CN114605370A - Myrobalan-chebula cleavage acid compound in myrobalan and preparation method and application thereof - Google Patents
Myrobalan-chebula cleavage acid compound in myrobalan and preparation method and application thereof Download PDFInfo
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- CN114605370A CN114605370A CN202210304920.7A CN202210304920A CN114605370A CN 114605370 A CN114605370 A CN 114605370A CN 202210304920 A CN202210304920 A CN 202210304920A CN 114605370 A CN114605370 A CN 114605370A
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- C07—ORGANIC CHEMISTRY
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- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/76—Benzo[c]pyrans
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
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- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention belongs to the technical field of substance extraction, and provides a myrobalan cracking acid compound in myrobalan, and a preparation method and application thereof. The myrobalan cleaving acid compound in the myrobalan has a structure shown in a formula I. The data of the examples show that: the myrobalan lyase compound provided by the invention has excellent activity of inhibiting sucrase and maltase, and can be applied to preparation of hypoglycemic drugs.
Description
Technical Field
The invention relates to the technical field of substance extraction, in particular to a myrobalan cracking acid compound in myrobalan and a preparation method and application thereof.
Background
The Tibetan medicine myrobalan is dry mature fruit of Terminalia chebula (Terminalia chebula Retz.) of Combretaceae, which is called Terminalia chebula, Terminalia chebula and Terminalia cordata, originally produced in India, Burma and other countries, and distributed in Tibet, Yunnan, Guangdong, Guangxi and other places in China. The medicine has the functions of astringing intestine and astringing lung, lowering fire and relieving sore throat, and has antioxidant, neuroprotective, antitumor, antiviral, antibacterial and other pharmacological functions. In Tibetan medicine and Mongolian medicine, Terminalia chebula is most commonly used, and the frequency of use is almost equal to that of liquorice in Han medicine prescription, so that the Terminalia chebula is regarded as the king of Tibetan medicine. Myrobalan has various effects, wide clinical application and high medicinal value, but the research on myrobalan is less and more scattered at present, and the medicinal components of myrobalan are not clear enough, so that the systematic research on the chemical components and the related activity of myrobalan is urgently needed.
Disclosure of Invention
In view of the above, the invention aims to provide a myrobalan cracking acid compound in myrobalan, and a preparation method and application thereof. The myrobalan cracking acid compound in the myrobalan provided by the invention has an excellent blood sugar reduction effect.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a myrobalan cracking acid compound in myrobalan, which has a structure shown in a formula I:
the invention also provides a preparation method of the myrobalan cleaving acid compound in the technical scheme, which comprises the following steps:
extracting the myrobalan fruit by using an ethanol water solution to obtain an extract;
purifying the extract by a solid phase extraction column to obtain a purified substance; the eluent purified by the solid phase extraction column is water;
carrying out silica gel column chromatography coarse separation on the purified product to obtain a second component; the eluent for the silica gel column chromatography crude separation is a chloroform-methanol system; the volume ratio of chloroform to methanol in the chloroform-methanol system is 9: 1-0: 1;
purifying the second component by reverse phase silica gel column chromatography to obtain a myrobalan cracking acid compound in the myrobalan; the eluent for the reverse phase silica gel column chromatography purification is methanol water solution.
Preferably, the volume concentration of the ethanol water solution is 75-95%.
Preferably, the extraction is performed under reflux conditions; the extraction times are 3 times, and the extraction time is 2h each time.
Preferably, after the extraction, the resulting extract is distilled under reduced pressure.
Preferably, the packing of the solid phase extraction column purified by the solid phase extraction column comprises AB-8 macroporous adsorption resin.
Preferably, the eluent for the crude separation of the silica gel column chromatography is chloroform and methanol in a volume ratio of 9: 1. 8: 2. 7: 3. 6: 4. 1: 1 and 0: 1 chloroform-methanol system.
Preferably, the concentration of the methanol aqueous solution is 10% by volume.
Preferably, the elution mode of the reverse phase silica gel column chromatography purification is isocratic elution.
The invention also provides an application of the myrobalan-in-myrobalan acid cracking compound in the technical scheme or the myrobalan-in-myrobalan acid cracking compound obtained by the preparation method in the technical scheme in preparation of a hypoglycemic drug.
