CN110478437B - Dendrobium stem extract and preparation method thereof - Google Patents
Dendrobium stem extract and preparation method thereof Download PDFInfo
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Abstract
The invention provides a dendrobium nobile extract and a preparation method thereof, wherein, dried dendrobium nobile stems are taken as raw materials, and are soaked and percolated after being crushed to obtain percolate; adjusting pH with lime milk, loading onto macroporous resin column, eluting, adjusting pH of eluate to neutral, and concentrating under reduced pressure to obtain fluid extract; mixing the fluid extract with silica gel, subjecting to silica gel column distribution chromatography to obtain eluate, recovering solution, recrystallizing, drying, and pulverizing to obtain herba Dendrobii extract. The content of total alkaloids in the dendrobium nobile lindl extract is more than 50 percent. The method has the advantages of reducing the use of organic solvents, reducing the risk of harming the body of production technicians by toxic organic solvents, and reducing the production cost and energy consumption.
Description
Technical Field
The invention belongs to the field of traditional Chinese medicine extracts, and particularly relates to a dendrobium nobile lindl extract and a preparation method thereof.
Background
Herba Dendrobii is dried stem of herba Dendrobii (Dendrobium nobile Lindl) of Orchidaceae, and has effects of benefiting stomach, promoting fluid production, nourishing yin and clearing heat. Dendrobium nobile Lindl contains various alkaloids such as dendrobine, and modern research shows that the total alkaloids have activities of resisting diabetes, hyperlipemia and senile dementia (Huangqi, Dendrobium nobile Lindl total alkaloids have protective effect on diabetic rat model, Zunyi medical college, 2009; Chenjing, Dendrobium nobile Lindl total alkaloids to ApoE-/-Effects of mouse hyperlipidemia and vascular atherosclerosis Zunyi medical school, 2016; influences of dendrobium nobile total alkaloids on learning and memory abilities of APP/PS1 transgenic mice, Zhanyiacai, 2016,39(3): 246-; dendrobium nobile Total alkaloid improves rat hippocampal neuron injury caused by streptozotocin, China New drug and clinical journal, 2017,36(6):340-]Journal of agricultural and food chemistry,2017,65(18): 3665-. Therefore, the dendrobium nobile total alkaloids have good potential in the aspect of new drug development. According to the regulations of 'Chinese medicine and natural medicine registration classification and declaration data requirements' in annex 1 of 'medicine registration management method', the effective component content of the fifth kind of new medicine is more than 50% of extract. Therefore, the development of the method for extracting and purifying the dendrobium nobile total alkaloids is significant. Regarding the extraction and purification method of dendrobium nobile alkaloid, some technical proposals (CN 101653563B; CN 101537128B; CN 108175831A; CN 101735231B) exist, but the defects are that a large amount of organic solvent is adopted, the production cost is high, and the energy consumption is large; in addition, chloroform treatment (CN 101537128B; CN 104557956A; Zhaofkun, Wu celery, Yuanfu, etc. improvement of the extraction and content determination method of total alkaloids of dendrobium nobile Lindl. Zhanyi medical college bulletin 2015,38(5):532 + 535; Cao Yun Li, Pan from Red, Zhao Xiao Jun. dynamic analysis of alkaloid extraction from dendrobium nobile Lindl. Ping shan college bulletin 2017,32(5): 44-49) is adopted in some technical schemes, and the defects are overcomeChloroform has high toxicity and great risk of harming the bodies of production technicians.
Disclosure of Invention
The invention overcomes the defects of the prior art and provides a preparation method of the dendrobium nobile lindl extract, which mainly uses aqueous solvent for extraction and separation and uses a small amount of organic solvent for treatment, and the content of the dendrobium nobile lindl total alkaloids in the prepared extract reaches more than 50 percent.
