Quantification Of Chlorogenic Acid,As Well As Preparation And Bioactivity Assessment Of Its Analogues And Derivatives

Chlorogenic acid is natural product with diverse bioactivities.Its content is an important index for the quality control of the renowned traditional Chinese medicine,YinChen(the dried aerial part of Artemisia capillary Thunb.or Artemisia scoparia Waldst.et Kit.).YinChen was first recorded in the"Shennong’s herbal classic".It has the effect of clearing heat,promoting diuresis,benefiting bile and reducing jaundice.In addition,modern pharmacology study confirmed that it has the effect of anti-cancer,anti-inflammatory and anti-pyretic analgesic.YinChen contains a variety of chemical constituents,such as chlorogenic acids,flavonoids and coumarins.This paper established a rapid analysis method to determine the contents of six constituents,including chlorogenic acid by ultra-high performance liquid chromatography-mass spectrometry(UPLC-MS/MS).We measured the content changes of these six constituents in different parts of YinChen with different drying methods and different harvesting months.The results showed that the content of the chlorogenic acid was significantly higher than flavonoids in YinChen.In addition,among different plant parts and different drying methods,the orders of chlorogenic acid content were:leaf>stem>root,freeze-drying treatment>air dried.The content(10.823 μg/mg)of chlorogenic acid in the leaves collected in April and processed by freeze-drying was the highest among all samples.The contents of the flavonoid compounds,such as 3,5,3’,4’-Tetrahydroxy-6,7-dimethoxy flavone were the highest in June.The contents of cirsimaritin and cirsilineol increased gradually in leaves from April to July,while the contents of chrysoeriol and rhamnocitrin were very low in YinChen.This method was highly reproducible,of high sensitivity and resolution,and could serve as a reference for the quality control of YinChen.The major bioactive constituent of YinChen,such as Chologenic acid,is highly hydrophilic because it has four phenolic hydroxyl and one carboxyl groups in the structure.This experiment was carried out to modify its lipophilicity by enzymatic acetylating using porcine pancreatic lipase.We found out the optimal reaction conditions as methyl tert-buty1 ether as the solvent and vinyl acetate as the acetyl donor.Under this condition,when the ratio of chlorogenic acid to vinyl acetate was 1:6,lipase amount was 100 mg,and reaction temperature was 50 ℃,acetylation of chlorogenic acid reached the highest yield.Two acetylated compounds were obtained after separation and purification of the mixture using reverse phase column chromatography.Their structures were determined as 1-acetylchlorogenic acid and 1,5-diacetylchlorogenic acid using NMR and UPLC-MS/MS analysis.Chlorogenic acid which has significant biological activities is the main active ingredient and used for quality control of some important traditional Chinese medicines.However,because of the easy-breaking property of the ester bond in its structure,a large portion of chologenic acid are hydrolyzed with only a small part of intact one can be absorbed after oral administration and some activities disappeard.So it is urgent to study the synthesis of chlorogenic acid analogues with higher stability.In this study,we conducted the synthesis of chlorogenic acid analogues by substituting amide linkage for ester bond.Using a quinic bisacetonide as the starting material and after a series of reactions including oxidation,hydroxylamination and catalytic hydrogenation,chlorogenic acid analogue with an amide bond replacing the ester bond was synthesized.Its structure was determined by NMR(including NOE)and high resolution mass spectroscopic methods as 3a-caffeoylquinic acid amide.This compound retained the antioxidant and anti-hepatitis C virus activities of chlorogenic acid,and showed much higher stability than chlorogenic acid.In order to verify the biological activity of these compounds to find better chlorogenic acid derivatives or analogues,we compared their properties,including radical scavenging activity,inhibition of the production of reactive oxygen species(ROS),protective effect of DNA on oxidative damage induced by peroxynitrite,inhibition of a-glucosidase activity and DPP IV activity.The experimental results show that the newly synthesized compound,3 a-caffeoylquinic acid amide showed comparable or even better properties in DPPH radical scavenging activity and the inhibition of ROS than chlorogenic acid.The 3a-caffeoylquinic acid amide,1-acetylchlorogenic acid and 1,5-diacetylchlorogenic acid showed better protective effect of DNA on oxidative damage than chlorogenic acid.These compounds have good biological activity in vitro,but the ability to be absorbed by the small intestine needs further verification.Using the Caco-2 cell monolayer model which is widely used in research of the absorption properties of oral drugs,it is found that the absorption properties improved through changing the ester bond to amide bond,or through acetylation of chlorogenic acid.This result provides a theoretical basis for the development of new medicines from chlorogenic acid.