The invention provides a myrobalan cracking acid compound in myrobalan, which has a structure shown in a formula I:
the data of the examples show that: the myrobalan lyase compound provided by the invention has excellent activity of inhibiting sucrase and maltase, and can be applied to preparation of hypoglycemic drugs.
The invention also provides a preparation method of the myrobalan cracking acid compound in the technical scheme, which comprises the following steps: extracting the myrobalan fruit by using an ethanol water solution to obtain an extract; purifying the extract by a solid phase extraction column to obtain a purified substance; the eluent purified by the solid phase extraction column is water; carrying out silica gel column chromatography coarse separation on the purified product to obtain a second component; the eluent for the silica gel column chromatography crude separation is a chloroform-methanol system; the volume ratio of chloroform to methanol in the chloroform-methanol system is 9: 1-0: 1; purifying the second component by reverse phase silica gel column chromatography to obtain a myrobalan cracking acid compound in the myrobalan; the eluent for the reverse phase silica gel column chromatography purification is methanol water solution. According to the invention, the myrobalan fruit is extracted by using an ethanol water solution, so that active substances in the myrobalan fruit can be extracted to the maximum extent; removing sugar and protein from the extract by solid phase extraction column purification technology; performing silica gel column chromatography coarse separation on the obtained purified substance by using a chloroform-methanol system as an eluent to obtain a second component; and (3) carrying out reverse phase silica gel column chromatography purification on the second component by using a methanol water solution as an eluent to obtain the myrobalan cracking acid compound in the myrobalan. The preparation method provided by the invention is simple to operate, and the purity of the obtained myrobalan cracking acid compound is higher than 90%.
The invention also provides an application of the myrobalan-in-myrobalan acid cracking compound in the technical scheme or the myrobalan-in-myrobalan acid cracking compound obtained by the preparation method in the technical scheme in preparation of a hypoglycemic drug. The myrobalan lyase compound provided by the invention has excellent sucrase and maltase inhibition activity, and can be applied to preparation of hypoglycemic drugs.
Drawings
FIG. 1 shows a cleavage acid compound of myrobalan of the present invention1H-NMR spectrum (nuclear magnetic resonance hydrogen)Spectra);
FIG. 2 shows a cleavage acid compound of myrobalan of the present invention12C-NMR spectrum (nuclear magnetic resonance carbon spectrum);
FIG. 3 is a DEPT135 spectrum of a myrobalan cleaving acid compound of the present invention;
FIG. 4 is a DEPT90 spectrum of a myrobalan cleaving acid compound of the present invention;
FIG. 5 is an HSQC spectrum of a myrobalan cleaving acid compound of the present invention;
FIG. 6 is an HMBC spectrum of a myrobalan cleaving acid compound of the present invention;
FIG. 7 shows a cleavage acid compound of myrobalan of the present invention1H-1H COSY spectrogram;
FIG. 8 is a NOESY spectrum of a chebula cleaving acid compound of the present invention;
FIG. 9 shows a myrobalan lyase compound sucrase IC of the present invention50A value;
FIG. 10 shows a myrobalan cleaving acid compound maltase IC of the present invention50The value is obtained.
Detailed Description
The invention provides a myrobalan cracking acid compound in myrobalan, which has a structure shown in a formula I:
the invention also provides a preparation method of the myrobalan cleaving acid compound in the technical scheme, which comprises the following steps:
extracting the myrobalan fruit by using an ethanol water solution to obtain an extract;
purifying the extract by a solid phase extraction column to obtain a purified substance; the eluent purified by the solid phase extraction column is water;
carrying out silica gel column chromatography coarse separation on the purified product to obtain a second component; the eluent for the silica gel column chromatography crude separation is a chloroform-methanol system; the volume ratio of chloroform to methanol in the chloroform-methanol system is 9: 1-0: 1;
purifying the second component by reverse phase silica gel column chromatography to obtain a myrobalan cracking acid compound in the myrobalan; the eluent for the reverse phase silica gel column chromatography purification is methanol water solution.
In the present invention, the starting materials used in the present invention are preferably commercially available products unless otherwise specified.
The invention utilizes ethanol water solution to extract myrobalan fruit to obtain the extract.