The technical scheme of the invention is as follows:
the extract is prepared by using dried stem of Dendrobium nobile Lindl (Dendrobium nobile Lindl) of Orchidaceae as raw material according to the following steps:
(1) crushing dendrobium nobile into granules;
(2) taking the granular raw materials, adding a solvent to soak for 6-12 h, then percolating, and collecting percolate with the volume 10-50 times of the weight of the raw materials;
the solvent is a hydrochloric acid aqueous solution with the pH value of 1-4 and containing a mixture of 1-5% by mass of glycine and ammonium acetate, and the molar ratio of glycine to ammonium acetate is 1: 1-1: 3;
(3) adjusting the pH of the percolate to 6-7 by using lime milk, passing the percolate through a D101 or AB-8 or macroporous resin column with similar polarity, washing the percolate with pure water with the volume of 3 times of the volume of a column bed, discarding a washing liquid, eluting the percolate with hydrochloric acid aqueous solution with the pH of 1-4 with the volume of 3-6 times of the volume of the column bed, and collecting an eluent A;
(4) taking the eluent A, adjusting pH to neutral with saturated sodium carbonate aqueous solution, and concentrating under reduced pressure to obtain fluid extract;
(5) taking the clear paste, uniformly stirring the clear paste with 80-mesh silica gel in equal quantity, then carrying out silica gel column distribution chromatography, and eluting with ethyl acetate with the volume of 3-6 times of that of a column bed to obtain eluent B;
the distribution chromatography is carried out, wherein a supporting agent is 80-mesh silica gel, a stationary phase is a saturated sodium carbonate aqueous solution (saturated by ethyl acetate in advance), and an eluent is ethyl acetate;
(6) taking the eluent B, recovering the solvent, and treating with methanol for 1-3 times according to a recrystallization treatment method;
(7) and (4) taking the solid after recrystallization, drying at 60 ℃, and crushing to obtain the dendrobium nobile extract.
Preferably, in the step (2), the raw materials are soaked for 8 hours, and the percolate with the volume being 30 times of the weight of the raw materials is collected.
Preferably, in step (2), the solvent is 3% glycine and ammonium acetate mixture in 1:1 molar ratio, and the pH is 2 hydrochloric acid aqueous solution.
Preferably, in step (3), the pH is adjusted to 6.5 with lime milk, and the mixture is loaded on a D101 macroporous resin column and eluted with 4.5 column volumes of hydrochloric acid aqueous solution with pH 2.
Preferably, in step (5), the column is eluted with 4 bed volumes of ethyl acetate.
Preferably, in step (6), the treatment with methanol is carried out 2 times.
In the step (2), the percolation extraction may be assisted by microwave or ultrasonic treatment.
In the step (6), methanol may be replaced by ethanol or ethyl acetate.
The alkaloid components of the dendrobium nobile are mostly lipophilic, and the dendrobium nobile extract is extracted by salifying acid water and alkaloid; glycine and ammonium acetate which are hydrogen bond donors and hydrogen bond acceptors are added into the solvent, so that the dendrobium nobile alkaloid can form hydrogen bonds, and the dissolving assisting effect is achieved. The invention ensures that the dendrobium nobile alkaloids are in a salified state or a free state by adjusting the pH value, so as to facilitate the dendrobium nobile alkaloids to be dissolved or adsorbed. The invention can remove impurities such as saccharides, amino acids, proteins and the like by macroporous resin column chromatography and water washing. According to the invention, by silica gel column distribution chromatography with the stationary phase being saturated sodium carbonate aqueous solution, some phenolic acid impurities can enter the stationary phase to be separated from the dendrobium nobile alkaloid.
The amount of the organic solvent used in the invention is far lower than that of the prior art, the cost of the organic solvent is low, and the links needing heating are few, so the energy consumption is low.
The organic solvent used in the invention is only alcohol solvent and ethyl acetate, and common chloroform treatment method is not adopted, so that the risk of harming the body of production technicians is very small.
The macroporous resin column chromatography and the silica gel column distribution chromatography in the invention can be repeatedly used, thereby reducing the production cost.
The dendrobium nobile lindl extract has the advantages of total alkaloid content of more than 50 percent, higher active ingredient content and lower production cost, and is the outstanding advantage of the dendrobium nobile lindl extract.
Detailed Description
The present invention is further illustrated in detail by the following specific examples, it being understood that the specific examples described herein are for the purpose of illustration only and are not intended to limit the invention, and that various changes may be made within the scope of the invention as defined by the claims.
