Prunella In Two Kinds Of Triterpene Acid Separation Determination

Based on a large number of literature referred, triterpenoids - the medical bioactive ingredients of Prunella Vulgaris L. in Hubei were studied systematically. Oleanolic acid (OA) and ursolic acid (UA) were extracted, isolated and identified by means of UV, IR, ¹HNMR and ¹³CNMR. Details are as follows:(1) Extraction of OA and UA. The best solvent extraction process was obtained by orthogonal design. And the optimal conditions were: temperature 85℃; 1:15 of 95% (v/v) ethanol aqueous; 2 h for the first time and 1 h for the second. The extraction volumes of OA and UA were 1.24 mg/g and 2.45 mg/g respectively. On the contrary, ultrasonic-assisted extraction need less time, but extraction volumes were lower than that of solvent extraction.(2) Purification and isolation of OA and UA. Solvent extraction, alkali-solution and acid-isolation were chosen as the primary purification methods. Silica gel column chromatography and recrystallization were used for further purification. Product A and B were obtained finally. The content of UA in product A was 90.30% in contrast to 28.58% without further purification with the yield of 25.10%, whereas the content of triterpene acids in product B was 92.15% with the yield of 24.81%. Moreover, X-5# macroporous resins were also used for separation of OA and UA. Two triterpene acids coexisted with the total content of 83.30% and the yield of 70.10%.(3) Determination and identification of OA and UA. Method of RP-HPLC was established to determine the contents of OA and UA in extracts and progress of purification and isolation. The analysis conditions were as follows: Column, Hedera ODS-C₁₈ (250 mm×4.6 mm, 5 urn); Mobile phase, methanol - water - acetic acid = 85:15:0.2 (v/v); Detection wavelength,λ= 210 nm; Flow-rate, 0.8 mL/min; Column temperature, 30℃; Injection volumn, 10μL. The good linear relationships between the peak area and concentration were obtained in the ranges of 18-360μg/mL and 19.5-390μg/mL for OA and UA, respectively. The structure of product A was identified as UA by means of UV, IR, ¹HNMR and ¹³CNMR.(4) Separation of triterpene acids by cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC). A Method of CD-MEKC was established to separate and determine the contents of OA and UA in extracts. Two triterpene acids were baseline separated in the buffer (pH = 9.0) of 6 % (v/v) methanol containing 10 mmol/L disodium tetraborate, 10 mmol/L sodium dihydrogen phosphate, 50 mmol/L sodium dodecylsulfate, 15 mmol/L 2-hydroxypropyl-β- cyclodextrin within 15 min. And a comparative study was carried out, in which a CD-MEKC method was validated in samples and it was compared with HPLC with CDs as chiral mobile phase additive (CMPA-HPLC).