In the invention, the myrobalan fruit is preferably myrobalan fruit powder which is naturally dried in the shade. In the present invention, the volume concentration of the ethanol aqueous solution is preferably 75 to 95%, and more preferably 80 to 90%. In the present invention, the extraction is preferably performed under reflux conditions; the extraction times are preferably 3 times, and the time for each extraction is preferably 2 h.
After the extraction, the invention preferably also comprises the step of distilling the obtained extracting solution under reduced pressure; and distilling under reduced pressure to obtain ethanol for recycling.
After the extract is obtained, the extract is purified by a solid phase extraction column to obtain a purified substance; the eluent purified by the solid phase extraction column is water.
In the present invention, the packing of the solid phase extraction column purified by the solid phase extraction column preferably comprises AB-8 macroporous adsorption resin. In the invention, the eluent purified by the solid phase extraction column is water. In the invention, the eluent purified by the solid phase extraction column is preferably ethanol water solution; the volume concentration of the ethanol aqueous solution is preferably 95%. In the present invention, the solid phase extraction column purification preferably comprises: and placing the extract in a solid phase extraction column, and sequentially leaching and eluting. In the present invention, the extraction is preferably further subjected to dissolution before being subjected to solid phase extraction; the dissolved reagent preferably comprises water, which preferably comprises purified water.
After the purification of the solid phase extraction column, the invention preferably further comprises the step of carrying out reduced pressure concentration on the obtained eluent, wherein the concentrate obtained by the reduced pressure concentration is the purified product.
In the invention, the eluent purified by the solid phase extraction column is water, and can remove sugar and protein in the extract.
After a purified substance is obtained, the purified substance is subjected to silica gel column chromatography coarse separation to obtain a second component; the eluent for the silica gel column chromatography crude separation is a chloroform-methanol system; the volume ratio of chloroform to methanol in the chloroform-methanol system is 9: 1-0: 1.
in the invention, the loading mode of the silica gel column chromatography coarse separation is preferably dry loading; specifically, it is preferable that: mixing the purified product with silica gel, and drying in the air to obtain a silica gel sample; placing the silica gel sample in a silica gel column; the particle size of the silica gel is preferably 100 meshes; the mass ratio of the purified product to the silica gel is preferably 1: 1.5. in the present invention, the particle size of the silica gel in the silica gel column is preferably 100 mesh; the mass ratio of the purified product to the silica gel in the silica gel column is preferably 1: 10.
in the invention, the eluent for the coarse separation of the silica gel column chromatography is a chloroform-methanol system; the volume ratio of chloroform to methanol in the chloroform-methanol system is 9: 1-0: 1. in the invention, the eluent for the silica gel column chromatography crude separation is chloroform and methanol in a volume ratio of 9: 1. 8: 2. 7: 3. 6: 4. 1: 1 and 0: 1 chloroform-methanol system; specifically, chloroform and methanol are sequentially used, and the volume ratio is 9: 1. 8: 2. 7: 3. 6: 4. 1: 1 and 0: 1, eluting the silica gel column by using a chloroform-methanol system, monitoring the obtained eluent by using a thin layer plate, and combining the same parts to obtain a first component, a second component, a third component, a fourth component, a fifth component and a sixth component.
After the second component is obtained, carrying out reverse phase silica gel column chromatography purification on the second component to obtain a myrobalan cracking acid compound in the myrobalan; the eluent for the reverse phase silica gel column chromatography purification is methanol water solution.
In the present invention, the stationary phase of the reverse phase silica gel column purified by the reverse phase silica gel column chromatography is preferably RP 18. In the present invention, the concentration by volume of the aqueous methanol solution is preferably 10%. In the present invention, the elution mode of the purification by reverse phase silica gel column chromatography is preferably isocratic elution.
In the present invention, it is preferable to repeat the purification by reverse phase silica gel column chromatography to obtain the cleavage acid compound of myrobalan with purity of 92.0% or more.
The invention also provides an application of the myrobalan-in-myrobalan acid cracking compound in the technical scheme or the myrobalan-in-myrobalan acid cracking compound obtained by the preparation method in the technical scheme in preparation of a hypoglycemic drug.
The myrobalan cracking acid compound in the myrobalan has excellent sucrase and maltase inhibiting activity, so that the myrobalan cracking acid compound can be applied to the preparation of hypoglycemic drugs.
