CN110041392A - A kind of preparation method of cucurbitacin D - Google Patents
A kind of preparation method of cucurbitacin D Download PDFInfo
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Abstract
The invention belongs to pharmaceutical technology fields, and in particular to a kind of preparation method of cucurbitacin D, this method step are as follows: take rat or mouse blood, isolate blood plasma, anti-coagulants is added in blood plasma, saves backup；Cucurbitacin B ethanol solution is prepared, is dissolved in treated blood plasma, is vortexed and mixes, be stored at room temperature reaction；Ethyl acetate is added in solution after above-mentioned reaction, is vortexed after extraction, isolates supernatant and subnatant, the same step of subnatant repeats extraction and separation, supernatant merges, and the total liquid of supernatant is obtained, after being dried with nitrogen, methanol is added, after ultrasonic dissolution, supernatant is isolated, is concentrated, drying is to get product cucurbitacin D；Present invention firstly provides the blood plasma in rat or mouse blood metabolic response occurs with Cucurbitacin B, produces cucurbitacin D, generates without the by-product of cucurbitacin salt, the high income and purity is high of cucurbitacin D product.
Description
Technical field
The invention belongs to pharmaceutical technology fields, and in particular to a kind of preparation method of cucurbitacin D.
Background technique
Cucurbitacin (Cucurbitacins) is a kind of cucurbitane type tetracyclic triterpenes chemical combination extracted from Chinese medicine muskmelon pedicel
Object is conventionally used to the diseases such as treatment sputum place food, wind phlegm epilepsy, jaundice with damp-heat pathogen, four limbs edema.Research shows that such compound has
The bioactivity such as cytotoxicity, protect liver, antitumor, anti-inflammatory.Wherein, Cucurbitacin B is most important in effective component in muskmelon pedicel
Compound, but the content highest in muskmelon pedicel are easy to extract from muskmelon pedicel isolated.
Cucurbitacin D (cucurbitacin D, CuD) is also one of cucurbitacin compounds of group, and pharmacological action is extensive, except tool
There is the pharmacological action similar with other cucurbitacin compounds of group (such as: cell toxicant, to people's colon, mammary gland, lung and central nervous system
Inhibiting effect, anti-inflammatory effect of growth of cancer cells etc.), moreover it is possible to induced activation fetal hemoglobin gene and as treatment sickle
The drug candidate of cell anemia, its attention is higher and higher in recent years.But cucurbitacin D content in muskmelon pedicel is very low, mentions
Take preparation difficult.
A kind of method that cucurbitacin D is prepared by Cucurbitacin B hydrolysis of the patent report of Publication No. CN103804451A,
Method is that Cucurbitacin B is dissolved in organic solvent, obtains organic phase；Secure ph is greater than 9 lye or buffer, obtains water phase；
By the organic phase and water phase hybrid reaction；The disadvantages of the method are as follows under alkaline condition, cucurbitacin salt easily generated influences cucurbitacin
The yield and purity of D product.
In the prior art, Cucurbitacin B and blood plasma the relevant report that metabolic response generates cucurbitacin D is not subjected to.
Summary of the invention
It is an object of the invention to overcome the deficiencies in the prior art, establish the novel fabrication method of cucurbitacin D a kind of, should
Preparation method simple process, cucurbitacin D product yield and purity is high.
The technical solution adopted by the present invention to solve the technical problems is:
A kind of preparation method of cucurbitacin D, method includes the following steps:
(1) rat or mouse blood are taken, blood plasma is isolated, a certain amount of anti-coagulants is added in blood plasma, is placed in 0~4 DEG C of ring
It is saved backup under border；
(2) certain density Cucurbitacin B ethanol solution is prepared, takes and is dissolved in the blood plasma that step (1) obtains in right amount, is vortexed mixed
It is even, it is stored at room temperature reaction a period of time；
(3) be added ethyl acetate into the solution after step (2) reaction, be vortexed after extraction, be centrifugated out supernatant and
Subnatant, the same step of subnatant repeat extraction and separation one to three times, and supernatant merges, and obtains the total liquid of supernatant；
(4) to get product cucurbitacin D after the total liquid of supernatant that step (3) obtains being dried with nitrogen.
