Solid phase extraction followed by high performance liquid chromatography for the determination of bioactive lignans in Schisandra chinensis in vitro cultures

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Year of publication 2005
Type Article in Proceedings
Conference 11th international Symposium on Separation sciences
MU Faculty or unit

Faculty of Medicine

Field Analytic chemistry
Keywords spe; hplc; lignans; Schisandra; in vitro cultures
Description Schisandra chinensis is well-known medicinal plants of traditional Chinese medicine. The fruits and seeds have been used for centuries as tonic and antitusic. Active principles are lignans with unusual structure derived from dibenzo[a,c]cyclooctadiene. These lignans have been shown to possess a broad range of biological effects, including antiviral, antioxidative and hepatoprotective activities [1, 2]. Content of lignans in Schisandra fruits is relatively low (~1 %) and is composed of at least thirty lignans [3]. Moreover, contents of individual lignans considerably vary from plant to plant. Plant cell culture may represent a solution of the cost-effective supply of these lignans. Calluses of Schisandra chinensis derived from endosperm and immature zygotic embryo were established. A simple method for quantitative analysis of bioactive lignans in Schisandra chinensis in vitro solid-phase extraction cultures and media samples has been developed as a tool for selection of high-yielding clones. An analytical method based on solid-phase extraction (SPE) and followed by liquid chromatographic separation and ultraviolet detection (HPLC-UV) is proposed for the determination of five structurally similar lignans (schizandrin, gomisin A, deoxyschizandrin, gomisin N and wuweizisu C) present in Schisandra chinensis. Methanol was found to be most efficient solvent for the extraction of lignans from freeze-dried samples. Methanolic extracts and media samples were cleaned by solid-phase extraction with Strata C18-E (Phenomenex) cartridges. After loading of the samples, the cartridges were washed with 40 % aqueous methanol (v/v) to remove an interfering compounds coeluting with the lignans. The lignans were eluted from the cartridges with methanol and examined by HPLC. The use of solid-phase extraction is not only necessary for clean-up of lignan fraction, but also for concentration of lignans from liquid media. The chromatographic separation was carried out on Chromolith Performance RP-18e monolith column (100 x 4.6 mm, Merck) using isocratic mobile phase of acetonitrile and water in the ratio 50:50 (v/v). The base line separation of major lignans was achieved within short time (18 minutes) owing to the relatively high flow rate of mobile phase (2 ml/min). The UV detector was set at 220 nm. The quantitative analysis was performed by comparing the peak areas of the samples with those of authentic lignans, isolated from the seeds of Schisandra chinensis as described previously [3]. The performance of the method was assessed by the evaluation of absolute recovery (generally higher than 89 % excluding highly unpolar lignan wuweizisu C with 35% absolute recovery). Our results showed that the concentration of lignans in plant cell cultures was mostly lower than that in fruits and leaves. The lignans were also detected in culture media, but the amount of lignans in the corresponding cell culture was always higher. Interestingly, the main lignans in the cell cultures were deoxyschizandrin, gomisin N and wuweizisu C, whereas the major lignans in the plant organs (e.g. fruits or leaves) were a bit polar hydroxyderivatives, schizandrin and gomisin A. A few method of HPLC determination of lignans in Schisandra seeds or fruits have been published, for review see ref. [4], however no investigation has been focused on determination of lignans in cell cultures and culture media. The method described here should be applicable for the monitoring the level of lignans in numerous samples of Schisandra chinensis. The method requires only 50 mg of samples and gives satisfactory separation, absolute recovery and rapid analysis time.
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