The myrobalan cleavage acid compound provided by the invention and the preparation method and application thereof are described in detail in the following examples, but the compounds are not to be construed as limiting the scope of the invention.
Example 1
The molecular formula of the myrobalan cleaving acid compound C18H20O11The structural formula is as follows:
the preparation method of the myrobalan lyase compound comprises the following steps:
(1) pulverizing 3kg of naturally dried fructus Chebulae fruit, sieving, reflux-extracting with 95% ethanol water solution for 3 times (2 hr each time), mixing extractive solutions, and concentrating under reduced pressure to recover ethanol to obtain extract.
(2) Dissolving the extract obtained in the step (1) with purified water, loading the extract on an AB-8 macroporous resin column, leaching with water, and removing sugar and protein; then, the mixture was eluted with an ethanol aqueous solution having a volume concentration of 95%, and the resulting eluate was concentrated under reduced pressure to obtain 320g of a concentrate.
(3) Mixing the concentrate obtained in the step (2) with 1.5 times of 100-mesh silica gel by weight, drying in the air, then carrying out silica gel column chromatography coarse separation on the 10 times of 100-mesh silica gel by weight, and segmenting by using a chloroform-methanol system, wherein the volume ratio of chloroform to methanol in the chloroform-methanol system is 9: 1. 8: 2. 7: 3. 6: 4. 1: 1 and 0: 1; the same fractions were combined for thin layer plate monitoring to obtain six fractions of a first fraction (fr.1), a second fraction (fr.2), a third fraction (fr.3), a fourth fraction (fr.4), a fifth fraction (fr.5) and a sixth fraction (fr.6).
Subjecting Fr.2 to reverse phase silica gel column (stationary phase is RP18), and isocratic eluting with 10% methanol water solution; silica gel column purification was repeated 1 time to obtain 21mg of 92.0% purity chebula cleavant compound.
Characterization test
Performing structural analysis on the obtained fructus Chebulae acid-cleaving compound, and obtaining the fructus Chebulae acid-cleaving compound1The H-NMR spectrum is shown in figure 1; preparation of the resulting chebula cleaving acid compound13The C-NMR spectrum is shown in FIG. 2; the DEPT135 spectrum of the obtained new compound of chebulariac cleavant is shown in figure 3; the DEPT90 spectrum of the obtained new compound is shown in figure 4; the HSQC spectrogram of the obtained novel compound of the chebula dehiscent acid is shown in figure 5; the HMBC spectrum of the obtained novel compound of the chebulariac cleavant is shown in figure 6; preparation of the novel compound of chebulagic acid1H-1The H COSY spectrum is shown in figure 7; the NOESY spectrum of the obtained novel compound of chebulariac acid is shown in FIG. 8.
The specific data are as follows: 13, 14-dimethyl-15-ethyl chebulariasplit acid and amorphous powder.
Activity assay
The myrobalan cleavage acid compound obtained in example 1 was used for the study of sucrase and maltase inhibitory activity.
The specific experimental method comprises the following steps:
(1) extraction and activity determination of rat small intestine sucrase and maltase: the rats are fasted for 12 hours, cervical vertebrae are removed for sacrifice, the small intestine is immediately taken out and placed on an ice bench, the small intestine is cut open and the intestinal mucosa is turned over to expose, washed by PBS buffer with precooling at 4 ℃ and wiped dry, the intestinal mucosa is scraped by a glass slide, and the weight ratio of the small intestine to the volume ratio of 1: 5 adding 4 deg.C precooled PBS buffer solution, homogenizing, centrifuging at 4 deg.C 8000r/min for 20min, collecting supernatant, subpackaging, and storing at-20 deg.C for use. Adding 0.35mL PBS buffer solution into 0.1mL of supernatant enzyme solution, water bathing at 37 deg.C for 10min, adding 50 μ L of 0.25mol/L sucrose (maltose) solution, immediately putting into ice water bath for 5min after 20min, and continuously adding 0.1mol/LNa2CO3The reaction was stopped with 0.5mL of the solution. The 3-well parallel assay was performed, and 1. mu. moL of glucose produced per minute in 1L of the solution at 37 ℃ and pH 6.8 was defined as 1 enzyme activity unit. The enzyme activity is glucose concentration × 2 × 1000/20.