Preferably, in the step (1), anti-coagulants is one or both of heparin or hirudin, wherein anti-coagulants
Concentration in blood plasma is 0.1mg/mL.
Preferably, concentration of the Cucurbitacin B in ethanol solution is less than in the Cucurbitacin B ethanol solution of the step (2)
0.6mg/mL, because of the poorly water-soluble of Cucurbitacin B, poor dispersion when being dissolved in blood plasma, therefore it is molten to prepare Cucurbitacin B ethyl alcohol
Liquid is dissolved in blood plasma and obtains uniform solution, and wherein blood plasma is the important component of blood, contains water, plasma protein, electrolysis
The nutrition compositions such as matter, nutrient, enzyme, hormone and cholesterol, the present invention are metabolized using the nutrition composition and Cucurbitacin B of blood plasma
Reaction generates cucurbit rope D, then carries out separating-purifying, obtains the higher cucurbitacin D product of purity.
Preferably, the volume of blood plasma is 5 times or more of Cucurbitacin B volumes of aqueous ethanol, because of second in the step (2)
The excessively high protein component Precipitation easily led in blood plasma of determining alcohol is not easy Cucurbitacin B metabolism instead.
Preferably, the reaction time is 30~60min in the step (2).
Preferably, centrifuge separation condition is revolving speed 8000r/min, disengaging time in step (3)~(4)
10min。
It can be with Cucurbitacin B the beneficial effects of the present invention are: (1) present invention firstly provides the blood plasma in rat or mouse blood
Metabolic response occurs, produces cucurbitacin D, reacts milder, is generated without the by-product of cucurbitacin salt, cucurbitacin D product
High income；(2) ethyl acetate extraction and separation are utilized, cucurbitacin D is enriched with into supernatant, and plasma protein, enzyme in blood plasma,
The nutriments such as hormone and cholesterol are enriched with into subnatant, avoid the separating difficulty of product cucurbitacin D, process is to product calabash
The rate of recovery height of Lu Su D and obtained cucurbitacin D product purity are high, and in blood plasma excess, gained cucurbitacin D purity is greater than
95%.
Detailed description of the invention
The ESI-MS of Fig. 1 cucurbitacin D schemes；
Fig. 2 cucurbitacin D's1H-NMR (500MHz) figure；
Fig. 3 cucurbitacin D's13C-NMR (125MHz) figure；
The high-efficient liquid phase chromatogram of Fig. 4 cucurbitacin D.
Specific embodiment
The present invention and its specific embodiment are described in further detail with reference to the accompanying drawings and examples, but it is not intended that
Limit the scope of the invention.
