Preparation method of dencichine

Abstract

The invention relates to a preparation method and application of dencichine, and belongs to the field of Chinese herbal medicine extraction and separation. The method comprises the following specific steps: taking pseudo-ginseng, pseudo-ginseng flowers, pseudo-ginseng stems and leaves, a whole pseudo-ginseng plant, decoction dregs after notoginsenoside extraction, rhizoma panacis majoris and American ginseng as raw materials, performing coarse crushing or direct feeding, performing water extraction, concentrating an extracting solution, performing centrifugation, enabling centrifugate to pass through an ultrafiltration membrane, concentrating a membrane-passing solution, purifying a concentrated solution through cation exchange resin, collecting a dencichine-containing segment, and performing freeze drying to obtain the pseudo-ginseng extract. And concentrating and drying to obtain dencichine. And selecting an ultrafiltration membrane to remove impurities and enrich dencichine. And purifying the dencichine by selecting ion exchange resin to obtain the dencichine with the content of more than 95%. The method has the advantages that the operation steps are simple and easy to repeat, the conditions are easy to control, the preparation amount is easy to amplify, the separation material can be repeatedly used, the environmental pollution is small, the obtained final product does not contain organic solvent residues, and the content can reach 95% or above.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a method for extracting and preparing notoginseng extract from Panax notoginseng, Panax notoginseng flowers, Panax notoginseng stems and leaves, the whole Panax notoginseng plant, the residue after extracting notoginseng saponins, Panax notoginseng, and American ginseng. Background Technology

[0002] Panax notoginseng (Burkill) FH Chen ex CH Chow is a perennial herb belonging to the genus Panax in the family Araliaceae. It is native to China and distributed in Yunnan and Guangxi provinces, primarily through cultivation. Panax notoginseng is warm in nature and has a sweet and slightly bitter taste. It enters the liver and stomach meridians. It possesses the effects of nourishing blood, reducing inflammation, promoting blood circulation, and removing blood stasis. It also has antioxidant and anti-aging properties. Panax notoginseng can dilate blood vessels, reduce coronary artery resistance, increase coronary blood flow, strengthen and improve coronary microcirculation, increase nutritional myocardial blood flow, lower arterial pressure, slightly reduce heart rate, and thus significantly reduce myocardial oxygen consumption.

[0003] Dencichine, also known as Panax notoginseng amino acid, is the main hemostatic active ingredient in Panax notoginseng, a precious traditional Chinese medicine belonging to the Araliaceae family. This component has also been found in other ginseng, such as American ginseng, Korean ginseng, and Panax quinquefolius. Dencichine, chemically named β-oxalyl-L-α,β-diaminopropionic acid (β-ODAP), has the molecular formula C5H8N2O5 and a relative molecular mass of 176.13. It is a water-soluble non-protein amino acid. Modern medical research shows that dencichine possesses various pharmacological activities, including hemostasis, neuroprotection, anti-inflammation, hypoglycemia, and mitigation of diabetic nephropathy damage.

