Schisandra is a magnolia plant. It is one of the most nourishing traditional Chinese medicines used in Zui. It has the functions of benefiting Qi, nourishing the kidney, concentrating the lungs, calming the nerves, etc. It is mainly used to treat lung deficiency, cough, night sweats, dysentery, nocturnal emission, neurasthenia, palpitations, chronic liver fire, etc. disease. Schisandra incarnata is distributed in the western part of Hubei Province. It is commonly known as “Northern Schisandraâ€, but it is different from the “Northern Schisandra†contained in the 2000 edition of the Chinese Pharmacopoeia. Schisandrin A (1) is one of the main active ingredients of Schisandra, which has obvious effects on promoting hepatic glycogen production, reducing transaminase and protecting liver cells. Therefore, this paper extracted and separated 1 of Xingshan Schisandra to expand the source of Schisandra.
There are mainly three extraction methods for Schisandra chinensis. (1) reflux extraction method, such as alcohol extraction, water extraction, etc., simple operation, low cost, easy to expand production; (2) supercritical CO2 extraction method, high equipment requirements; (3) phytosol solvent method The operation is complicated, the equipment capacity is only 100g, and it is not easy to expand production. In this paper, the reflux extraction method of the literature was used, and the RP- HPLC method was established to analyze the extract.
1 Instruments and reagents
Waters high performance liquid chromatography , including 2690 high pressure pump, 996 diode array detector. Xingshan Schisandra (Hubei Yichang Chinese Herbal Medicine Company, identified by Hubei Yichang Drug Testing Institute): 1 Control substance (China National Institute for the Control of Pharmaceutical and Biological Products, content greater than 98%).
2 Methods and results
2.1 Separation and extraction Take 20g of Xingshan Schisandra, grind it, soak it in water for 24h, then dry it, add methanol reflux to extract. Since the methanol concentration (A), the extraction time (B), the number of extractions (C), and the ratio of the methanol-sample (D) have an influence on the extraction rate, the orthogonal experiment (see the table of factors) is used for process optimization. The results showed that the factors A, B and D3 had a great influence on the separation and extraction of Schisandra. The good condition of zui is A2B1C2D3 (orthogonal result storage and editing department is available).
The extract obtained by the optimized process is evaporated to dryness, dissolved in a small amount of chloroform, and subjected to an alumina column, followed by elution with petroleum ether (30-60 ° C) and chloroform, and the chloroform eluate is collected, evaporated to dryness, and then dissolved in methanol. And make up to 10ml, shake well, as a test solution.
2.2 Chromatographic conditions optimization of mobile phase: methanol-water (85:15, 70:30, 60:40) with different ratios were used for separation and determination. The results show that as the proportion of methanol decreases, the retention time of 1 is prolonged, but other impurity peaks are significantly more. Methanol-water (85:15) was used as the mobile phase, and the linear relationship and reproducibility were good, and the separation effect was good when the flow rate was controlled at 1 ml/min.
Column temperature: The column temperature has a large effect on the chromatographic behavior of 1. If the column temperature is too high or too low, the separation of 1 and Schisandrin B from other impurities will be reduced, and even the baseline separation will not be achieved. The test uses a column temperature of 30 ° C, and the separation effect is good.
Detection wavelength: UV scanning in the wavelength range of 190-400 nm, 1 has large absorption at 220 nm, and the response is strong, so 220 nm is selected as the detection wavelength.
The optimized chromatographic conditions were: column Nova-Pak C18 (3.9 mm x 150 mm, 4 μm); mobile phase methanol-water (85:15); flow rate 1 ml/min: column temperature 30 ° C: detection wavelength 220 nm. Under this chromatographic condition, 1 retention time is approximately 3 min.
2.3 Linear test Weigh accurately to dryness to a constant weight of 1 control 3mg, placed in a 10ml volumetric flask, add methanol to dissolve and volume, shake. The measurements were accurately measured by taking 3, 5, 7, 9, 11 and 15 μ1 respectively. Taking the peak area (A) as the ordinate and the injection amount Q (μg) as the abscissa, a standard curve is drawn. A regression equation of A = 3.81 × 1000000 - 4.43 × 10000 (r = 0.9999).
2.4 Precision test 10 μl of a reference solution of 0.3 mg/ml was accurately weighed and measured continuously for 6 times in 1 d to obtain an RSD of 0.25%.
