CN111892634A - Glucoside compound from raw materials, extraction method and application thereof - Google Patents
Glucoside compound from raw materials, extraction method and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of glycoside compounds, in particular to a glycoside compound from raw materials and the like, an extraction method and application thereof, wherein the glycoside compound is a carbohydrate derivative containing carbocycle, the structural formula of the glycoside compound is shown as a formula (I), and the preparation method comprises the following steps: decocting raw materials in water, filtering, and concentrating to obtain extract; decocting and concentrating the extract to obtain a semi-finished dry extract, standing the semi-finished dry extract, taking out, and drying to obtain crude dry extract; extracting the crude dry extract with alcohol solvent, sequentially separating with macroporous resin, reversed phase column chromatography, and preparative high performance liquid chromatography. The biogenic glycoside compound has a remarkable uric acid reducing effect, can restore the high blood uric acid level of an animal model mouse to a normal level only by a low dose, can effectively inhibit the proliferation of liver cancer cells, and can be applied to a pharmaceutical preparation for inhibiting the proliferation of the liver cancer cells.
Description
Technical Field
The invention relates to the technical field of glycoside compounds, in particular to a glycoside compound from biogenesis and the like, an extraction method and application thereof.
Background
Raw and the like are generally understood as dry xylem (called core material) of stem of Rhamnella gilgiata Mansf. etMelch.) belonging to Rhamnaceae family of Cat genus, which is a Tibetan medicine material with Tibetan transliteration of "forest et al", "raw et al", "climbing", "Mulberry et al". The original plants grow in mountain shrubs or sparse forests with elevation 2600-2900 m, are mainly distributed in southeast of Tibet, northwest of Yunnan and southwest of Sichuan and are carried by Tibetan medicine standards, and modern medical research shows that the plants can cool blood, astringe dry yellow water and relieve swelling. Mainly treats rheumatic arthritis, leprosy and high mountain hyperemia, and has the functions of detumescence and sore toxin treatment by being decocted into paste for external use. Degree mother grass: can astringe the yellow water; the four medical classics: cooling blood, astringing dry yellow water; the book of Tibetan medicine: dispelling pathogenic wind and removing dampness, astringing dry yellow water, relieving swelling and pain, cooling blood, and treating rheumarthritis, yellow water disease, and hyperplastic blood disease.
At present, the medicinal mode of raw materials and the like is mainly to prepare raw extract by a decoction method and then use the raw extract to mix with other medicinal materials for pharmacy. For example, the extract of raw materials is used to prepare the Tibetan medicine twenty-five-ingredient tea pills.
However, the crude extract extracted in the prior art is troublesome to store, easy to breed microorganisms, easy to deteriorate, not beneficial to storage and transportation and control of dosage, and the stability and effectiveness of the medicine need to be improved. Therefore, how to improve the stability, effectiveness and availability of raw medicines and make the products convenient for storage and transportation is a technical problem to be solved at present.
The above background disclosure is only for the purpose of assisting understanding of the inventive concept and technical solutions of the present invention, and does not necessarily belong to the prior art of the present patent application, and should not be used for evaluating the novelty and inventive step of the present application in the case that there is no clear evidence that the above content is disclosed at the filing date of the present patent application.
Disclosure of Invention
In view of the above, the present invention aims to provide a glycoside compound with uric acid reducing activity derived from raw materials and the like, which has a significant uric acid reducing effect, can restore the level of hyperuricemia in animal model mice to a normal level with only a low dose, and can effectively inhibit proliferation of liver cancer cells, so that the glycoside compound can be applied to a pharmaceutical preparation for inhibiting proliferation of liver cancer cells.
In order to achieve the above object, the present invention provides the following technical solutions.
[1] A glucoside compound with uric acid reducing activity, which is derived from raw materials and the like, is a carbohydrate derivative containing carbocycle, is named as 2-hydroxy-5-carboxyl-4-isopropylbenzene-O-beta-D-glucoside, and has a structural formula shown as a formula (I):
the glycoside compound is a white powder, tasteless, readily soluble in methanol, soluble in water, and has the chemical formula C16O9H22Relative molecular mass 358.13.
The glucoside compound is taken from Tibetan medicine raw materials and the like, and through deep research on the blood uric acid level of a hyperuricemia mouse, the glucoside compound has a remarkable uric acid reducing effect, can restore the blood uric acid level of an animal model mouse to a normal level only by a low dose (25mg/Kg), can be used for preparing medicines for treating hyperuricemia, gout and the like, and shows a huge development prospect.
[2] A process for producing the glycoside compound of item [1], which comprises the steps of:
s1: decocting raw materials in water, filtering, and concentrating to obtain extract;
s2: decocting and concentrating the extract to obtain a semi-finished dry extract, standing the semi-finished dry extract, taking out, and drying to obtain crude dry extract;
s3: extracting the crude dry extract with alcohol solvent, sequentially separating with macroporous resin, reversed phase column chromatography, and preparative high performance liquid chromatography.