Example 1
The preparation method of the dendrobium nobile extract comprises the following steps in sequence:
(1) taking 200g of dendrobium nobile medicinal material, and crushing into granules;
(2) taking the granular raw materials, adding a solvent, soaking for 8 hours, then percolating, and collecting percolate with the volume being 30 times of the weight of the raw materials;
the solvent is hydrochloric acid aqueous solution with the pH value of 2 and containing 3% of mixture of glycine and ammonium acetate, and the molar ratio of the glycine to the ammonium acetate is 1:1.
(3) Adjusting pH of the percolate with lime milk to 6.5, separating with D101 macroporous resin column, washing with 3 times of pure water, discarding water washing solution, eluting with 4.5 times of hydrochloric acid aqueous solution of pH 2, and collecting eluate A;
(4) taking the eluent A, adjusting pH to neutral with saturated sodium carbonate aqueous solution, and concentrating under reduced pressure to obtain fluid extract;
(5) mixing the fluid extract with 80 mesh silica gel, performing silica gel column distribution chromatography, and eluting with 4 times of column bed volume of ethyl acetate to obtain eluate B;
the distribution chromatography is carried out by using 80-mesh silica gel as a supporting agent, using saturated sodium carbonate aqueous solution (saturated by ethyl acetate in advance) as a stationary phase and using ethyl acetate as an eluent.
(6) Collecting eluate B, recovering solvent, and treating with methanol for 2 times according to recrystallization method;
(7) and (4) taking the solid after recrystallization, drying at 60 ℃, and crushing to obtain the dendrobium nobile extract.
Example 2
The preparation method of the dendrobium nobile extract comprises the following steps in sequence:
(1) taking 200g of dendrobium nobile medicinal material, and crushing into granules;
(2) taking the granular raw materials, adding a solvent, soaking for 12 hours, then percolating, and collecting percolate with the volume 50 times of the weight of the raw materials;
the solvent is a hydrochloric acid aqueous solution with the pH value of 1.5 and containing 4% of a mixture of glycine and ammonium acetate, and the molar ratio of the glycine to the ammonium acetate is 1: 1.5.
(3) Adjusting pH of the percolate to 7 with lime milk, loading onto D101 macroporous resin column, washing with 3 times of pure water, discarding water washing solution, eluting with 6 times of hydrochloric acid aqueous solution with pH of 1.5, and collecting eluate A;
(4) taking the eluent A, adjusting pH to neutral with saturated sodium carbonate aqueous solution, and concentrating under reduced pressure to obtain fluid extract;
(5) mixing the fluid extract with 80 mesh silica gel, performing silica gel column distribution chromatography, and eluting with 5 times of column bed volume of ethyl acetate to obtain eluate B;
the distribution chromatography is carried out by using 80-mesh silica gel as a supporting agent, using saturated sodium carbonate aqueous solution (saturated by ethyl acetate in advance) as a stationary phase and using ethyl acetate as an eluent.
The subsequent treatment was the same as in example 1.
Comparative example 1
Compared with example 1, the difference is only that: in the step (2), the solvent is hydrochloric acid aqueous solution with pH 2, and glycine and ammonium acetate are not added.
Comparative example 2
Compared with example 1, the difference is only that: in the step (3), the percolate is taken and treated with lime milk to adjust the pH to 6.5, and then the step (4) "decompression concentration to obtain clear paste" is directly carried out without macroporous resin column chromatography.
Comparative example 3
Compared with example 1, the difference is only that: the step (5) is not included, and the clear paste in the step (4) directly jumps to the step (6) according to a recrystallization treatment method.
Comparative example 4
Compared with example 1, the difference is only that: in the step (6), after the solvent is recovered, the process directly goes to the step (7) "drying at 60 ℃ and grinding".
The results of the examples and the comparative examples show the comparison of the yield and total alkaloid content of the extract, as shown in Table 1.
The yield is the weight percentage of the extract to the raw materials.
The method for measuring the content of the total alkaloids comprises the following steps: the content of polysaccharide and total alkaloid is determined by reference to a literature method (Shidongjun, Yang wenyu, Tangsheng, etc. Dendrobium nobile in the process of decoction and ointment decoction, Shizhen Chinese medicine 2016,27(2):355 + 358).
