Analyses Of Phenolic Compounds In Hawthorn Leaves By Using HPLC

Hawthorn is a dual-purpose plant resource of herb and food, many researches showthat hawthorn has many physiological and pharmacological functions, such as improvingcardiovascular system, which is closely related to its phenolic compounds, includingprocyanidins，flavonoid and so on. There are relatively high contents of flavonoid in theleaves of Hawthorn, and it also contains a certain amount of procyanidins and phenolicacids, so it has high value of development and application. It was shown by our previousstudy in our laboratory that we found Eucomic acid for the first time, which is unique tothe hawthorn, and it belongs to secondary metabolite of phenolic acids. We carry out thefollowing researches on the basis of prophase studies in our laboratory.1.In the present research, a suit of analysis method was established by using a highperformance liquid chromatographic apparatus, which was used for analysing severalactive components in the fruits and leaves of hawthorn, including procyanidins, flavonoidand phenolic acids. The contents of chlorogenic acid, eucomic acid, epicatechin,procyanidin B2, procyanidin C1, procyanidin D1, vitexin-2″-O-rhamnoside, hyperosideand isoquercitrin were determined by the following chromatographic condition: Column:Hypersil BDS C18(250mm×4.6mm id,5μm); temperature:45℃; mobile phase, A:methanol/acetonitrile=1:2containing500μL/L formic acid; B:500μL/L formic acid; thegradient elution condition (min/A%) was:0/8,26/20,30/50,35/50,37/8,47/8; flow rate:0.8mL/min; detection: diode array detector (DAD) at280nm and350nm; injectionquantity:10μL. The external standard calibration curves were used in quantification. Thepolyphenols compounds in the leaves of hawthorn were separated and determined well bythis method. The relative standard deviations (RSD) of those components were between0.77%~2.06%, the recovery was93.7%~116.2%. The linearity ranges were between20ng~5000ng. The lowest detection limit was10~15ng. The correlation coefficients (r) werebetween0.9994~0.9999. These9kinds of polyphenols were separated and determined wellin35min.The contents of chlorogenic acid, epicatechin, procyanidin B2, procyanidin B5,procyanidin C1, procyanidin C2, procyanidin D1, procyanidin E1, hyperoside andisoquercitrin were determined by the following chromatographic condition: Column:Hypersil BDS C18(250mm×4.6mm id,5μm); temperature:35℃；mobile phase, A:methanol/acetonitrile=3:7containing500μL/L formic acid；B:500μL/L formic acid; thegradient elution condition (min/A%) was:0/13,10/14,15/22,23/30,26/100,31/100,33/13, 43/13; flow rate:0.8mL/min; detection: diode array detector (DAD) at280nm and350nm; injection quantity:5μL. The external standard calibration curves were used inquantification. The polyphenols compounds in the fruits of hawthorn were separated anddetermined well by this method. The relative standard deviations (RSD) of thosecomponents were between0.24%~1.63%，the recovery was91.3%~107.6%. The linearityranges were between20ng~5000ng. The lowest detection limit was10~15ng. Thecorrelation coefficients (r) were between0.9994~0.9999. These10kinds of polyphenolswere separated and determined well in30min.2. By this method, we analyzed the phenols content of the leaves of hawthorn duringthe various growing stage and different leaf arrangement. In hawthorn leaves, flavonolsand C-glycoside flavonoid are the major phenolic compounds. Meanwhile, theprocyanidins and phenolic acids in hawthorn leaves owns considerable content. Thecontent of these components with a slow growth trend with the growth, and reached themaximum in October when the fruits are ripe. The total content of flavonoids in leaves of3species,2kinds of phenolic acids and3kinds of proyanidins were8.01,4.57,5.35mg/gD.W respectively. The total phenolic content of the same branch of hawthorn leaf ofdifferent leaf arrangement changed little, between16.4~22.7mg/g D.W, but the proportionof each constituents were different. The basilar leaf has higher content of the C-glycosideflavonid. The top leaves has higher content of the O-glycoside flavonoid.3. By this method, we analyzed the main phenols content of the leaves, flowers andfruits of29kinds of the representative hawthorn, which are native to seven Chineseprovinces and cities. The results showed that there was significant differences on thephenols content among the different species and organs, however, they have the same kindsof phenols.The total phenols content in the leaves of hawthorn was24.2~36.2mg/g D.W,and the main components in them was flavonoid, followed by phenolic acids. The contentof total flavonoids in the date of flowering was higher than other periods, and C-glycosideflavonoid was the main type. In terms of phenolic acid, Eucomic acid was the primarycomponent and its content was about5.6~6.3mg/g D.W. The content of total phenolics inthe flowers was19.2mg/g D.W, the main components in them was procyanidins, followedby phenolic acids. The content of total phenolics in the fruits was10.5mg/g F.W, andprocyanidins accounted for96%. At the same time, we found that there was differentdegree of correlation among the same variety of hawthorn leaves, flowers and fruits.