Preferably, in S1 the first step of the method,
adding 6-10 times of water into crude drugs for first decoction, and filtering by a 100-mesh sieve to obtain residue I and liquid I;
adding 4-6 times of water into the residue I for second decoction, and filtering with a 100-mesh sieve to obtain a residue II and a liquid medicine II;
and combining the liquid medicine I and the liquid medicine II to obtain the liquid medicine.
Preferably, the decocting temperature of step S1 is 185-190 ℃.
Preferably, in the decocting process of step S1, stirring is carried out for 1-2 min at a speed of 15-30 r/min every 15-20 min.
Preferably, the concentrated liquid medicine in step S1 means concentrating the liquid medicine at 185-190 ℃, stirring for 1-2 min at a speed of 15-30 r/min every 15-20 min, and concentrating to obtain an extract with a density of 0.95 g/mL.
Preferably, in the process of decocting and concentrating the extract in the step S2, the extract is stirred at the speed of 10-30 r/min, and the extract is stirred once every 10min when the density is lower than 1.19 g/mL; when the density of the extract reaches 1.19g/mL, the stirring frequency is continuous stirring.
Preferably, the decocting and concentrating temperature of the extract in the step S2 is 185-190 ℃, the decocting temperature is adjusted according to the density change of the extract, and the decocting and concentrating are finished when no steam emerges from the extract.
Further preferably, the step S2 of adjusting the decocting temperature according to the density change of the extract specifically includes:
when the density of the extract reaches 0.95g/mL, controlling the decoction temperature to be 190 ℃;
when the density of the extract reaches 1.06g/mL, controlling the decoction temperature to be 180 ℃;
when the density of the extract reaches 1.15g/mL, controlling the decoction temperature to be 170 ℃;
when the density of the extract reaches 1.17g/mL, controlling the decoction temperature to be 160 ℃;
when the density of the extract reaches 1.19g/mL, controlling the decoction temperature to be 120 ℃;
when the density of the extract reaches 1.22g/mL, controlling the decoction temperature to be 90 ℃;
and when the extract does not emit steam, decocting and concentrating to finish.
Preferably, the standing time of the dry paste semi-finished product of the step S2 is not less than 12h, and the drying mode is natural shade drying.
Further preferably, the electric heating blast air drying is carried out after the natural drying in the shade of 8-10 d in the step S2, the drying temperature is 60-80 ℃, the drying time is 7-8 d, and finally the vacuum drying is carried out, wherein the drying temperature is 60 ℃, the pressure is 0.05Mpa, and the drying time is 1-12 h.
Preferably, step S3 specifically includes:
1) adding 6-10 times of water solution containing ethanol and glycerol into the crude dry paste for reflux extraction, concentrating under reduced pressure until no alcohol smell exists, and sequentially extracting with petroleum ether, chloroform, ethyl acetate and water saturated n-butanol;
2) separating the ethyl acetate part by using column chromatography silica gel (200-300 meshes), performing gradient elution by using chloroform-methanol-water, and collecting elution fractions 1-5;
3) and (3) passing fraction 5 (chloroform-methanol-water 5:5:1 elution fraction) through macroporous resin, ODS medium-low pressure reversed phase liquid chromatography column, preparative high performance liquid chromatography, and acetonitrile-water to obtain glycoside compound.
More preferably, in the aqueous solution containing ethanol and glycerol in the step 1), the mass content of the ethanol is 50-80%, and the mass content of the glycerol is 0.5-2.0%;
the reflux extraction temperature of the step 1) is 82-85 ℃, and the extraction is carried out for at least 3 times, and each time lasts for at least 3 hours;
the chloroform-methanol-water eluent in the step 2) is prepared from 9:1:1, 8:2:1, 7:3:1, 6:4:1 and 5:5:1 in volume ratio;
the macroporous resin in the step 3) is selected from one of HP-21, SP850, D101, XDA1, X-5, NKA-2 or ADS-2;
the eluent used for the macroporous resin chromatography of the step 3) is acetone solution;
the mobile phase of the low-pressure reversed-phase liquid chromatography in the ODS of the step 3) is an aqueous solution of an organic solvent, and the organic solvent is methanol or acetonitrile;
the elution of the low-pressure reversed-phase liquid chromatography in the ODS of step 3) is gradient elution, and 7 elution fractions (A-G) are obtained by eluting with water and organic solvents in the volume ratio of 20:80, 30:70, 40:60, 50:50, 60:40, 70:30 and 80:20 respectively;
the mobile phase of the preparative high performance liquid chromatography of the step 3) is acetonitrile-water solution;
the preparative high performance liquid chromatography of the step 3) selects the fraction D for elution, and the chromatographic conditions are as follows: ODS-C18 chromatographic column (30mm × 250mm, 10 μm), acetonitrile-water volume ratio of 50:50 as mobile phase, flow rate of 10-15 mL/min, retention time of 52min, and separating to obtain the glycoside compound.