Embodiment 1:
The qualitative analysis of cucurbitacin D:
A kind of preparation method of cucurbitacin D, method includes the following steps:
(1) rat blood is taken, blood plasma is isolated, heparin is added in blood plasma, makes concentration 0.1mg/ of the heparin in blood plasma
ML is placed under 0~4 DEG C of environment and saves backup；
(2) compound concentration is the Cucurbitacin B ethanol solution of 0.5mg/mL, and 200 μ L is taken to be dissolved in the blood that 1mL step (1) obtains
It in slurry, is vortexed and mixes, be stored at room temperature reaction 60min；
(3) 2mL ethyl acetate is added into the solution after step (2) reaction, is vortexed after extraction 1min, is placed in centrifuge separation
In machine, centrifugation rate 8000r/min is centrifuged 10min, isolates supernatant and subnatant, and the same step of subnatant repeats
Extraction and separation are primary, and supernatant merges twice, obtain the total liquid of supernatant；
(4) the total liquid of supernatant that step (3) obtains is placed in after being dried with nitrogen, be added 500 μ L methanol, after dissolution, be placed in from
In centrifugal separator, centrifugation rate 8000r/min is centrifuged 10min, isolates supernatant, carries out liquid chromatogram separation, liquid chromatogram
Separation condition are as follows: Sepax GP-C18 chromatographic column (21.2mm × 250mm), mobile phase methanol and water volume ratio are 58: 42, flow velocity
3mL/min, sampling volume 1mL in sample separation process, collect the eluent that chromatographic retention is 60~70min and carry out
ESI-MS、1H-NMR and13C-NMR analysis, ESI-MS analysis, there are molecular ion peak [M+H]+, m/z 517.3, another combination1H-
NMR and13C-NMR modal data determines that compound molecule formula is C30H44O7, degree of unsaturation 9；
1H-NMR (MeOD) high field region has multiple methyl singlets signals, 0.94 (3H, s, 18-CH3), 1.07 (3H, s, 19-
CH3), 1.30 (3H, s, 28-CH3), 1.32 (3H, s, 29-CH3), 1,35 (3H, s, 26-CH3), 1.35 (3H, s, 27-CH3),
1.41 (3H, s, 30-CH3), 1.42 (3H, s, 21-CH3) and 6.86 (1H, d, J=15.0) and 7.00 (1H, d, J=15.0)
Trans double bond and double bond 5.84 (1H, m), can determine that the basic structure of the metabolin does not change compared with Cucurbitacin B；
13C-NMR spectrum provides 30 C signals, 3 carbonyl C signals, and 4 olefinic carbon signals find its most of signal and cucurbit
Plain B is almost the same, but with Cucurbitacin B13C-NMR spectrum is compared to a few carbonyl C signal and methyl C signal；
In conjunction with ESI-MS,1H-NMR,13C-NMR modal data, it may be determined that the compound in collected eluent is cucurbitacin D,
I.e. Cucurbitacin B and blood plasma metabolic response generate cucurbitacin D；
Wherein, ESI-MS,1H-NMR (500MHz) and13C-NMR (125MHz) analysis is as a result, as shown in Figure 1, Figure 2 respectively, Fig. 3
It is shown.
Embodiment 2:
Cucurbitacin D is verified in the separating effect of liquid chromatogram:
A: taking rat blood, isolates blood plasma, heparin is added in blood plasma, makes concentration 0.1mg/ of the heparin in blood plasma
ML is placed under 0~4 DEG C of environment and saves backup, and takes in the treated blood plasma of 1mL, is vortexed and mixes, is stored at room temperature 1h；
B:(1 rat blood) is taken, blood plasma is isolated, heparin is added in blood plasma, makes concentration of the liver rope in blood plasma
0.1mg/mL is placed under 0~4 DEG C of environment and saves backup；(2) the cucurbitacin D ethyl alcohol that mass volume ratio is 0.7: 1mg/mL is prepared
Solution takes 200 μ L to be dissolved in the blood plasma that 1mL step (1) obtains, and is vortexed and mixes, and is stored at room temperature reaction 1h；
C:(1 rat blood) is taken, blood plasma is isolated, heparin is added in blood plasma, makes concentration of the heparin in blood plasma
0.1mg/mL is placed under 0~4 DEG C of environment and saves backup；(2) it is molten to prepare the Cucurbitacin B ethyl alcohol that mass volume ratio is 1: 1mg/mL
Liquid takes 200 μ L to be dissolved in the blood plasma that 1mL step (1) obtains, and is vortexed and mixes, and is stored at room temperature reaction 1h；
Liquid phase analysis detection is carried out to tri- kinds of solution of A, B, C, chromatographic condition is Agilent C18Chromatographic column (4.6mm ×
250mm, 5 μm), mobile phase volume is than methanol: water=61: 39,25 DEG C of column temperature, and flow velocity 1mLmin-1, 30 μ L of sampling volume；Figure
Compose result as shown in figure 4, analysis the result shows that, under the chromatographic condition, target substance cucurbitacin D separating degree is good, and in blood plasma
Property substance in source is noiseless to measuring.