[0004] Literature review indicates that notoginsenosides are mainly isolated from Panax notoginseng, Panax notoginseng residue, or notoginsenoside extract. Liang Ruian et al. (Method for extracting and separating notoginsenoside R-(1), ginsenoside Re, and notoginsenoside from Panax notoginseng powder [P], Chinese Patent, CN1166679C, 2004-09-15), Jia Wei et al. (Method for extracting notoginsenoside from Panax notoginseng [P], Chinese Patent, CN100336801C, 2007-09-12), Yu Fadong (An apparatus and extraction method for extracting notoginsenoside from Panax notoginseng [P]. Chinese Patent, CN108675939A, 2018-10-19), Li Mingjin (Preparation method and apparatus for notoginsenoside diol, notoginsenoside triol saponins and notoginsenoside [P]. Chinese Patent, CN110845565A, 2020-02-28), Chen Tong (Extraction method and application of notoginsenoside and extraction method of notoginseng amino acids [P]. Chinese patents CN107162926B (2021-05-14), Yang Lingjuan (Method for separating and purifying notoginseng from legumes [P]), and Zhu Yunrui (A method for recovering notoginseng from total saponin extract of Panax notoginseng [P]), mainly employ the following methods: extraction with methanol or ethanol, followed by water extraction of the residue, or water extraction followed by alcohol precipitation, or alcohol extraction followed by macroporous adsorption resin, or water extraction followed by addition of inorganic salts. The notoginseng is then enriched using cation exchange resin, and finally purified by crystallization or dextran gel column chromatography. The methods described above all involve some impurity removal before, after, or after water extraction or alcohol extraction. However, amino acids, peptides, and water-soluble proteins with similar properties to notoginseng are not removed during this process. These substances are the main factors affecting the utilization rate of ion exchange resins and the purification effect of notoginseng. Therefore, how to effectively remove these substances from the extract and improve the utilization rate of ion exchange resins and the purification effect of notoginseng has become an urgent technical problem to be solved. This patent proposes a feasible method for this purpose. This invention uses an ultrafiltration membrane to remove most of the amino acids, polypeptides, and water-soluble proteins that are similar to notoginseng, making it easier for notoginseng to bind to the ion sites in the cation exchange resin. This eliminates the possibility of similar substances competing for ion sites in the ion exchange resin and also eliminates the possibility of similar substances being indiscriminately adsorbed. This allows notoginseng to be directly purified after passing through the cation exchange resin column, eliminating the need for further purification. Compared with previous patents, this invention greatly reduces the purification steps. Furthermore, the main solvents used in the preparation of notoginseng in this invention are water and ammonia, making the process simple and feasible, suitable for large-scale industrial production, with low toxicity. The ultrafiltration membrane and ion exchange resin used can be reused, resulting in less environmental pollution, and the final product is basically free of organic solvent residues. Summary of the Invention

[0005] The present invention aims to provide a method and application for extracting and preparing notoginseng from plants or medicinal residues containing notoginseng (including notoginseng rhizomes, notoginseng flowers, notoginseng stems and leaves, whole notoginseng plant, Panax notoginseng, and American ginseng). The structure of the notoginseng is shown below:

[0006]

[0007] This invention aims to utilize ultrafiltration membranes for pretreatment of notoginseng extract to improve the utilization rate of ion exchange resins and the purification effect of notoginseng. The specific principle is as follows: Notoginseng has a molecular weight of 176.13. The extract containing notoginseng is passed sequentially through ultrafiltration membranes with molecular weights of 150 and 200-3000. This concentrates the molecular weight of the solute in the extract between 150-200 and 3000, enriching notoginseng while maximally removing amino acids, peptides, and water-soluble proteins with similar properties to notoginseng. This improves the specificity of the ion exchange process, increases the utilization rate of the ion exchange resin, and enhances the purification effect of notoginseng. It also eliminates the need for subsequent purification steps such as crystallization or dextran gel column chromatography, simplifying the preparation process of notoginseng.

[0008] A method for preparing high-purity Panax notoginseng extract, characterized in that the preparation method includes the following steps:

[0009] 1) Coarse powder of Panax notoginseng or raw materials containing Panax notoginseng (Panax notoginseng flowers, stems and leaves, whole plant of Panax notoginseng, residue after extraction of Panax notoginseng saponins, Panax ginseng, American ginseng, etc.) are extracted with water at a material-to-liquid ratio of 1:5 to 1:50, 1 to 5 times, each time for 30 to 300 minutes, at an extraction temperature of 50℃ to 100℃. The extract is filtered, concentrated to a material-to-liquid mass-to-volume ratio of 1:5 to 1:35, centrifuged, and set aside.

[0010] The above water extraction methods include, but are not limited to, hot reflux, percolation, and cold soaking.

[0011] 2) The concentrate obtained in step 1) is separated using a membrane separation device. The ultrafiltration membrane (with a molecular weight of 150) is washed with 1 to 10 times the volume of the concentrate with water to remove impurities with smaller molecular weights. The retentate is collected for later use.