According to the technical scheme, raw medicinal materials are added with water for decoction and filtration to obtain filtered liquid medicine; stirring and concentrating the liquid medicine to obtain an extract; decocting and concentrating the extract, and controlling the decocting temperature according to the density change of the extract until no steam emerges from the extract, so as to obtain a dry extract semi-finished product; and standing the semi-finished dry paste, taking out, and drying to obtain crude dry paste. The crude dry paste prepared by the invention can replace crude extractum for use, is convenient to store and transport, can accurately control the dosage, has a simple preparation method, is easy to popularize, and can improve the stability and effectiveness of the medicine.
Further, on the basis of the crude dry paste, the ethyl acetate extract part is subjected to column chromatography silica gel separation, appropriate fractions are collected and then sequentially subjected to macroporous resin, ODS low-pressure reversed-phase liquid chromatography column and preparative high performance liquid chromatography to obtain the purified glycoside compound, the yield of the target product is high, and the excellent uric acid reducing effect can be used for preparing medicines for treating hyperuricemia, gout and the like. The inventor of the invention further researches the glycoside compound, and finds that the glycoside compound also has good antitumor activity and can inhibit the proliferation of liver cancer cells, and based on the antitumor activity, the glycoside compound can be applied to a pharmaceutical preparation for inhibiting the proliferation of liver cancer cells, so that the application field of crude dry paste such as crude dry paste is expanded to a great extent.
[3] A drug for treating hyperuricemia, wherein the drug contains the glycoside compound of [1] or [2] or an extract or a mixture containing the compound as a pharmaceutically effective ingredient.
Preferably, the extract or mixture includes, but is not limited to, raw equal extract, raw equal dry extract, raw equal alcohol extract.
Preferably, the medicine is prepared into tablets, capsules, injections, powder injections, granules, emulsions, microcapsules, dropping pills, ointments and other formulations by the glucoside compound or the extract or the mixture containing the compound and pharmaceutically acceptable carriers.
[4] A medicine for resisting liver cancer or liver tumor cells, the effective components of the medicine comprise the glycoside compound [1] or [2] or the extract or the mixture containing the compound.
Preferably, the extract or mixture includes, but is not limited to, raw equal extract, raw equal dry extract, raw equal alcohol extract.
Preferably, the medicine is prepared into tablets, capsules, injections, powder injections, granules, emulsions, microcapsules, dropping pills, ointments and other formulations by the glucoside compound or the extract or the mixture containing the compound and pharmaceutically acceptable carriers.
Preferably, the liver cancer or liver tumor cell is in particular HepG2 or Hep 3B.
The above-described preferred conditions may be combined with each other to obtain a specific embodiment, in accordance with common knowledge in the art.
The raw materials or reagents involved in the invention are all common commercial products, and the operations involved are all routine operations in the field unless otherwise specified.
The invention has the beneficial effects that:
1) the raw dry paste can replace raw extractum for use, is convenient to store and transport, can accurately control the dosage, has simple and easily-popularized preparation method, and can improve the stability and effectiveness of the medicine, the raw dry paste can replace the raw extractum in the prior art for use or be mixed with other medicines for pharmacy, for example, the raw dry paste provided by the application replaces the raw extractum for preparing the Tibetan medicine twenty-five-ingredient tea pills and the like;
2) on the basis of crude dry paste, the purified glycoside compound is prepared by alcohol solution reflux extraction and separation, has obvious effect of reducing uric acid, can restore the high uric acid level of an animal model mouse to a normal level only by a lower dose (25mg/Kg), and can be used for preparing medicaments for treating hyperuricemia, gout and the like;
3) further research also finds that the glycoside compound also has good antitumor activity and can effectively inhibit the proliferation of liver cancer cells, so that the glycoside compound can be applied to a medicinal preparation for inhibiting the proliferation of liver cancer cells, and the application field of crude dry paste and the like is expanded to a greater extent.
The invention adopts the technical scheme for achieving the purpose, makes up the defects of the prior art, and has reasonable design and convenient operation.