TABLE 1 Total alkaloid content test results table
| Extract sample | Dendrobium nobile Lindl weight | Extract weight | Yield of | Total alkaloid content |
| Example 1 | 200g | 1.2653g | 0.63％ | 76.7％ |
| Example 2 | 200g | 1.2047g | 0.60％ | 72.18％ |
| Comparative example 1 | 200g | 1.0878g | 0.54％ | 76.36％ |
| Comparative example 2 | 200g | 2.5145g | 1.26％ | 35.85％ |
| Comparative example 3 | 200g | 1.8416g | 0.92％ | 46.54％ |
| Comparative example 4 | 200g | 1.4275g | 0.71％ | 58.61％ |
In example 1, the yield of the extract and the total alkaloid content were slightly reduced when the process parameters were changed from the preferred parameters, as compared with example 2. Comparing the comparative examples 1-4 with the example 1, it can be seen that the addition of 4 treatments of the mixture of glycine and ammonium acetate to the extraction solvent, macroporous resin column chromatography, silica gel column distribution chromatography, and recrystallization has a certain beneficial effect on the yield/total alkaloid content of the dendrobium nobile extract.
It should be understood that the above-described embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (2)
1. A preparation method of dendrobium nobile lindl extract is characterized by comprising the following steps:
(1) crushing the dendrobium stem into granules as a raw material;
(2) taking the granular raw materials, adding a solvent to soak for 6-12 h, then percolating, and collecting percolate with the volume 10-50 times of the weight of the raw materials;
the solvent is a hydrochloric acid aqueous solution with the pH value of 1-4 and containing a mixture of 1-5% by mass of glycine and ammonium acetate, and the molar ratio of glycine to ammonium acetate is 1: 1-1: 3;
(3) adjusting the pH of the percolate to 6-7 by using lime milk, passing the percolate through a D101 or AB-8 or macroporous resin column with similar polarity, washing the percolate with pure water with the volume of 3 times of the volume of a column bed, discarding a washing liquid, eluting the percolate with hydrochloric acid aqueous solution with the pH of 1-4 with the volume of 3-6 times of the volume of the column bed, and collecting an eluent A;
(4) taking the eluent A, adjusting pH to neutral with saturated sodium carbonate aqueous solution, and concentrating under reduced pressure to obtain fluid extract;
(5) taking the clear paste, uniformly stirring the clear paste with 80-mesh silica gel in equal quantity, then carrying out silica gel column distribution chromatography, and eluting with ethyl acetate with the volume of 3-6 times of that of a column bed to obtain eluent B;
the distribution chromatography is carried out, wherein a supporting agent is 80-mesh silica gel, a stationary phase is a saturated sodium carbonate aqueous solution which is saturated by ethyl acetate in advance, and an eluent is ethyl acetate;
(6) taking the eluent B, recovering the solvent, and treating with methanol, ethanol or ethyl acetate for 1-3 times according to a recrystallization treatment method;
(7) and (4) taking the solid after recrystallization, drying at 60 ℃, and crushing to obtain the dendrobium nobile extract.
2. A dendrobium nobile extract characterized by being prepared by the preparation method of claim 1.
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| CN102477040A (en) * | 2010-11-25 | 2012-05-30 | 苏州宝泽堂医药科技有限公司 | Method for preparing dendrobine |
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| CN100427485C (en) * | 2006-04-19 | 2008-10-22 | 中国医学科学院放射医学研究所 | Process of preparing sophoridine, oxidized sophoridine and salts from sophora alopecuroide |
| CN106822599B (en) * | 2017-01-09 | 2021-03-30 | 中山大学 | Dendrobium loddigesii Rolfe extract and anti-diabetic application thereof |
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| CN102477040A (en) * | 2010-11-25 | 2012-05-30 | 苏州宝泽堂医药科技有限公司 | Method for preparing dendrobine |
| CN106220480A (en) * | 2016-06-24 | 2016-12-14 | 江西九草铁皮石斛科技协同创新有限公司 | Tank assembles the technique of dendrophnol in dynamic countercurrent extraction Herba Dendrobii |
| CN108477612A (en) * | 2018-02-06 | 2018-09-04 | 刘利勇 | A kind of dendrobium nobile nutritive powder and preparation method including dendrobium candidum purified extract |
| CN109620787A (en) * | 2019-01-30 | 2019-04-16 | 广州诗美化妆品有限公司 | A kind of moisturizing golden hairpin dendrobium extract and the preparation method and application thereof |
Non-Patent Citations (2)
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