[0012] 3) The retentate obtained in step 2) is separated using a membrane separation device. The ultrafiltration membrane (with a molecular weight of 200-3000) is eluted with 1-10 times the volume of the retentate with water to mainly remove large molecular weight peptides, proteins and other components. The membrane solution is collected and concentrated to obtain a crude extract rich in Panax notoginseng.

[0013] 4) The crude extract obtained in step 3) is eluted with the treated cation exchange resin, water and ammonia solution, and the ammonia eluent is collected and concentrated under reduced pressure to dryness to obtain Panax notoginseng extract.

[0014] The ultrafiltration membrane used in step 2) is an ultrafiltration membrane with a molecular weight of 150.

[0015] The ultrafiltration membrane used in step 3) is an ultrafiltration membrane with a molecular weight of 200 to 3000.

[0016] The ion exchange resin used in step 4) is a cation exchange resin, model D001 or 001×7.

[0017] In step 4), the concentration of the ammonia solution is 0.2 mol / L.

[0018] 5) The content of notoginsenoside was detected by high performance liquid chromatography (HPLC). The detection method included:

[0019] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the packing material; 0.3% tetrabutylammonium hydroxide (pH adjusted to 4.0 with phosphate buffer) was used as mobile phase A, and acetonitrile was used as mobile phase B, with gradient elution performed according to the specifications in Table 1; column temperature was 30℃; flow rate was 0.8 ml / min; detection wavelength was 220 nm.

[0020]

[0021] Preparation of reference solution: Take an appropriate amount of notoginseng reference standard, accurately weigh it, add water to prepare a solution containing 0.2 mg of notoginseng per ml.

[0022] Preparation of the test solution: Take an appropriate amount of the powder of this product, accurately weigh it, add water to prepare a solution containing 0.4 mg per 1 ml.

[0023] Assay: Accurately pipette 10 μl each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0024] Step 5) HPLC analysis: The sample prepared in step 4) was analyzed by HPLC using a high performance liquid chromatograph.

[0025] The method for preparing notoginseng extract yields notoginseng extract with a purity greater than 95% and a transfer rate of 59% to 82% as detected by HPLC. Attached Figure Description

[0026] Figure 1 This is the high-performance liquid chromatogram of notoginseng reference standard.

[0027] Figure 2 This is a high-performance liquid chromatogram of the notoginseng sample obtained in this invention.

[0028] Figure 3 This is a chromatogram showing the purity of the notoginseng chromatogram peak in the notoginseng sample obtained by this invention. Detailed Implementation

[0029] The present invention will be further described in detail below through embodiments. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters according to the specific actual equipment to implement the invention. In particular, all similar substitutions or modifications are obvious to those skilled in the art and are also considered to be included in the present invention. The method of the present invention has been described in detail for the preparation of Panax notoginseng. Those skilled in the art can modify or appropriately change and combine the methods described herein without departing from the content, spirit and scope of the present invention to implement and apply the method of the present invention. However, the scope of protection of the present invention is not limited to the content described herein. Unless otherwise specified, the methods in the embodiments are conventional methods, and the reagents used are conventional commercially available reagents or reagents prepared according to conventional methods.

[0030] Example 1:

[0031] 1) Panax notoginseng, 50 kg, extracted by reflux with water, material-to-liquid ratio 1:10, extracted 3 times, 60 minutes each time, extraction temperature 100℃, the extract was filtered, combined and vacuum concentrated to material-to-liquid mass-to-volume ratio 1:35, centrifuged, and the supernatant was used for later use.

[0032] 2) The supernatant obtained in step 1) is separated using a membrane separation device. The ultrafiltration membrane (with a molecular weight cutoff of 150) is eluted with 5 times the volume of concentrated liquid and the retentate is collected.

[0033] 3) The retentate obtained in step 2) is separated using a membrane separation device. The ultrafiltration membrane (with a molecular weight cutoff of 1000) is eluted with 5 times the volume of the retentate in water. The filtered liquid is collected, concentrated, and then the Panax notoginseng concentrate is obtained.