Drawings
The foregoing and/or other objects, features, advantages and embodiments of the invention will be more readily understood from the following description taken in conjunction with the accompanying drawings in which:
FIG. 1 is a schematic structural diagram of the glycoside compounds of the present invention;
FIG. 2 is a flow chart of the process for preparing raw equal extract of the present invention;
FIG. 3 is a flow chart of the process for preparing the raw equal dry paste of the invention;
FIG. 4 is a graph showing the change of density of the extract with temperature in example 1 of the present invention; the abscissa is density (unit: g/ml), and the ordinate is temperature (unit: DEG C);
FIG. 5 is a statistical chart of the weight of glycoside compounds in examples 2 to 12 of the present invention; the abscissa is the example number and the ordinate is the yield (unit: mg);
FIG. 6 is a schematic diagram of the uric acid lowering effect of the glycoside compound of the present invention; the ordinate is blood uric acid (unit: mg/L); a is blank group, B is model group, C is positive drug group, D is low dose group, E is medium dose group, F is high dose group; ("#" means significant relative to the blank group, "# #" means very significant relative to the blank group, "+" means significant relative to the model group, "+" means very significant relative to the model group).
Detailed Description
Those skilled in the art can appropriately substitute and/or modify the process parameters to implement the present disclosure, but it is specifically noted that all similar substitutes and/or modifications will be apparent to those skilled in the art and are deemed to be included in the present invention. While the products and methods of making described herein have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the products and methods of making described herein may be made and utilized without departing from the spirit and scope of the invention.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The present invention uses the methods and materials described herein; other suitable methods and materials known in the art may be used. The materials, methods, and examples described herein are illustrative only and are not intended to be limiting. All publications, patent applications, patents, provisional applications, database entries, and other references mentioned herein, and the like, are incorporated by reference herein in their entirety. In case of conflict, the present specification, including definitions, will control.
All percentages, parts, ratios, etc., are by weight unless otherwise indicated; additional instructions include, but are not limited to, "wt%" means weight percent, "mol%" means mole percent, "vol%" means volume percent.
When an amount, concentration, or other value or parameter is given as either a range, preferred range or a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when a range of "1 to 5(1 to 5)" is described, the described range is understood to include ranges of "1 to 4(1 to 4)", "1 to 3(1 to 3)", "1 to 2(1 to 2) and 4 to 5(4 to 5)", "1 to 3(1 to 3) and 5", and the like. Where numerical ranges are described herein, unless otherwise stated, the ranges are intended to include the endpoints of the ranges, and all integers and fractions within the ranges.
When the term "about" is used to describe a numerical value or an end point value of a range, the disclosure should be understood to include the specific value or end point referred to.
Furthermore, "or" means "or" unless expressly indicated to the contrary, rather than "or" exclusively. For example, condition a "or" B "applies to any of the following conditions: a is true (or present) and B is false (or not present), a is false (or not present) and B is true (or present), and both a and B are true (or present).
In addition, the indefinite articles "a" and "an" preceding an element or component of the invention are intended to mean no limitation on the number of occurrences (i.e., occurrences) of the element or component. Thus, "a" or "an" should be understood to include one or at least one and the singular forms of an element or component also include the plural unless the singular is explicitly stated.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation. The use of the phrase "comprising one of the elements does not exclude the presence of other like elements in the process, method, article, or apparatus that comprises the element.
The materials, methods, and examples described herein are illustrative only and not intended to be limiting unless otherwise specified. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.
The crude drugs are added with water for decoction and filtration to obtain filtered liquid medicine, the filtered liquid medicine is stirred and concentrated to obtain extract, the extract is decocted and concentrated, the decoction temperature is controlled according to the density change of the extract until no steam emerges from the extract to obtain a semi-finished dry paste, and the semi-finished dry paste is taken out after standing and dried to obtain the crude dry paste. The crude dry paste prepared by the invention can replace crude extractum for use, is convenient to store and transport, can accurately control the dosage, has a simple preparation method, is easy to popularize, and can improve the stability and effectiveness of the medicine. The raw dry paste provided by the invention can replace the raw extract in the prior art or be mixed with other medicines for preparing medicines, for example, the raw dry paste provided by the application can replace the raw extract to prepare the Tibetan medicine twenty-five-ingredient tea pills. It mainly has the following advantages; 1, storage advantage: the crude dry paste can be stored conveniently to a great extent, the storage time is prolonged, and the utilization rate of the raw medicinal materials is improved; the defects of troublesome storage of extractum, easy breeding of microorganisms, easy deterioration and the like are avoided; 2, transportation advantages: the crude dry paste can be conveniently transported, and the loss in the transportation process is reduced. Meanwhile, the transportation distance and the transportation time can be increased on the premise of ensuring the quality of the medicinal materials; 3, accurate medicine application: the crude dry paste can be accurately taken according to a prescription, and various parameters such as the dosage, the medication concentration and the like can be accurately controlled, so that the uniformity and the stability of the quality of the medicine can be ensured. Above several advantages all are favorable to guaranteeing stability and the validity of Tibetan medicine medicinal material, improve product quality, guarantee that medicine quality is homogeneous stable to the preparation method of crude dry paste such as that this application provided is simple easily promotes.
