CN110251504B - Preparation method and novel medical application of sanggenon D - Google Patents
Preparation method and novel medical application of sanggenon D Download PDFInfo
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- CN110251504B CN110251504B CN201910617148.2A CN201910617148A CN110251504B CN 110251504 B CN110251504 B CN 110251504B CN 201910617148 A CN201910617148 A CN 201910617148A CN 110251504 B CN110251504 B CN 110251504B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
- C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
- C07D493/04—Ortho-condensed systems
Abstract
The invention discloses a preparation method for extracting sanggenon D from cortex mori, ramulus mori and folium mori and a new medical application, in particular to an application of the sanggenon D with a single component for inhibiting xanthine oxidase, which can be applied to the preparation of anti-gout and anti-hyperuricemia drugs. The compound has the advantages of obvious curative effect, convenient use, lower cost, no obvious toxic or side effect and important clinical value. Provides a new direction for improving the phenomenon of large side effect of the existing gout medicine.
Description
Technical Field
The invention relates to a preparation method for extracting sanggenon D from cortex mori radicis, ramulus mori and folium mori and a new medical application, in particular to an application of the sanggenon D with a single component for inhibiting xanthine oxidase, which can be applied to the preparation of anti-gout and anti-hyperuricemia medicines, and also relates to a medicinal preparation of the medicines, belonging to the technical field of medicines.
Background
Gout is a common disease caused by purine metabolic disorder to form uric acid, and is listed as one of the 21 st century 20 persistent ailments by the united nations. Recent epidemiological studies show that the gout incidence rate of China rises year by year and is higher than the average level of the world, and the traditional anti-gout drugs, namely uricosuric drugs (such as probenecid, benzbromarone and the like) are often accompanied by side effects of rash, fever, kidney damage and the like. Xanthine oxidase inhibitors can reduce stress reactions or damage to tissues caused by free radicals, and also reduce the formation of uric acid in the human body. Researches find that the inhibitor taking xanthine oxidase as an action target has good anti-gout effect, is an important medicament for treating gout and can reduce some fatal gout complications.
The xanthine oxidase inhibitor is taken as an anti-gout drug, mainly allopurinol which is marketed in the 60 th century, although the xanthine oxidase inhibitor is used up to now, the xanthine oxidase inhibitor has a lot of side effects, and after the xanthine oxidase inhibitor is applied, patients can have side effects such as fever, allergic rash, abdominal pain, diarrhea, leukopenia and thrombocytopenia, and even liver function damage (the research progress of the xanthine oxidase inhibitor, the foreign medical and pharmaceutical handbook, 2006, 33(5), 351-. Therefore, the research of the novel xanthine oxidase inhibitor is of great significance. We have found that a novel xanthine oxidase inhibitor, sanggenon D, is extracted and separated from cortex Mori, ramulus Mori and folium Mori of Morus.
Extracts of mulberry twig have been reported in literature to have hypolipidemic effects (swiftlet, university of southwest, 2017, master thesis); the zhanxianli study reported that the extract of mulberry twig has the activity of inhibiting lipase (chinese traditional medicine, 2012, volume 37, phase 9, page 1323); CN103156869A discloses sanggenon C and sanggenon D extracted from cortex mori radicis, mulberry twigs and mulberry leaves and the new medical application of a composition taking the sanggenon C and the sanggenon D as main components, in particular to the application of the single components of the sanggenon C and the sanggenon D and the composition consisting of the sanggenon C and the sanggenon D, mulberroside, oxyresveratrol, resveratrol and deoxynojirimycin in the preparation of alpha-glucosidase inhibitor medicines; the Wangcolong research shows that the mulberry twig extract has the function of regulating organic ion transport and improving hyperuricemia, is a crude extract, does not have the chemical components of the crude extract and is a complex mixture.
The invention discovers that sanggenon D extracted from Morus plants has the activity of inhibiting xanthine oxidase for the first time. Before the completion of the invention, reports about the application of sanggenon D as a xanthine oxidase inhibitor in the aspects of preventing and treating gout are not found, and the invention has wide application prospect.
Disclosure of Invention
The invention aims to discover that sanggenon D extracted from cortex mori, mulberry twigs and mulberry leaves has the activity of inhibiting xanthine oxidase for the first time, has new application in preparing anti-gout and anti-hyperuricemia medicines, and has obvious innovation and technical progress.
Another object and feature of the present invention is: the sanggenon D has the effect of inhibiting xanthine oxidase and has more obvious pharmacological activity with the mulberry twig extract reported in the past literature, and the extract reported in the past is a crude extract, so the specific active ingredients are not clear; the invention has definite components and obvious technical progress and innovation.
