HPLC Determination Of Echinacoside in Cistanche Cistanche

HPLC determination of echinacoside in Cistanche Cistanche

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Objective: HPLC determined the amount of echinacoside in Cistanche ground in liquid nitrogen condition as well as in naturally air-dried condition, comparing it in wild cistanche and artificially cultivated Cistanche.

Methods: the lc-20a HPLC system (Shimadzu, Japan) was used. The column was YMC inertsil ods-sp (4.6 mm × 150 mm，5μm；YMC Co Ltd，Japan)； Mobile phase: acetonitrile-methanol-1% formic acid (10:15:75); Detection wavelength 334 nm; Flow rate 0.7 mg · ml-1. Column temperature 30 ° C.

Results: different treatment conditions had a very clear effect on the echinacoside content, with generally higher levels of echinacoside in Cistanche ground under liquid nitrogen than in those ground under natural air-dried conditions; The active ingredient content of wild Cistanche was much higher than that of artificial culture.

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Cistanche is an endangered species, a parasitic plant that inhabits the roots of Clostridium, Salix rubrum. In China, Cistanche is mainly distributed in Inner Mongolia, Ningxia, Gansu, and Xinjiang, with an excellent reputation for " desert ginseng ", which is of great medicinal value. Its main medicinal ingredients are phenylethanolic glycosides, which can be divided into echinacoside, leaf cohoside, Cistanche, and others depending on their substituents. Among them, echinacoside has improved sexual function, antioxidant, and free radical scavenging effects, but also has some anti-apoptotic effects. Since the 1980s, HPLC technology, which provides a simple and rapid method for the separation of phenylethanolic glycosides, has been widely used in the separation of natural products. An HPLC method was chosen to analyze the contents of Cistanche, the major active constituent of wild and artificially cultivated Cistanche, at different growth periods, providing a scientific basis for the rational development and utilization of Cistanche and for the effective evaluation of its quality.

Instruments and reagents

Shimadzu, Japan lc-20a HPLC apparatus, lc-20at tandem double plunger pump, cto-20a column oven, spda-20a UV Vis dual wavelength detector; Kunshan kq-100 ultrasonic cleaner; Acetonitrile (chromatographically pure, Tianjin Guangfu Chemical Research Institute); Methanol (chromatographically pure, Tianjin Guangfu Chemical Research Institute); Formic acid (chromatographically pure, Tianjin Guangfu Chemical Research Institute); distilled water

Control: echineoside, content ≥ 98.43% (Shanghai Yuanye Bio Technology Co., Ltd.).

Five samples of Cistanche: artificially planted for one year with the following Cistanche, artificially planted for Cistanche, artificially dried Cistanche, wild Cistanche, and artificially planted for more than three years with Cistanche.

2 methods and results

2.1 determination of chromatographic conditions

In order to separate the peak of the measured echineoside in the LC chromatogram from other substances, as well as to control the pump pressure and the time table to be within a suitable range, the experimental modification of the chromatographic conditions was carried out, and after optimization experiments with mobile phase, it was concluded that the higher the temperature, the shorter the retention time and the lower the concentration, the shorter the pump pressure and the lower the retention time, but the peak height became lower, which was unfavorable for the measurement. The mobile phase was finally determined to be acetonitrile-methanol-1% formic acid (10:15:75), the detection wavelength 334 nm, the flow rate 0.7 mL / min, and the column temperature 30 ° C, according to the objective conditions of room temperature and the maximum bearing pressure of the pump, which resulted in abundant information with sharp peak shapes and good separation of the constituents。

2.2 determination of columns

YMC-Inertsil ODS-SP(4.6 mm × 150 mm，5 μ m；YMC Co Ltd，Japan).

2.3 experimental solution preparation

2.3.1 preparation of standard solutions

An appropriate amount of echineoside standard was accurately weighed and spiked with mobile phase acetonitrile-methanol-1% formic acid (10:15:75) to make 0.06 mg · ML - 1, 0.05 mg · ML - 1, 0.04 mg · ML - 1, 0.03 mg · ML - 1, 0.02 mg · ML - 1, 0.01 mg · ML - 1, and 0.005 mg · ML - 1 solutions of echinacoside standard.

2.3.2 preparation of test solution

Appropriate amounts of artificially planted fresh Cistanche (under one year), artificially fresh Cistanche, and wild fresh Cistanche were ground to powder using a mortar under liquid nitrogen; Also, an appropriate amount of the above 3 Cistanche was cut into blocks, air dried naturally in shade ventilation, and after drying, it was pulverized and ground into powder. Artificial dry cistanche and wild over three years dry Cistanche were ground directly into the powder. 0.5 g of Cistanche powder prepared above was taken and placed into a centrifuge tube, 25 ml of mobile phase was added, weighed and soaked for 30 min, sonicated for 40 min, cooled and then weighed again to make up the missing weight with mobile phase, shaken and centrifuged, and the supernatant was taken for 0.45 μ M microporous filter membrane filtration.

2.4 plots of regression curves for the standard sample of echinacoside

The prepared standard samples of echineoside were injected into the LC apparatus separately, and the concentration of the control (mg · L-1) was used as the abscissa, and the corresponding peak areas were ordinated to plot the standard curve, resulting in the regression equation: y = 66 867 100x-46 360.83, R2 = 0.98637, which showed the echineoside at 0.1 ~ 1.2 μg within a good linear relationship.

3 Discussions

Echinacoside is one of the main active components of Cistanche, which can well represent the chemical basis of the pharmacological effect of Cistanche, making the quality evaluation of the medicinal material and its pharmacological effect correspond, and improving the pertinence of Cistanche quality evaluation.

The different treatment conditions had a clear effect on the echinacoside content, with generally higher levels of echinacoside in Cistanche ground under liquid nitrogen than in those ground under natural air-dried conditions. Common air drying decreased the amount of echineoside, which might have been consumed during air drying or converted. In order to prevent the decrease of active ingredient content and guarantee the efficacy of Cistanche, an appropriate treatment should be selected.

Consistently, most believed that the artificial cultivation environment of Cistanche as well as growth habits were indistinguishable from the wild with no difference in quality, tended to place only emphasis on yield, and did not analyze quality. The present study showed that the amount of artificially cultivated Cistanche echinacoside was lower than that of wild Cistanche echinacoside. The highest was 0.3360% of wild cistanche and the lowest was 0.007 0% of artificial Cistanche. Therefore, it still needs to be studied in cultivation technology to improve the content of active ingredients.

The quality of Cistanche depends on the amount of its active ingredient, echinacoside, which in turn has a strong relationship with the season, time, and method of harvesting, so harvesting of Cistanche should take into account the accumulation status of the active ingredient and the stage of growth and development. Consider both quality and yield.

The method mentioned in this paper is simple, precise, reproducible, and accurate for determining echineoside. Useful for Cistanche determination.