The present invention is described in detail below.
Because the technical problems that raw extractum extracted according to raw and the like is not favorable for storage and transportation and control of dosage exist in the prior art, the invention provides raw and the like dry paste, which specifically comprises the following steps:
example 1: a raw and other dry paste:
as shown in fig. 2 and 3, the crude dry paste is prepared by the following steps:
(1) cleaning 20kg crude drugs, slicing, adding 120L water into a 500L jacketed kettle, decocting at 190 deg.C for 90min, stirring every 20min for 1min at 15 r/min; after decocting, carrying out first filtration (100-mesh sieve) to obtain medicine residue I and medicine liquid I;
(2) adding 80L of water into the residue I, and decocting for the second time at 190 deg.C for 120min, stirring once every 20min, and stirring at 15r/min for 1 min; filtering for the second time (100 mesh sieve) after decocting to obtain residue II and medicinal liquid II, and discarding residue II;
(4) mixing the liquid medicine I and the liquid medicine II together to obtain filtered liquid medicine;
(5) adding the filtered liquid medicine into a 300L jacketed kettle for concentration, stirring once every 20min at the concentration temperature of 190 ℃, stirring for 1min at 15r/min, measuring the density of the filtered liquid medicine, increasing the density of the liquid medicine along with the continuous concentration of the filtered liquid medicine, and finishing the concentration when the density of the filtered liquid medicine reaches 0.95g/mL to obtain an extract;
(6) adding the extract into a 300L jacketed kettle, decocting and concentrating, setting the decocting temperature to 190 ℃, stirring once every 20min, stirring for 1min at 15r/min, detecting the density of the extract, wherein the higher the density of the extract is, the faster the stirring frequency is, the stirring frequency can be properly adjusted according to the density, and when the density of the extract reaches 1.19g/mL, the stirring frequency is continuous stirring; the decoction temperature is adjusted downwards according to the density change of the extract, the density and temperature control table of the extract is shown in Table 1, and the curve graph of the density of the extract along with the temperature change is shown in figure 4;
TABLE 1 control table of density and temperature of extract
In particular, the method comprises the following steps of,
when the density of the extract reaches 0.95g/mL, controlling the decoction temperature to be 190 ℃;
when the density of the extract reaches 1.06g/mL, controlling the decoction temperature to be 180 ℃;
when the density of the extract reaches 1.15g/mL, controlling the decoction temperature to be 170 ℃;
when the density of the extract reaches 1.17g/mL, controlling the decoction temperature to be 160 ℃;
when the density of the extract reaches 1.19g/mL, controlling the decoction temperature to be 120 ℃;
when the density of the extract reaches 1.22g/mL, controlling the decoction temperature to be 90 ℃;
when the extract has no steam, decocting and concentrating to obtain semi-finished dry extract;
(7) standing the semi-finished dry paste for 12h, taking out, naturally drying in the shade for 7d, and collecting paste after drying to obtain crude dry paste.
The raw dry paste provided by the application can replace the raw extract in the prior art or be mixed with other medicines for preparing medicines, for example, the raw dry paste provided by the application can replace the raw extract to prepare the Tibetan medicine twenty-five-ingredient tea pills. It mainly has the following advantages; 1, storage advantage: the crude dry paste can be stored conveniently to a great extent, the storage time is prolonged, and the utilization rate of the raw medicinal materials is improved; the defects of troublesome storage of extractum, easy breeding of microorganisms, easy deterioration and the like are avoided; 2, transportation advantages: the crude dry paste can be conveniently transported, and the loss in the transportation process is reduced. Meanwhile, the transportation distance and the transportation time can be increased on the premise of ensuring the quality of the medicinal materials; 3, accurate medicine application: the crude dry paste can be accurately taken according to a prescription, and various parameters such as the dosage, the medication concentration and the like can be accurately controlled, so that the uniformity and the stability of the quality of the medicine can be ensured. Above several advantages all are favorable to guaranteeing stability and the validity of Tibetan medicine medicinal material, improve product quality, guarantee that medicine quality is homogeneous stable to the preparation method of crude dry paste such as that this application provided is simple easily promotes.
It should be noted that the density and temperature control table of the extract is a preferred embodiment of the present application, and can be adjusted according to specific implementation scenarios, and other ways of adjusting the decocting temperature according to the density change of the extract all belong to the protection scope of the present application.