(2) Screening of sucrase inhibitory activity: mu.L of the enzyme containing solution at 17.5U/mL and 50. mu.L of the 5mg/mL sample were added to a 48-well plate and preincubated at 37 ℃ for 10 min. Then 50. mu.L of a 0.5mol/L sucrose solution was added and the mixture was incubated at 37 ℃ for 20 min. Immediately putting into ice water bath for 5min, reducing enzyme activity, and continuously adding 0.1mol/LNa2CO3The reaction was stopped with 50. mu.L of the solution and repeated three times. And (3) determining the concentration of glucose by adopting a glucose kit, and calculating the inhibitory activity of the sample on the sucrase.
(3) Screening of maltase inhibitory activity: 50 μ L of enzyme containing 11.56U/mL and 50 μ L of 5mg/mL sample were added to a 48-well plate and preincubated at 37 ℃ for 10 min. Then, 50. mu.L of 1.39mmol/mL maltose solution was added and the mixture was incubated at 37 ℃ for 20 min. Immediately putting into ice water bath for 5min, reducing enzyme activity, and continuously adding 0.1mol/LNa2CO3The reaction was stopped with 50. mu.L of the solution and repeated three times. And (3) determining the glucose concentration by adopting a glucose kit, and calculating the inhibitory activity of the sample on maltase.
The results are shown in FIGS. 9 and 10. As can be seen from fig. 9 and 10: sucrase inhibitory activity IC of myrobalan cleaving acid compound50Value of 475.6. mu.M, half inhibition IC of maltase50The value was 427.5. mu.M.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A myrobalan cleaving acid compound in myrobalan is characterized by having a structure shown as a formula I:
2. the method for preparing the myrobalan cleaving acid compound of claim 1, comprising the steps of:
extracting the myrobalan fruit by using an ethanol water solution to obtain an extract;
purifying the extract by a solid phase extraction column to obtain a purified substance; the eluent purified by the solid phase extraction column is water;
carrying out silica gel column chromatography coarse separation on the purified product to obtain a second component; the eluent for the silica gel column chromatography crude separation is a chloroform-methanol system; the volume ratio of chloroform to methanol in the chloroform-methanol system is 9: 1-0: 1;
purifying the second component by reverse phase silica gel column chromatography to obtain a myrobalan cracking acid compound in the myrobalan; the eluent for the reverse phase silica gel column chromatography purification is methanol water solution.
3. The method according to claim 2, wherein the ethanol aqueous solution has a volume concentration of 75 to 95%.
4. The method of claim 3, wherein the extraction is performed under reflux conditions; the extraction times are 3 times, and the extraction time is 2 h.
5. The method according to claim 2 or 4, wherein after the extraction, the obtained extract is subjected to distillation under reduced pressure.
6. The method of claim 2, wherein the packing of the solid phase extraction column purified solid phase extraction column comprises an AB-8 macroporous adsorbent resin.
7. The preparation method according to claim 2, wherein the eluent for the crude separation by silica gel column chromatography is chloroform and methanol in a volume ratio of 9: 1. 8: 2. 7: 3. 6: 4. 1: 1 and 0: 1 chloroform-methanol system.
8. The method according to claim 2, wherein the aqueous methanol solution has a concentration of 10% by volume.
9. The method according to claim 2 or 8, wherein the reverse phase silica gel column chromatography is performed in a isocratic elution manner.
10. The use of the myrobalan-in-myrobalan lyase compound of claim 1 or the myrobalan-in-myrobalan lyase compound obtained by the preparation method of any one of claims 2-8 in the preparation of a hypoglycemic medicament.
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| CN104292278A (en) * | 2014-05-29 | 2015-01-21 | 内蒙古大学 | Myrobalan tannin compounds, and preparation method and applications thereof |
| WO2021204018A1 (en) * | 2020-04-09 | 2021-10-14 | 中国热带农业科学院热带生物技术研究所 | Compounds possessing ptp1b inhibitory activity, and application thereof |
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| CN104292278A (en) * | 2014-05-29 | 2015-01-21 | 内蒙古大学 | Myrobalan tannin compounds, and preparation method and applications thereof |
| WO2021204018A1 (en) * | 2020-04-09 | 2021-10-14 | 中国热带农业科学院热带生物技术研究所 | Compounds possessing ptp1b inhibitory activity, and application thereof |
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