chemical composition of Youguiyin 1)Qualitative characterization of Youguiyin water decoction:?It determines the source of the medicinal materials of 80 mass spectrum peaks in Youguiyin water decoction.?The structure of 85 chemical components in Youguiyin water decoction was determined.Among them,there were 7 iridoids and 2 tannins derived from Corni fructus,3 phenylpropanoids and 5 flavonoids derived from Lycii fructus.There were 1 iridoid and 7 lignan components derived from Eucommiae cortex.There was 1 phenylpropanoid derived from Cinnamomi cortex.There were 6 phenylpropanoids and 6 iridoids derived from Rehmanniae radix praeparata.There were 2 phenylpropanoids,8 flavonoids and 18 triterpene saponins derived from Glycyrrhizae radix et rhizoma praeparata cum melle.There were 19 alkaloids derived from Aconiti lateralis radix praeparata.2)Qualitative characterization of Youguiyin formula granule liquid:?It determines the source of medicinal materials of 232 mass spectrum peaks in Youguiyin formula granule liquid.?It determined the structure of 29 chemical components in the granule liquid of Youguiyin formula.Among them,there were 3 iridoids and 1 tannin derived from Corni fructus.There were 2 flavonoids derived from Lycii fructus.There were 2 lignans derived from Eucommiae cortex.There were 2 phenylpropanoids derived from Cinnamomi cortex.There were 2 phenylpropanoid components derived from Rehmanniae radix praeparata.There were 1 flavonoid and 4 triterpene saponins derived from Glycyrrhizae radix et rhizoma praeparata cum melle.There were 12 alkaloids derived from Aconiti lateralis radix praeparata.2.Selection of Quantitative Components of Two Formulations of Youguiyin:?There are 16 chemical components with the determined structure in Youguiyin waterdecoction and formula granules.It including aconitine,mesaconitine,hypaconitine,benzoylhypacoitine,benzoylaconitine,benzoylmesaconine,cinnamaldehyde,verbascoside,isoverbascoside,morroniside,loganin,glycyrrhizin,glycyrrhizic acid,pinoresinol diglucoside,geniposide and mesaconine.The top 15 were index components for the determination of the content of the 2015 Pharmacopoeia.?The results of network pharmacological prediction was showed the 28 of most effective ingredients for the treatment of osteoporosis that was including:sophocarpine,songorine,verbascoside,isoverbascoside,loganin,glycyrrhizin,glycyrrhizic acid,pinoresinol diglucoside,geniposide,gallic acid and cinnamaldehyde and so on3.The quantitative analysis of the chemical composition for Youguiyin It was used the multi-reaction monitoring mode(MRM)to simultaneously detect the contents of 18 chemical components in Youguiyin water decoction and formula granule liquid,such as aconitine,mesaconitine,hypaconitine,benzoylhypacoitine,benzoylaconitine,benzoylmesaconine,cinnamaldehyde,verbascoside,isoverbascoside,morroniside,loganin,glycyrrhizin,glycyrrhizic acid,pinoresinol diglucoside,geniposide and mesaconine.The methodological verification indicators including specificity,linearity(r?0.9947),repeatability(?1.08),precision(?1.34%),stability(?1.73%)and the rate of average recovery(89.49?106.67%)were meeted the contents determination requirements of the 2015 Chinese Pharmacopoeia4.The KM mice was observed no significantly acute toxicity by administered the maximum dosage of Youguiyin formula granule liquid Compared with the blank group,the mice in the Youguiyin 720 g·kg-1 group had the following changes:?The mice in the Youguiyin group had normal signs and no poisoning.There was no significant change in body weight of mice(*P>0.05).?The appearance of the main organs such as brain,heart,spleen and other organs in Youguiyin group did not change significantly(*P>0.05).?The blood routine indexes of WBC,HGB,MCH,MCHC,HCT,MCV and PLT of mice in Youguiyin group were not statistically different(*P>0.05).?The content of liver function indexes that were ALT,AST,ALP,TP,GLU,T-CHO and TG in Youguiyin group did not change significantly(*P>0.05).?The contents of CREA,UREA and CK in the kidney function indexes of Youguiyin group had no significant changes(*P>0.05)5.The SD rats showed no significant long-term toxicity by administered the Youguiyin formula granule liquid solution in 90 days and recovery observation for 30 days Compared with the blank group,the rats in the Youguiyin 60 g·kg-1 group had the following changes:?There were no abnormal changes in the general signs of the animals in the Youguiyin group,and was any rat died.There was no significant difference in weekly body weight of rats.?The gross morphology and organ index of major organs have not changed significantly.?The HGB,RBC,HCT,MCH,MCHC and PLT in blood routine indexes had no statistical difference.?After 90 days of administration and 30 days after drug withdrawal,the levels of liver function indexes that were ALT,AST,ALP,ALB,TP,TG,GLU and T-CHO in the serum of Youguiyin group were not significantly different.But the levels of liver function indexes ALT,AST,ALP,TP,GLU and T-CHO in serum were decreased significantly in recovery observation for 30 days.?The CREA in serum was no significantly different in Youguiyin group.But the UREA wae decreased after 90 days of administration and 30 days of recovery.The CK was increased significantly in after 90 days of administration,that was significant decreased after 30 days of recovery(*P<0.05)Conclusions1.Youguiyin water decoction contains at least 85 well-defined chemical components that have been qualitatively tested by UPLC-QTOF-MS/MS.Youguiyin formula granule liquid contains at least 29 well-defined chemical components.They have a total of 16 chemical components2.We established the quantitative detection method of UPLC-QqQ-MS/MS can quickly detect the contents of 15 predicted medicinal components(8 index components)in Yougui drinking water decoction or formula granule liquid and Three are the content of toxic ingredients3.Acute toxicity and long-term toxicity tests has showed that Youguiyin formula granule liquid is safe for oral administration.