Determination of oleanolic and ursolic acids in different parts of Perilla frutescens by high-performance liquid chromatography

Abstract

A Perilla frutescens (L.) Britt.(Lamiaceae) é uma planta famosa usada na medicina tradicional chinesa para o tratamento de diversas doenças. A fim de avaliar a qualidade da P. frutescens de forma simples, rápida e precisa foi desenvolvido um método de cromatografia líquida de alta performance (CLAE) para caracterização dos dois ácidos triterpenos bioativos: o ácido oleanólico (OA) e o ácido ursólico (UA). Foi usada uma coluna Kromasil 100 C18 RP, com metanol e solução de ácido fosfórico (H 3 PO 4 ) como fase móvel e detecção a 210 nm. O método foi preciso, com um desvio-padrão relativo para esses dois componentes variando entre 0,3-0,6% (análises feitas no mesmo dia) e 0,6-1,2% (análises em dias diferentes). As quantidades de OA e UA na P. frutescens foram determinados por recuperação, variando de 96,7 a 102,0%. Os teores desses dois fitoquímicos encontrados em diferentes partes da P. frutescens cultivada em cinco locais da China, foram determinados para verificar a eficácia do método.Perilla frutescens (L.) Britt.(Lamiaceae), a famous traditional Chinese medicine, has been used for the treatment of various diseases. To evaluate the quality of P. frutescens, a simple, rapid and accurate high-performance liquid chromatography (HPLC) method was developed for the assessment of two bioactive triterpene acids: oleanolic acid (OA) and ursolic acid (UA). The HPLC system used an Kromasil 100 C 18 RP column with methanol and aqueous H 3 PO 4 as the mobile phase and detection at 210 nm. The method was precise with relative standard deviations for these two constituents that ranged between 0.3-0.6 % (intraday) and 0.6-1.2 % (interday). The contents of the OA and UA in P. frutescens were determined with recoveries ranging from 96.7 to 102.0%. The content of these two phytochemicals in different parts of P. frutescens growing at five different locations of China were determined to establish the effectiveness of the method.Keywords: Perilla frutescens, Lamiaceae, HPLC, oleanolic acid, ursolic acid IntroductionPerilla frutescens (L.) Britt. (Lamiaceae) is known as the wild/weedy form, which is found in China, Korea and Japan. It is used as a spicy vegetable and as an oil crop. 1 P. frutescens, which is a traditional Chinese medicinal herb, has been used in China for centuries to treat various diseases including depression, anxiety, tumor, cough, bacterial and fungal infections, allergy, intoxication and some intestinal disorders.2-5 It has been reported that triterpenoid compounds isolated from the leaves of P. frutescens, the two main bioactive constituents were identified spectroscopically and chemically as oleanolic acid (OA) and ursolic acid (UA) (Figure 1). 6,7 OA and UA were both reported to have the anti-inflammatory activity. 8,9 Furthermore, OA possesses anti-ulcer 10 and hepatoprotective activities, 11 and UA exerts its anti-tumor action through enhancing the production of both nitric oxide and tumor necrosis factor-.12 Needless to say, a sufficiently high content of the bioactive phytochemicals is crucial for therap...

Application of Response Surface Methodology for Optimisation of Simultaneous UHPLC-PDA Determination of Oleanolic and Ursolic Acids and Standardisation of Ericaceae Medicinal Plants

“…Considering the earlier studies, C18 is the stationary phase that is most commonly used in the LC separation of OA and UA [8,[15][16][17][18][19][20][21]26,38]. This is hardly surprising, as RP-18 columns are also the most popular ones when it comes to plant material standardisation in general and, therefore, easily available in laboratories performing such studies.…”

“…According to the literature [16,[18][19][20][21]28,[39][40][41][42], isocratic binary elution systems containing acetonitrile or methanol are preferred in analysis of native triterpenoid acids as preventing baseline drifts often observed in gradient modes at detection wavelengths set below 220 nm, the region which the both organic modifiers exhibit also the highest absorption in. In the corresponding mobile phases, the concentrations of both solvents are similar, usually 80%-95% (v/v) for methanol and 85%-90% (v/v) for acetonitrile.…”

“…Although some significant effects of organic solvents and pH of the mobile phase on selectivity have been reported [16], the stationary phase efficiency is believed to be the most important factor determining the resolution of the overlapping bands of native acids. Subsequently, even for relatively simple plant matrices, the use of long chromatographic columns and analysis time of over 15 min, and sometimes over 30 min, are usually required for satisfactory separation [15][16][17][18][19]. As most of the methods utilize HPLC equipment with ultraviolet (UV) detectors, reduced sensitivity may be also an issue, as triterpenes, in general, exhibit low absorption of ultraviolet light.…”

Identification of the Plant Steroid α-Spinasterol as a Novel Transient Receptor Potential Vanilloid 1 Antagonist with Antinociceptive Properties

“…Reverse-phase chromatographic analyses were carried out under isocratic conditions by using a C 18 column (4.6 ϫ 250 mm) packed with 5-m diameter particles. The mobile phase was methanol/0.5% aqueous H 3 PO 4 (88:12, v/v), following the method described by Zou and Chen (2008) with slight modifications. The chromatography peak was confirmed by comparing its retention time with that of a reference standard and by diode array detector spectra (190 -400 nm).…”

Gas phase isomeric differentiation of oleanolic and ursolic acids associated with heptakis‐(2,6‐di‐O‐methyl)‐β‐cyclodextrin by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

“…For the need of quality control of medicinal herbs, it is necessary to develop methods to effectively differentiate OA and UA. Usually direct isomeric discrimination of OA and UA can be achieved by chromatographic12–16 or capillary electrophoretic17, 18 methods. Another strategy for isomeric determination of OA and UA is to form non‐covalent complexes with selective host molecules, providing a specific chemical environment where the structural difference between isomers can be amplified to a detectable level 19…”