CN104892687B - The method that high speed adverse current chromatogram isolates and purifies monomeric compound in Chinese mahonia leaf - Google Patents

Abstract

The invention discloses a method for rapidly separating and purifying monomeric compounds from the leaves of shigonggong, which comprises the following steps: taking the dried and pulverized shigonggong leaves, adding ethanol solution for ultrasonic extraction, filtering, and concentrating the filtrate under reduced pressure to obtain shigonggong Leaf extract; after the extract is extracted with different polar solvents, the ethyl acetate extraction section is separated by high-speed countercurrent chromatography, and the corresponding peak components are collected according to the chromatogram, concentrated and dried under reduced pressure to obtain high-purity chlorogenic acid and quercetin ‑3‑O‑β‑D‑glucoside and isorhamnetin‑3‑O‑β‑D‑glucoside overcome the shortcomings of traditional preparation methods such as complex operation, sample dead adsorption loss, and low yield. The method has the advantages of high efficiency, simple operation, large preparation amount, low comprehensive cost and good popularization and use value.

Description

Method for Separating and Purifying Monomer Compounds in Shigongong Leaves by High Speed Countercurrent Chromatography

technical field

The invention belongs to the field of natural medicinal chemistry, in particular to a rapid separation and purification of chlorogenic acid, quercetin-3-O-β-D-glucoside, isorhamnetin-3- O-β-D-glucoside method.

Background technique

The dried leaves of Mahonia bealei (Fort.) Carr. are the dried leaves of Mahonia bealei (Fort.) Carr., which has the functions of clearing heat and detoxifying, reducing inflammation and relieving pain, drying dampness and purging fire, clearing liver and improving eyesight. It is also widely used as Kudingcha in the provinces south of the Qinling Mountains. It is widely used among the people as an indication for dizziness, tinnitus, soreness of the waist and knees, damp-heat diarrhea, stomach fire and toothache, icteric hepatitis and other diseases. Modern pharmacological research has found that Shigongo has antibacterial, anti-inflammatory, anti-viral, anti-tumor, anti-proliferation and hypotensive activities, and is a medicinal plant with development value.

Most of the research on the chemical constituents of Shigonggong focused on its alkaloids, and there were no reports in the literature on the isolation of chlorogenic acid, quercetin-3-O-β-D-glucoside, and isomycin from the leaves of Shigonggong. Chlorogenic acid, quercetin-3-O-β-D-glucoside, and isorhamnetin-3 have been isolated and purified from other Chinese herbal medicines as reported in existing literature -O-β-D-glucoside and other methods often use column chromatography. Chai Xingyun et al. [Study on phenolic acids in Flos Japonica, China Natural Medicines, No. 6, 2004] with 95%, 90%, 85%, 70% ethanol reflux, ethyl acetate extraction and silica gel column, SephadexLH-20 column The method of purification and separation is to separate and purify, and identify its structure according to the physical and chemical characteristics and spectral data of the compound, and isolate compounds such as chlorogenic acid from the mountain silver flower. Quercetin-3-O-β-D-glucoside and isorhamnetin-3-O-β-D-glucoside are distributed in most higher plants. Chemical composition research, Chinese Journal of Traditional Chinese Medicine, No. 2, 2009] with 70% ethanol at room temperature extraction, ethyl acetate solvent extraction and macroporous adsorption resin, SephadexLH-20, ODS and normal phase silica gel column and other chromatographic means to separate, isolate Quercetin-3-O-β-D-glucoside. Zhao Xiaoliang et al. [Research on the chemical constituents of lotus leaves, Chinese Journal of Traditional Chinese Medicine, No. 5, 2013] used silica gel column, macroporous adsorption resin column, Sephadex LH-20 column and preparative thin-layer chromatography to analyze chemical constants and spectra. Isorhamnetin-3-O-β-D-glucoside was isolated from the n-butanol fraction of lotus leaf.

These components are usually subjected to repeated column chromatography, which is complicated, time-consuming, consumes a lot of solvent, and the sample loss is large, and the repeat effect is not ideal.