Example 2: a glycoside compound:
on the basis of the embodiment 1, the crude and other dry paste obtained by the method is used for preparing glucoside compounds, and the specific steps are as follows:
1) taking the crude dry paste of example 1, adding 10 times of aqueous solution containing 65% ethanol and 1% glycerol, mixing uniformly, heating to 85 ℃, extracting for 3 times each time for 3 hours, combining the extracting solutions, concentrating under reduced pressure until no alcohol smell exists, and extracting with petroleum ether (60-90 ℃), trichloromethane, ethyl acetate and water saturated n-butanol in sequence;
2) separating the ethyl acetate part obtained in the step 1) by using column chromatography silica gel (200-300 meshes), eluting by using chloroform-methanol-water gradient, eluting by using mixed solution with volume ratio of 9:1:1, 8:2:1, 7:3:1, 6:4:1 and 5:5:1 respectively, and correspondingly collecting elution fraction 1-5;
3) sampling fraction 5 (chloroform-methanol-water 5:5:1 elution fraction) on SP850 macroporous resin, wherein the sample is eluted by 30vol% acetone according to the volume ratio of 1: 4;
4) gradient eluting 30vol% acetone fraction with ODS low pressure reversed phase liquid chromatography column (50 × 500mm), and eluting with methanol-water solution (volume ratio of 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20) to obtain fractions A-G;
5) taking fraction D (methanol-water volume ratio 50:50) to perform preparative high performance liquid chromatography, wherein the chromatographic conditions are as follows: ODS-C18 chromatographic column (30X 250mm, 10 μm), acetonitrile-water volume ratio 50:50 as mobile phase, flow rate 15mL/min, retention time 52min, and final separation to obtain 232.8mg of the glycoside compound.
The glycoside compound is white powder, and is easily dissolved in methanol. HR-ESI-MSm/s: 362.1554[ M + NH4]+(calculated 358.1432), knotCombination of Chinese herbs1H-NMR spectrum and13C-NMR spectrum, and determination that the molecular formula is C16O9H22The calculated unsaturation is 6.1H-NMR(500MHz，DMSO-d6) In the spectrum of the light source,H7.14, 7.51 are proton signals for two aryls, suggesting tetra-substitution on the phenyl ring, and both are unimodal suggesting that they are in the para position.13C-NMR(125MHz，DMSO-d6) In the spectrum of the light source,C152.1, 150.3, 138.4, 126.0, 115.6, 114.3 are a set of aromatic carbon signals suggesting the presence of 1,2,4, 5-tetrasubstituted benzene ring in the molecule.1In the H-NMR spectrum, the molecular weight of the compound,H4.52 is the terminal proton signal of the sugar,H3.65-3.40 are other proton signals of sugars.13In the C-NMR spectrum, the molecular weight of the compound,C109.5, 81.5, 76.8, 73.4, 71.5, 62.2 are the 6 carbon signals of group 1 pyranose, suggesting that the compound is a glucopyranoside compound.1In the H-NMR spectrum, the molecular weight of the compound,H1.1 is that 6 proton signals are proton signals on 2 equivalent methyl groups, while high fields are still presentH3.1, an isopropyl group is presumed to be present;13methylene carbon signals are also observed in the C-NMR spectrumC29.1 and two methyl carbon signalsC23.3, and a carboxyl signalC172.0, it was found that there were one isopropyl group substitution and one carboxyl group substitution in the benzene ring mechanism. In HMBC spectra, 6 methyl hydrogen signalsH1.1 withC138.4 andCcarbon related, carboxyl hydrogen signal of 29.1H11.0 withC172.0 andCa carbon of 126.0 is associated with,H5.35 withC152.1 of the carbon content of the polymer,H5.88 withC150.3 carbon is associated and glucose is presumed to be attached to the 1-carbon of the phenyl ring. In conclusion, the structural formula of the glycoside compound disclosed by the application is shown in figure 1.
Examples 3 to 12: a glycoside compound:
on the basis of the embodiment 1, the crude and other dry paste obtained by the method is used for preparing glucoside compounds, and the specific steps are as follows:
1) taking the crude dry paste of example 1, adding 10 times of water solution, uniformly mixing, heating to 85 ℃, extracting for 3 times, each time for 3 hours, combining the extracting solutions, concentrating under reduced pressure, and sequentially extracting with petroleum ether (60-90 ℃), trichloromethane, ethyl acetate and water saturated n-butyl alcohol;
2) the same as example 2;
3) the same as example 2;
4) the same as example 2;
5) the glycoside compound is finally separated in the same way as in example 2; wherein
The composition of the aqueous solution of step 1) is specifically shown in table 2.
Table 2, alcohol extract composition of examples 2 to 12
Counting the weight of glycoside compounds obtained in examples 2-12, the results are shown in fig. 5, and it can be seen that the glycoside compounds obtained by extracting the crude dry paste with deionized water are the least (example 3), while the glycoside compounds can not be efficiently extracted without adding glycerol in the alcohol extract solution (examples 4-7), and it is also seen that the extraction of glycoside compounds is not facilitated when the alcohol extract solution contains too little or too much glycerol (examples 8 and 10), and correspondingly the extraction efficiency of glycoside compounds is reduced when the alcohol content in the alcohol extract solution is low (examples 11 and 12), on the basis of crude dry paste, the glycoside compounds are purified by refluxing extraction with an aqueous solution containing a certain amount of ethanol and glycerol, then separating the ethyl acetate extraction part with column chromatography silica gel, collecting appropriate fractions, sequentially passing through macroporous resin, ODS medium-low pressure reversed phase liquid chromatography column and preparative high performance liquid chromatography, the yield of the glucoside compound is higher.