2.5 Determination of the precision of 1 in Xingshan Schisandra 1 Take the test solution for the test solution under “2.1â€. The average content of 1 in Xingshan Schisandra was calculated to be 0.22% (n=5).
references:
[1] Yang Xiaoling, Li Aimin, Schisandra Research Overview [J], Shizhen Guo Medicine, 1999, 10 (4): 300-301.
[2] Lu Jinqing, He Zehua, Zhang Baojun, et al. Study on the extraction process of Schisandra chinensis [J]. Chinese patent medicine, 1998, 20 (12): l one 2
There are mainly three extraction methods for Schisandra chinensis. (1) reflux extraction method, such as alcohol extraction, water extraction, etc., simple operation, low cost, easy to expand production; (2) supercritical CO2 extraction method, high equipment requirements; (3) phytosol solvent method The operation is complicated, the equipment capacity is only 100g, and it is not easy to expand production. In this paper, the reflux extraction method of the literature was used, and the RP- HPLC method was established to analyze the extract.
1 Instruments and reagents
Waters high performance liquid chromatography , including 2690 high pressure pump, 996 diode array detector. Xingshan Schisandra (Hubei Yichang Chinese Herbal Medicine Company, identified by Hubei Yichang Drug Testing Institute): 1 Control substance (China National Institute for the Control of Pharmaceutical and Biological Products, content greater than 98%).
2 Methods and results
2.1 Separation and extraction Take 20g of Xingshan Schisandra, grind it, soak it in water for 24h, then dry it, add methanol reflux to extract. Since the methanol concentration (A), the extraction time (B), the number of extractions (C), and the ratio of the methanol-sample (D) have an influence on the extraction rate, the orthogonal experiment (see the table of factors) is used for process optimization. The results showed that the factors A, B and D3 had a great influence on the separation and extraction of Schisandra. The good condition of zui is A2B1C2D3 (orthogonal result storage and editing department is available).
The extract obtained by the optimized process is evaporated to dryness, dissolved in a small amount of chloroform, and subjected to an alumina column, followed by elution with petroleum ether (30-60 ° C) and chloroform, and the chloroform eluate is collected, evaporated to dryness, and then dissolved in methanol. And make up to 10ml, shake well, as a test solution.
2.2 Chromatographic conditions optimization of mobile phase: methanol-water (85:15, 70:30, 60:40) with different ratios were used for separation and determination. The results show that as the proportion of methanol decreases, the retention time of 1 is prolonged, but other impurity peaks are significantly more. Methanol-water (85:15) was used as the mobile phase, and the linear relationship and reproducibility were good, and the separation effect was good when the flow rate was controlled at 1 ml/min.
Column temperature: The column temperature has a large effect on the chromatographic behavior of 1. If the column temperature is too high or too low, the separation of 1 and Schisandrin B from other impurities will be reduced, and even the baseline separation will not be achieved. The test uses a column temperature of 30 ° C, and the separation effect is good.
Detection wavelength: UV scanning in the wavelength range of 190-400 nm, 1 has large absorption at 220 nm, and the response is strong, so 220 nm is selected as the detection wavelength.
The optimized chromatographic conditions were: column Nova-Pak C18 (3.9 mm x 150 mm, 4 μm); mobile phase methanol-water (85:15); flow rate 1 ml/min: column temperature 30 ° C: detection wavelength 220 nm. Under this chromatographic condition, 1 retention time is approximately 3 min.
2.3 Linear test Weigh accurately to dryness to a constant weight of 1 control 3mg, placed in a 10ml volumetric flask, add methanol to dissolve and volume, shake. The measurements were accurately measured by taking 3, 5, 7, 9, 11 and 15 μ1 respectively. Taking the peak area (A) as the ordinate and the injection amount Q (μg) as the abscissa, a standard curve is drawn. A regression equation of A = 3.81 × 1000000 - 4.43 × 10000 (r = 0.9999).
2.4 Precision test 10 μl of a reference solution of 0.3 mg/ml was accurately weighed and measured continuously for 6 times in 1 d to obtain an RSD of 0.25%.
2.5 Determination of the precision of 1 in Xingshan Schisandra 1 Take the test solution for the test solution under “2.1â€. The average content of 1 in Xingshan Schisandra was calculated to be 0.22% (n=5).
references:
[1] Yang Xiaoling, Li Aimin, Schisandra Research Overview [J], Shizhen Guo Medicine, 1999, 10 (4): 300-301.
[2] Lu Jinqing, He Zehua, Zhang Baojun, et al. Study on the extraction process of Schisandra chinensis [J]. Chinese patent medicine, 1998, 20 (12): l one 2
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