High-speed counter-current chromatography (High-Speed Counter-Current Chromatography, HSCCC) is a new type of continuous and efficient liquid-liquid distribution chromatography invented by Ito et al. It is based on a special dynamic equilibrium phenomenon - unidirectional fluid A separation method on top of a dynamic equilibrium (HDES) system. Compared with the conventional solid-liquid chromatography separation method, since the liquid is used as the stationary phase carrier, the target compound can be separated in the two incompatible phases due to the difference in distribution coefficient, which solves the problem of dead adsorption, denaturation, and peak of the traditional solid carrier sample. In order to solve problems such as shape tailing, using high-speed countercurrent chromatography not only greatly improves the separation speed, but also can collect samples well and repeatedly according to the ultraviolet absorption chromatogram. It has the advantages of high preparation efficiency, large preparation volume, and low cost. People's attention has been widely used in the fields of biomedicine, natural products, environmental analysis, food and cosmetics, especially in the natural product industry has been considered as an effective new separation technology.

Contents of the invention

The object of the present invention is: to overcome the deficiencies of the prior art, to provide an efficient and rapid separation and purification of chlorogenic acid, quercetin Method for cortex-3-O-β-D-glucoside, isorhamnetin-3-O-β-D-glucoside.

The technical solution of the present invention is to separate and purify chlorogenic acid, quercetin-3-O-β-D-glucoside and isorhamnetin-3-O-β-D-glucoside from the leaves The method comprises the following process steps:

(1) Preparation of the leaf extract of Shida Gonggong: Dry the leaves of Dabai Gonggong in the shade, pulverize them, use pure ethanol as solvent for ultrasonic extraction, the solid-liquid ratio is 1:20, the extraction time is 2 h, filter, and repeat the treatment of the filter residue twice; combine the filtrates And concentrated under reduced pressure in vacuum to obtain the crude extract of the leaves of Shida Gonggong;

(2) Disperse the crude extract in step 1 with water, extract with normal hexane, dichloromethane, ethyl acetate and n-butanol respectively, filter the extract, concentrate under reduced pressure to obtain normal hexane, dichloromethane and ethyl acetate respectively Ester, n-butanol and water extraction section, 4 ° C refrigerator for future use;

(3) Separation of monomers: using n-hexane-methanol-ethyl acetate water as the solvent system, the upper phase is the stationary phase, the lower phase is the mobile phase, and the separation column rotation speed is 700-900 rpm. HSCCC separation is carried out in the ester extraction section, and the ultraviolet detector is monitored online. Different fractions are collected and dried under reduced pressure to obtain chlorogenic acid, quercetin-3-O-β-D-glucoside, and isorhamnetin-3-O - beta-D-glucoside.

The volume ratio of n-hexane-methanol-ethyl acetate-water in step (3) is 1:1:1:1-1:8:1:8, preferably 1:5:1:5.

The flow rate of the mobile phase in step (3) was 2 mL/min.

The advantages of the present invention are: after the separation of the ten major meritorious service leaf extracts, the purity of each fraction can reach more than 85% through HPLC, and each fraction can be further purified to obtain more than 95% pure product chlorogenic acid, quercetin-3-O- β-D-glucoside, isorhamnetin-3-O-β-D-glucoside, the method is suitable for one-step purification and preparation of high-purity chlorogenic acid, quercetin-3-O -β-D-glucoside, Isorhamnetin-3-O-β-D-glucoside. Good stability and easy operation.