Experimental example 1: uric acid reduction experiment:
experimental animals: 20-22 g of SPF male mice are adaptively raised for one week and then divided into 6 groups, wherein each group comprises 10 mice and the groups respectively comprise: blank group, model group, positive medicine group, and low, medium and high dose group of experimental samples.
Sample preparation: all samples were prepared 1h before the start of the experiment, the glycoside compound described in example 2 was prepared at 0.5g/mL with sterile water for injection, and the low, medium and high dose groups were gavaged with 25mg/Kg, 50mg/Kg and 100mg/Kg, respectively, and the positive drug was the same daily before the assay.
Hyperuricemia modeling agent: potassium oteroxide, prepared with hot sterilized water for injection at 1.5g/100mL and treated with ultrasound for 10min, and is injected into the abdominal cavity at 270mg/kg 1h before intragastric administration.
Positive drug: 0.1g/100mL allopurinol is prepared by sterilizing water for injection, and the mixture is subjected to ultrasonic treatment for 10min and then is subjected to 10mg/Kg intragastric administration before use.
Observing the seventh day, taking the medicine for 1h, dislocating and killing the medicine, fasting for 12h before killing without water, digging eye beads to take blood, placing the medicine at room temperature for 2h, centrifuging at 2000r for 5min, taking supernate, centrifuging at 3000r for 5min, taking serum, and keeping at-30 ℃ for later use.
Blood uric acid measurement results: the results are shown in fig. 6, and the model group has significant difference with the blank group as the reference, indicating that the molding is successful. By taking a model group as a reference, the medium and high dose groups of the glucoside compound have very significant differences, wherein the uric acid reducing effect of the high dose group is equivalent to that of the positive drug group, which shows that the glucoside compound has significant uric acid reducing effect, only low dose is needed to restore the hyperuricemia level of an animal model mouse to a normal level, and the glucoside compound can be used for preparing drugs for treating hyperuricemia, gout and the like.
Experimental example 2: in vitro inhibition of cancer cell proliferation assay:
experimental materials: human hepatoma cells HepG2, Hep3B, 96-well plates, cell counting plates, Countstar automatic cell counting instrument, DMEM/High Glucose culture medium, BioTek multifunctional enzyme labeling instrument, penicillin-streptomycin mixed solution, pancreatin, PBS buffer solution, CCK-8, DMSO, FBS, positive control drug crizotinib, and the glycoside compound described in experimental drug example 2.
The experimental method comprises the following steps: complete medium 1640 with 10% FBS, 100U/mL penicillin, 100. mu.g/mL streptomycin in 5% CO2Culturing at 37 deg.C to logarithmic phase, collecting tumor cells at logarithmic phase, digesting with pancreatin to obtain single cell suspension, counting with Countstar automatic cell counter, and counting at 4 × 103cells/well diluted were plated in 96-well plates at 100. mu.L/well. The same volume of medium was used as a blank to add cell suspension only, not cell suspensionThe drug was set as a negative control. To the test wells, different concentrations of the glycoside compound described in example 2 of the test drug to be tested were added, 4 replicates per concentration were set, and 100. mu.L of the medium was added to each of the blank control wells and the negative control wells. Respectively at 37 deg.C and 5% CO2The incubation period of (1) was 72h, 100. mu.L of each well was discarded, 10. mu.L of CCK-8 was added, the incubation period was 37 ℃ for 2h, the OD at 570nm was measured with a BioTek multifunctional microplate reader, the inhibition was calculated with the formula (II), the experiment was repeated at least 3 times, IC50The values are averaged. The results of the experiment are shown in table 3.
TABLE 3 inhibition of hepatoma cell proliferation in vitro by glycoside compounds described herein
As can be seen from Table 3, the glycoside compound disclosed by the application has a good inhibition effect on human liver cancer cells HepG2 and Hep3B, is equivalent to or even better than that of the known medicament crizotinib, and therefore can be used for effectively treating liver cancer or liver tumor.
Experimental example 3: in vitro inhibition of c-Met kinase assay:
the assay used to measure c-Met kinase activity is based on an enzyme-linked immunosorbent assay (ELISA).
