| Hovenia acerba lindl.is a member of Rhamnaceae family. The seeds of Hovenia acerba lindl. is widely used in traditional Chinese herbal medicine which has functions of clearing heat, diuresis, slaking thirst, dissolving the annoyance, relieving alcoholism etc. and has been used to cure Polydipsia, hiccups, vomiting, alcoholism and other diseases.The chemical constituents of Hovenia acerba lindl consist of flavones, triterpenoids, saponins, phenylpropanoids, alkaloids etc. Ten compounds were isolated from80%ethanol extract of the seeds of Hovenia acerba lindl by using the silica gel, ODS column chromatography, preparative chromatograph and crystallization, and the chemical structures of which were identified by spectral methods such as MS, NMR combining with their physical and chemical properties. The ten compounds were identified as Hovenitin I, dihydromyricetin,(2R,3R)-3,3’,5’,5,7-pentahydroflava none, myricitin, acerboside B,hodulosideIH,3-O-[(1â†'2)-β-D-quinovopyranosyl (1â†'3)-6-acetyl-β-D glucopyranosyl]-β-L-arabinopyranosyl jujubogenin, hovenidulcioside A1, hovenidulcioside B1and daucosterol. Among the ten compounds,3-O-[(1â†'2)-β-D-quinovopyranosyl (1â†'3)-6-acetyl-β-D glucopyranosyl]-β-L-arabinopyranosyl jujubogenin was gained from the seeds of Hovenia acerba lindl for the first time.This paper has also established HPLC method for content determination of Hovenitin I and (2R,3R)-3,3’,5’,5,7-pentahydroflavanone in the seeds of Hovenia acerba lindl.1. Extraction, isolation and structural identification of the chemical constituents in the seeds of Hovenia acerba lindl.1.1ExtractionTen kilograms dried seeds of Hovenia acerba lindl were crushed firstly, then were extracted by heat reflux in80%ethanol for three times, the dosage of the solvent were10,10and8times (V/W) in trun, and the extracting time were2.0h,1.0h,1.0h. respectively. Put the extracted solution together before filerted, then concentrated with ratary evaporators, the crude extracts of the seeds of Hovenia acerba lindl were obtained finally.1.2Isolation and purificationThe crude extracts were separated by silica gel column chromatography with mobile phase I and ten parts A,B,C,D,E,F,QH,I,J were obtained.(1) Fraction B was separated into three parts (a, b, c) by silica gel column chromatography, compound J-1was obtained by silica gel column chromatography from part a. and compound J-3and J-4were gained by using ODS column chromatography repeatly from the part b and c respectively.(2) Fraction C was separated into two parts (d and e) by silica gel column chromatography. White floc producted from part d, named compound J-5. compound ZJZ-A was obtained by recrystallization from part e.(3) Fraction D was isolated by using silica gel column chromatography, ODS column chromatography, and preparative liquid chromatography, then received compounds ZJZ-E1and ZJZ-E2.(4) Fraction H was separated into three parts (h, i and j), compound ZJZ-a and ZJZ-b were obtained by using ODS column chromatography repeatly from part h and i. compound ZJZ-F and ZJZ-G were gained from part1by using silica gel column chromatography, ODS column chromatography, and preparative liquid chromatography.1.3Structural identificationBy means of various spectroscopy methods such as MS and NMR, ten compounds were identified as Hovenitin I, dihydromyricetin,(2R,3R)-3,3’,5’,5,7-pentahydroflavanone, myricitin, hoduloside III,3-O-[(1â†'2)-(3-D-quinovopyranosyl (1â†'3)-6-acetyl-β-D glucopyranosyl]-β-L-arabinopyranosyl jujubogenin,hovenidulcioside B1, hovenidulcioside A1, acerboside B and daucosterol. 2. The contents determination of two flavonoids in the seeds of Hovenia acerba lindl by HPLC2.1Instruments and chromatographic conditions Chromatography:Agilent1100HPLC (Detector:VWD); Column:Agilent HC-C8(4.6×150mm,5μm) Mobile phase:methanol-water (0.1%phosphoric acid)16:84Flow rate:1.0mL/min Chromatograpy column temperature:25℃Wave length:290nm Injection volumn:20μL.2.2Methodology Investigation2.2.1The linear testThe sample concentration of the two compounds Hovenitin I (J-1) and (2R,3R)-3,3’,5’,5,7-pentahydroflavanone (J-3) had a good linear relationship within the range of0.025～2μg and0.375-30μg respectively. The linear equations and its coefficients of correlation were showed as follows: J-1:y=70812x+30.094, r=0.9997J-3:y=57744x+575.17, r=0.99962.2.2LOD and LOQThe LODs of compound J-1and J-3were2.5ng and1.04ng respectively; The LOQs of compound J-1and J-3were5.0ng and1.88ng respectively.2.2.3precisionThe solution of the J-1and J-3were analysed by HPLC. The result showed that the intra day RSDs of peak areas of compound J-1and J-3were1.05%and1.01%respectively(n=6); the intraday RSDs of peak areas compound of J-1and J-3were0.83%and1.44%respectively(n=6). It illustrated that the equipment is in good precision.2.2.4Repeatability testSix copies of the sample solution were measured by HPLC and the RSDs of the contens were1.05%and1.22%(n=6)for compound J-1and J-3respectively, which showed the method was in good precision.2.2.5Stability testThe sample solution was analysed by HPLC each two hours for six times, the RSDs of compound J-1and J-3were1.19%and0.72%respectively. It showed that the sample solution had a good stability within10hours.2.2.6Recovery testThe average recoveries of compound J-1and J-3were99.4%and100.1%respectively, and the RSDs were1.55%and1.59%, respectively(n=6).3. Contents determinationFour batches of the seeds of Hovenia acerba lindl were determined and the average contents of compound J-1and J-3were0.029mg/g and1.056mg/g respectively.4. SummaryThe chemical constituents of Hovenia acerba lindl have been deeply studied in this experiment. Ten compounds were isolated and identified from the seeds of Hovenia acerba lindl. The contents determination method of Hovenitin I (J-1) and (2R,3R)-3,3’,5’,5,7-Pentahydroflavanone (J-3) was established by HPLC. The study further improved the establishment of quality standards, and provided favorable scientific basis for research, utilization and pharmacology activity of Hovenia acerba lindl. |
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