[0034] 4) The Gynostemma pentaphyllum concentrate obtained in step 3) was eluted on a pretreated D001 cation exchange resin column with 3 column volumes of water, and then eluted with 8 column volumes of 0.2 mol / L ammonia solution. The ammonia eluent was collected and concentrated to dryness under reduced pressure to obtain the desired Gynostemma pentaphyllum. The Gynostemma pentaphyllum was detected by HPLC and the content was 98.8% with 1.3% moisture.

[0035] Example 2:

[0036] 1) 100 kg of Panax notoginseng stems and leaves were extracted by water reflux at a ratio of 1:50. The extraction was performed once for 300 minutes at a temperature of 95°C. The extracts were filtered, combined, and concentrated under vacuum until the ratio of material to liquid mass was 1:10. The extracts were then centrifuged and the supernatant was kept for later use.

[0037] 2) The supernatant obtained in step 1) is separated using a membrane separation device. The ultrafiltration membrane (with a molecular weight cutoff of 150) is eluted with 7 times the volume of concentrated liquid and the retentate is collected.

[0038] 3) The retentate obtained in step 2) is separated using a membrane separation device. The ultrafiltration membrane (with a molecular weight cutoff of 200) is eluted with water at a volume of 7 times that of the retentate. The membrane solution is collected, concentrated, and then the Panax notoginseng concentrate is obtained.

[0039] 4) The 001×7 cation exchange resin column on which the ginsenoside concentrate obtained in step 3) was prepared was eluted with 4 column volumes of water and then with 6 column volumes of 0.2 mol / L ammonia solution. The ammonia eluent was collected and concentrated to dryness under reduced pressure to obtain the desired ginsenoside. The ginsenoside content was 96.1% and the water content was 1.4% as determined by HPLC.

[0040] Example 3:

[0041] 1) 50 kg of Panax notoginseng flowers were extracted by water reflux at a ratio of 1:20. The extraction was performed twice, each time for 120 minutes at a temperature of 50°C. The extracts were filtered, combined, and concentrated under vacuum until the mass-to-volume ratio of the extract to the liquid was 1:5. The extracts were then centrifuged and the supernatant was kept for later use.

[0042] 2) The supernatant obtained in step 1) is separated using a membrane separation device. The ultrafiltration membrane (with a molecular weight cutoff of 150) is eluted with 4 times the volume of concentrated liquid and the retentate is collected.

[0043] 3) The retentate obtained in step 2) is separated using a membrane separation device. The ultrafiltration membrane (with a molecular weight cutoff of 500) is eluted with water at a volume of 4 times that of the retentate. The membrane solution is collected, concentrated, and then the Panax notoginseng concentrate is obtained.

[0044] 4) The Gynostemma pentaphyllum concentrate obtained in step 3) was eluted on a pretreated D001 cation exchange resin column with 2 column volumes of water, and then eluted with 7 column volumes of 0.2 mol / L ammonia solution. The ammonia eluent was collected and concentrated to dryness under reduced pressure to obtain the desired Gynostemma pentaphyllum. The Gynostemma pentaphyllum was detected by HPLC and the content was 97.5% with 1.8% moisture.

[0045] Example 4:

[0046] 1) Take 100 kg of residue after extracting Panax notoginseng saponins, coarsely crush it, extract it by water reflux at a ratio of 1:5, extract it 5 times, 30 minutes each time, at an extraction temperature of 80℃, filter it, combine the filtrates, concentrate it under vacuum until the mass-to-volume ratio of the material to the liquid is 1:6, centrifuge it, and keep the supernatant for later use.

[0047] 2) The supernatant obtained in step 1) is separated using a membrane separation device. The ultrafiltration membrane (with a molecular weight cutoff of 150) is eluted with water at a volume of 9 times that of the concentrate, and the retentate is collected.

[0048] 3) The retentate obtained in step 2) is separated using a membrane separation device. The ultrafiltration membrane (with a molecular weight cutoff of 3000) is eluted with 6 times the volume of the retentate in water. The membrane solution is collected, concentrated, and then the Panax notoginseng concentrate is obtained.