Experimental materials:
a glycoside compound of example 2;
50pM c-Met (His-tagged recombinant human Met (amino acid 974-terminus), expressed by baculovirus);
assay buffer: 25mM MOPS, pH7.4, 5mM MgCl2，0.5raM MnCl 2100 μ M sodium orthovanadate, 0.01% Triton X-100, 1mM DTT, final DMSO concentration of 1 vol%.
The method comprises the following specific operations:
1. incubation of the glycoside compound described in example 2, 50pM c-Met and 5. mu.M ATP in assay buffer for 20min at room temperature on 0.25mg/mL PGT coated plates;
2. the reaction mixture was removed by washing and the phosphorylated polymer substrate was detected with 0.2 μ g/mL of horseradish peroxidase (HRP) conjugated phosphotyrosine specific monoclonal antibody (PY 20);
3. after the color development was stopped by adding 1M phosphoric acid, the color of the developed substrate (TMB) was quantified spectrophotometrically at 450 nm. The results are shown in Table 4.
TABLE 4 in vitro c-Met kinase inhibition of glycoside compounds described herein
The glycoside compound originated from the raw materials and the like has certain inhibition effect on the c-Met kinase, so that the glycoside compound or the extract or the mixture containing the glycoside compound of the item [1] or [2] can be applied to the preparation of the c-Met kinase inhibitor medicine.
Conventional techniques in the above embodiments are known to those skilled in the art, and therefore, will not be described in detail herein.
In view of the numerous embodiments of the present invention, the experimental data of each embodiment is huge and is not suitable for being listed and explained herein one by one, but the contents to be verified and the final conclusions obtained by each embodiment are close. Therefore, the contents of the verification of the respective examples are not described herein, and the excellent points of the present invention will be described only by examples 1 to 12 and experimental examples 1 to 3.
The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.
While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.
While the above detailed description has shown, described, and pointed out novel features as applied to various embodiments, it will be understood that various omissions, substitutions, and changes in the form and details of the device or method illustrated may be made without departing from the spirit of the disclosure. In addition, the various features and methods described above may be used independently of one another, or may be combined in various ways. All possible combinations and sub-combinations are intended to fall within the scope of the present disclosure. Many of the embodiments described above include similar components, and thus, these similar components are interchangeable in different embodiments. While the invention has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the invention extends beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and obvious modifications and equivalents thereof. Accordingly, the invention is not intended to be limited by the specific disclosure of preferred embodiments herein.
Claims (9)
1. A glucoside compound with uric acid reducing activity, which is originated from raw materials and the like, is characterized in that the glucoside compound is a carbohydrate derivative containing carbocycle, and the structural formula of the glucoside compound is shown as the formula (I):
2. a drug for treating hyperuricemia, wherein the drug contains the glycoside compound according to claim 1 as a pharmaceutically effective ingredient.
3. The medicament of claim 2, wherein the medicament is in the form of tablets, capsules, injections, powder injections, granules, emulsions, microcapsules, dripping pills or ointments.
4. A drug for anti-liver cancer or anti-liver tumor cells, wherein the drug contains the glycoside compound of claim 1 as a pharmaceutically effective ingredient.
5. The medicament according to claim 4, wherein the liver cancer or liver tumor cell is HepG2 or Hep 3B.
6. A process for preparing the glycoside compound of claim 1, comprising the steps of:
s1: decocting raw materials in water, filtering, and concentrating to obtain extract;
s2: decocting and concentrating the extract to obtain a semi-finished dry extract, standing the semi-finished dry extract, taking out, and drying to obtain crude dry extract;
s3: extracting the crude dry paste with alcohol solvent, sequentially separating with macroporous resin, reversed phase column chromatography, and preparative high performance liquid chromatography to obtain the glycoside compound.
7. The method according to claim 6, wherein the S3 specifically includes:
1) adding 6-10 times of water solution containing ethanol and glycerol into the crude dry paste for reflux extraction, concentrating under reduced pressure until no alcohol smell exists, and sequentially extracting with petroleum ether, chloroform, ethyl acetate and water saturated n-butanol;
2) separating the ethyl acetate part by using 200-300-mesh column chromatography silica gel, performing gradient elution by using chloroform-methanol-water, and collecting elution fractions 1-5;
3) and (3) eluting the fraction 5 by macroporous resin, an ODS medium-low pressure reversed phase liquid chromatography column and preparative high performance liquid chromatography in sequence to obtain the glycoside compound.
8. The method according to claim 7, wherein in the aqueous solution containing ethanol and glycerol in step 1), the mass content of ethanol is 50 to 80%, and the mass content of glycerol is 0.5 to 2.0%.
9. The raw and other dry paste is characterized by being prepared by the following steps:
s1: decocting raw materials in water, filtering, and concentrating to obtain extract;
s2: decocting and concentrating the extract to obtain a semi-finished dry extract, standing the semi-finished dry extract, taking out, and drying to obtain crude dry extract.
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