Embodiment 3:
The measuring method of the content of cucurbitacin D:
(1) drafting of standard curve: precision weighs cucurbitacin D reference substance (purity > 98%) in right amount, is dissolved in methanol, matches
It is 161.20 μ gmL that concentration, which is made,-1Control stock solution, take cucurbitacin D reference substance stock solution to be configured to mass concentration to be
5.373、4.478、3.582、2.687、1.791、0.896、0.179μg·mL-1Control series product solution；Utilize liquid chromatogram
It is detected, chromatographic condition is Agilent C18Chromatographic column (4.6mm × 250mm, 5 μm), mobile phase methanol: water=61: 39, column
25 DEG C of temperature, flow velocity 1mLmin-1, 30 μ L of sampling volume；Obtaining the regression equation between peak area Y and cucurbitacin D mass concentration X is
Y=24.24X+0.411, correlation coefficient r=0.9985
(2) cucurbitacin D content is detected: to the solution after Cucurbitacin B and plasma solutions metabolic response, according in step (1)
Liquid phase chromatogram condition carry out detecting its peak area Y, substitute into above-mentioned regression equation, calculate the content of cucurbitacin D:
The yield calculation formula of cucurbitacin D is as follows:
Cucurbitacin D yield (y%)=cucurbitacin D substance amount (mol)/Cucurbitacin B substance amount (mol) × 100%.
The purity of cucurbitacin D is with the calculating of liquid phase area normalization method.
Embodiment 4:
A kind of preparation method of cucurbitacin D, method includes the following steps:
(1) rat blood is taken, blood plasma is isolated, hirudin is added in blood plasma, makes concentration of the hirudin in blood plasma
0.1mg/mL is placed under 0~4 DEG C of environment and saves backup；
(2) compound concentration is the Cucurbitacin B ethanol solution of 0.5mg/mL, and 200 μ L is taken to be dissolved in the blood that 1mL step (1) obtains
It in slurry, is vortexed and mixes, be stored at room temperature reaction 30min；
(3) 2mL ethyl acetate is added into the solution after step (2) reaction, is vortexed after extraction 1min, is placed in centrifuge separation
In machine, centrifugation rate 8000r/min is centrifuged 10min, isolates supernatant and subnatant, and the same step of subnatant repeats
Extraction and separation are primary, and supernatant merges twice, obtain the total liquid of supernatant；
(4) the total liquid of supernatant that step (3) obtains is placed in after being dried with nitrogen, 500 μ L methanol of addition, after ultrasonic dissolution, is set
In centrifugal separator, centrifugation rate 8000r/min is centrifuged 10min, isolates supernatant, is concentrated, dry to get product calabash
Lu Su D, obtained product carry out liquid-phase chromatographic analysis, and the yield of measurement cucurbitacin D is 90.3%, purity 95.7%.
Embodiment 5:
Repeat embodiment 4 by the same steps, but in the step (2), Cucurbitacin B ethanol solution it is dense
Degree is 0.3mg/ml, and the metabolic response time that 10 times of blood plasma are added is 45min, and the yield of measurement cucurbitacin D is 93.6%, purity
It is 97.2%.
Embodiment 6:
Repeat embodiment 4 by the same steps, but in the step (2), the metabolism of Cucurbitacin B and blood plasma
Reaction time is 60min, and in the step (3), ethyl acetate extraction times are 3 times, and the yield of measurement cucurbit rope D is
96.3%, purity 97.4%.