Description of drawings

Fig. 1 is the chromatogram of the separation and purification of ten merit leaves by high-speed countercurrent chromatography;

Figure 2 is a high performance liquid chromatogram of the separated and purified chlorogenic acid;

Figure 3 is the HPLC chromatogram of the isolated and purified quercetin-3-O-β-D-glucoside;

Figure 4 is the HPLC chromatogram of the isolated and purified isorhamnetin-3-O-β-D-glucoside.

detailed description

Take 5.0 kg of Shibagongong leaves that were naturally dried in the shade, crush them, and extract pure ethanol as solvent ultrasonically, the solid-to-liquid ratio was 1:20, the extraction time was 2 h, filtered, and the filter residue was treated twice; the filtrates were combined and concentrated under reduced pressure to obtain ten The crude extract of Dagong leaves; the crude extract was dispersed with water, extracted with n-hexane, dichloromethane, ethyl acetate and n-butanol respectively, the extract was filtered, concentrated under reduced pressure to obtain n-hexane, dichloromethane and ethyl acetate respectively Ester, n-butanol and water extraction sections, stored in a 4°C refrigerator for later use. Use n-hexane-methanol-ethyl acetate water with a volume ratio of 1:5:1:5 as the solvent system, the flow rate of the mobile phase (lower phase) is 2 mL/min, the rotation speed of the separation column is 900 rpm, and the upper phase is the stationary phase. The HSCCC separation was carried out on the ethyl acetate extraction section of the top ten leaves, and the ultraviolet detector was monitored online. The detection wavelength was 254 nm, and the target components were collected. The fractions were concentrated and dried under reduced pressure to obtain the corresponding high-purity compounds, as shown in Figure 1.

The chemical structure identified by 1H-NMR and 13C-NMR is as follows,

The resulting monomeric compounds were 18.32 mg of chlorogenic acid, 20.46 mg of quercetin-3-O-β-D-glucoside, and 28.36 mg of isorhamnetin-3-O-β-D-glucoside. Calculated by the peak area normalization method, the purity was higher than 97%, as shown in Figure 2-Figure 4.

Claims (4)

1. A method for separating and purifying chlorogenic acid, quercetin-3-O-β-D-glucoside, and isorhamnetin-3-O-β-D-glucoside in Shigonggong leaves by high-speed countercurrent chromatography, including It is characterized in that it comprises the following process steps: (1) Preparation of the extract of the top ten contributive leaves: dry the ten contributive leaves in the shade, pulverize them, use pure ethanol as the solvent for ultrasonic extraction, the solid-to-liquid ratio is 1:20, the extraction time is 2 h, filter, and repeat the treatment of the filter residue twice; combine the filtrates And concentrated under reduced pressure in vacuum to obtain the crude extract of the leaves of Shida Gonggong; (2) Disperse the crude extract in step (1) with water, extract with n-hexane, dichloromethane, ethyl acetate, n-butanol respectively, filter the extract, concentrate under reduced pressure to obtain n-hexane, dichloromethane, Ethyl acetate, n-butanol and water extraction section, 4 ° C refrigerator for future use; (3) Separation of monomers: using n-hexane-methanol-ethyl acetate-water as the solvent system, the upper phase is the stationary phase, the lower phase is the mobile phase, and the separation column speed is 700-900 rpm. HSCCC is separated in the ethyl ester extraction section, and the ultraviolet detector is monitored online. Different fractions are collected and dried under reduced pressure to obtain chlorogenic acid, quercetin-3-O-β-D-glucoside, isorhamnetin-3- O-beta-D-glucoside. 2. Chlorogenic acid, quercetin-3-O-β-D-glucoside, and isorhamnetin-3-O-β-D in the leaves of Shigonggonggong for separation and purification by high-speed countercurrent chromatography according to claim 1 -The method of glucoside, it is characterized in that, the volume ratio of the n-hexane-methanol-ethyl acetate-water described in step (3) is 1:1:1:1-1:8:1:8. 3. Chlorogenic acid, quercetin-3-O-β-D-glucoside, and isorhamnetin-3-O-β-D in the leaves of Shigonggonggong for separation and purification by high-speed countercurrent chromatography according to claim 2 - The method of glucoside, characterized in that the volume ratio of n-hexane-methanol-ethyl acetate-water in step (3) is 1:5:1:5. 4. Chlorogenic acid, quercetin-3-O-β-D-glucoside, isorhamnetin-3- O-β-D-glucoside method, characterized in that the flow rate of the mobile phase in step (3) is 2 mL/min.