[0049] 4) The Gynostemma pentaphyllum concentrate obtained in step 3) was eluted on a pretreated D001 cation exchange resin column with 3 column volumes of water, and then eluted with 9 column volumes of 0.2 mol / L ammonia solution. The ammonia eluent was collected and concentrated to dryness under reduced pressure to obtain the desired Gynostemma pentaphyllum. The Gynostemma pentaphyllum was detected by HPLC and the content was 99.6% with 1.2% moisture.

[0050] Example 5:

[0051] 1) 150 kg of whole Panax notoginseng plant, coarsely crushed, extracted by water percolation, with a material-to-liquid ratio of 1:50. The extract was filtered, combined, and vacuum concentrated until the material-to-liquid mass-to-volume ratio was 1:10. After centrifugation, the supernatant was used for later use.

[0052] 2) The supernatant obtained in step 1) is separated using a membrane separation device. The ultrafiltration membrane (with a molecular weight cutoff of 150) is eluted with 5 times the volume of concentrated liquid and the retentate is collected.

[0053] 3) The retentate obtained in step 2) is separated using a membrane separation device. The ultrafiltration membrane (with a molecular weight cutoff of 200) is eluted with water at a volume of 7 times that of the retentate. The membrane solution is collected, concentrated, and then the Panax notoginseng concentrate is obtained.

[0054] 4) The 001×7 cation exchange resin column treated with the ginsenoside concentrate obtained in step 3) was eluted with 4 column volumes of water and then eluted with 6 column volumes of 0.2 mol / L ammonia solution. The ammonia eluent was collected and concentrated to dryness under reduced pressure to obtain the desired ginsenoside. The ginsenoside content was 98.6% and the moisture content was 1.6% as determined by HPLC.

[0055] The examples are summarized in Table 2.

[0056]

Claims

1. A method for preparing panaxin, characterized by the following steps: 1) using panax notoginseng, panax notoginseng flower, panax notoginseng stem and leaf, panax notoginseng whole plant, residue after extracting panax notoginseng saponin, panax japonicus, panax quinquefolium as raw materials, coarsely crushing or directly feeding, water extraction, solid-liquid ratio 1:5-1:50, extracting 1-5 times, each time 30 minutes-300 minutes, extraction temperature 50℃-100℃, filtering the extraction liquid, concentrating to solid-liquid mass ratio 1:5-1:35, centrifuging, and reserving; The water extraction includes but is not limited to hot reflux, percolation and cold soaking; 2) using membrane separation equipment to separate the concentrated liquid obtained in step 1), using 1-10 times volume of water to elute the ultrafiltration membrane with molecular weight 150, mainly removing impurities with small molecular weight, collecting the cut-off liquid, and reserving; 3) using membrane separation equipment to separate the cut-off liquid obtained in step 2), using 1-10 times volume of water to elute the ultrafiltration membrane with molecular weight 200-3000, mainly removing polypeptide and protein components with large molecular weight, collecting the membrane-passed liquid, and concentrating, thereby obtaining concentrated liquid rich in panaxin; 4) using the concentrated liquid obtained in step 3) to elute the treated cation resin, collecting the ammonia water eluate, and concentrating the ammonia water eluate to dryness under reduced pressure, thereby obtaining panaxin.

2. The method of claim 1, wherein the gypenoside is prepared by the steps of: The panax notoginseng is the root and rhizome of panax notoginseng; the panaxin-containing raw material is one or more of panax notoginseng flower, panax notoginseng stem and leaf, panax notoginseng whole plant, residue after extracting panax notoginseng saponin, panax japonicus and panax quinquefolium. ​ 3. The method of claim 1, wherein the gypenoside is prepared by the steps of: The membrane separation equipment in step 2) is WTM-1812G membrane separation equipment or WTM-4040G-3 organic membrane equipment or KM1812-UF membrane separation equipment. ​ 4. The method of claim 1, wherein the gypenoside is prepared by the steps of: The membrane in step 2) has a specification of 150 molecular weight and 200-3000 molecular weight. ​ 5. The method for preparing notoginseng extract according to claim 1, characterized in that, The cation resin in step 4) is D001 or 001×7.

6. The method for preparing notoginseng extract according to claim 1, characterized in that, The ammonia water solution in step 4) has a concentration of 0.2 mol / L.

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