Embodiment 7:
Repeat embodiment 4 by the same steps, but in the step (2), Cucurbitacin B ethanol solution it is dense
Degree is 0.4mg/ml, and the metabolic response time of Cucurbitacin B and blood plasma is 50min, in the step (3), ethyl acetate extraction time
Number is 3 times, and the yield of measurement cucurbitacin D is 97.2%, purity 98.1%.
Above-mentioned embodiment is only a preferred solution of the present invention, not the present invention is made in any form
Limitation, for the those familiar of this field, can readily realize other modification, without prejudice to claim and equivalency range
Defined by the case where universal, the present invention is not limited to specific details.
Claims (5)
1. a kind of preparation method of cucurbitacin D, which is characterized in that method includes the following steps:
(1) rat or mouse blood are taken, blood plasma is isolated, a certain amount of anti-coagulants is added in blood plasma, is placed under 0~4 DEG C of environment
It saves backup；
(2) certain density Cucurbitacin B ethanol solution is prepared, takes and is dissolved in the blood plasma that step (1) obtains in right amount, is vortexed and mixes,
It is stored at room temperature reaction a period of time；
(3) ethyl acetate is added into the solution after step (2) reaction, is vortexed after extraction, is centrifugated out supernatant and lower layer
Liquid, the same step of subnatant repeat extraction and separation one to three times, and supernatant merges, and obtains the total liquid of supernatant；
(4) to get product cucurbitacin D after being dried with nitrogen the total liquid of supernatant that step (3) obtains, purity is greater than 95%.
2. the preparation method of cucurbitacin D according to claim 1 a kind of, which is characterized in that anticoagulant in the step (1)
Agent is one or both of heparin or hirudin.
3. the preparation method of cucurbitacin D according to claim 1 a kind of, which is characterized in that the cucurbitacin of the step (2)
In B ethanol solution, concentration of the Cucurbitacin B in ethanol solution is less than 0.6mg/mL.
4. the preparation method of cucurbitacin D according to claim 1 a kind of, which is characterized in that in the step (2), blood plasma
Volume be 5~10 times of Cucurbitacin B volumes of aqueous ethanol.
5. the preparation method of cucurbitacin D according to claim 1 a kind of, which is characterized in that in the step (2), reaction
Time is 30~60min.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102659888A (en) * | 2012-03-02 | 2012-09-12 | 张南 | Cucurbitacin derivatives and preparation method thereof |
| CN103804451A (en) * | 2012-11-14 | 2014-05-21 | 沈阳药科大学 | Method for preparing cucurbitacine D by hydrolyzing cucurbitacine B |
| CN107857789A (en) * | 2017-12-15 | 2018-03-30 | 张南 | Cucurbitacin derivatives and preparation method thereof |
| CN108659087A (en) * | 2018-06-28 | 2018-10-16 | 湖州展舒生物科技有限公司 | A kind of extraction process of cucurbitacin D |
-
2019
- 2019-02-27 CN CN201910147876.1A patent/CN110041392A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102659888A (en) * | 2012-03-02 | 2012-09-12 | 张南 | Cucurbitacin derivatives and preparation method thereof |
| CN103804451A (en) * | 2012-11-14 | 2014-05-21 | 沈阳药科大学 | Method for preparing cucurbitacine D by hydrolyzing cucurbitacine B |
| CN107857789A (en) * | 2017-12-15 | 2018-03-30 | 张南 | Cucurbitacin derivatives and preparation method thereof |
| CN108659087A (en) * | 2018-06-28 | 2018-10-16 | 湖州展舒生物科技有限公司 | A kind of extraction process of cucurbitacin D |
Non-Patent Citations (3)
| Title |
|---|
| 关宴星 等: "H3-葫芦素B在动物体内的吸收、分布、排泄", 《中国医药工业杂志》 * |
| 吴小林 等主编: "《药物化学》", 31 December 2018 * |
| 李超英 等: "血浆中葫芦素B 的高效液相色谱测定方法研究", 《中国药学杂志》 * |
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Application publication date: 20190723 |