Another objective of the present invention is to provide a method for preparing sanggenon D: crushing cortex mori radicis, ramulus mori or folium mori into coarse powder, performing reflux extraction for 2-3 times by using 30-40% ethanol solution in an amount which is 6-10 times that of the coarse powder, directly passing an extracting solution through a macroporous resin column, eluting 1-3 column volumes by using 30-40% ethanol, discarding an eluent, eluting 2-5 column volumes by using 50-80% ethanol solution, collecting ethanol eluent, recovering ethanol, adding acid into a concentrated solution to adjust the pH value to 1-4, and standing for precipitation; refluxing the precipitate with 5-10 times of organic solvent, filtering while hot, adding into a silica gel column, carrying out gradient elution with an acetone aqueous solution, collecting effective components, recovering acetone, and standing for crystallization; filtering out the crude crystal, passing through C18 column with methanol-water (70-30) as mobile phase, collecting the effective components, and mixing to obtain sanggenon D.
The preparation method adopts the extracting solution to directly carry out macroporous resin separation without concentration for the first time, removes impurities, solves the problems that the extracting solution is concentrated to cause the separation of effective components and the loss is serious after the extracting solution passes through the macroporous resin, improves the yield of the effective components, and has obvious technical progress and innovation.
However, in the prior art, sanggenon D has only been reported to have an α -glucosidase inhibitory effect and a lipase inhibitory effect, but no xanthine oxidase inhibitory effect has been reported.
The purity of the sanggenon D is 95-98%, for example, 95%, 96%, 97%, 98% and the like. The sanggenon D provided by the invention can be safely and effectively applied within the purity range. Generally, the purity of sanggenon D is more than 90 percent, and the normal pharmacological effect can be achieved.
Preferably, the dosage form of the medicament comprises any one of suspension, granules, capsules, powder, tablets, emulsions, solutions, dripping pills, injections, suppositories, enemas, aerosols, patches or drops.
Preferably, the medicament further comprises pharmacologically acceptable excipients.
Preferably, the auxiliary materials comprise any one or a combination of at least two of a carrier, a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrating agent, an emulsifier, a cosolvent, a solubilizer, an osmotic pressure regulator, a surfactant, a coating material, a pH regulator, an antioxidant, a bacteriostatic agent or a buffering agent.
In the invention, the dosage form of the medicament comprises any one of suspension, granules, capsules, powder, tablets, emulsions, solutions, dripping pills, injections, suppositories, enemas, aerosols, patches or drops.
In the present invention, the administration mode of the drug can be selected from various modes. For example, oral, rectal, parenteral or topical administration, and the like may be used.
Pharmacodynamic experiments prove that the sanggenon D has stronger xanthine oxidase inhibiting effect (the sanggenon D inhibition rate is 78.69% and the ramulus mori extract inhibition rate is 10.18% at the concentration of 0.5 mg/ml) than the ramulus mori extract, and the result is also another obvious technical progress, innovation and outstanding contribution of the invention.
The invention discloses that the sanggenon D has the effect of inhibiting xanthine oxidase for the first time, so that the sanggenon D is prepared into a medicament independently or in combination with other active components or auxiliary materials, and the medicament is used for preventing and treating gout and hyperuricemia and belongs to the protection scope of the invention. The composition of the invention has the functions of preventing and treating gout and hyperuricemia when being prepared into any dosage form.
The application of sanggenon D provided by the invention has the following advantages: 1. the raw materials are cheap and easy to obtain: cheap cortex mori radicis, mulberry twigs and mulberry leaves are used as raw materials and are obtained by extraction, the process is simple and feasible, and the content of sanggenon D in the cortex mori radicis is high; 2. the inhibition activity is strong; 3. the safety is high: the cortex mori radicis, the mulberry twig and the mulberry leaf are approved products which can be used as health-care food, have good safety and avoid the side effect of the prepared medicine for treating gout.
The effect of sanggenon D in inhibiting xanthine oxidase is confirmed by the following pharmacodynamic experiments.
1. Principle of experiment
Xanthine oxidase catalyzes xanthine to form uric acid. If the compound has an inhibitory effect on xanthine oxidase, the amount of xanthine catalyzed by xanthine oxidase decreases, uric acid produced accordingly decreases, and absorbance accordingly decreases.
2. Experimental Material
The sanggenon D and the mulberry twig extract used in the pharmacodynamic test are both prepared by self, and allopurinol, xanthine and xanthine oxidase are purchased from sigma company. KH (Perkin Elmer)2PO4、K2HPO4NaOH, dimethyl sulfoxide (DMSO) as analytical grade, distilled water.
3. In vitro activity test method
1) Preparing a buffer solution: 0.9560g KH are precisely weighed2PO4，5.3020g K2HPO4Adding distilled water for dissolving, and diluting to 500mL to obtain (PBS).
2) Preparation of a substrate: 3.65mg of xanthine is precisely weighed, a small amount of 0.5M NaOH solution is added for dissolving, and PBS is used for fixing the volume to 50mL to obtain 0.48mM substrate solution which needs to be prepared for use.
3) Preparation of enzyme solution: taking 100 μ L xanthine oxidase with liquid transfer gun, adding PBS, and diluting to 25mL to obtain 0.04 U.mL-1The xanthine oxidase solution is prepared in situ.
4) Preparation of a test sample: a proper amount of sample is precisely weighed, a small amount of DMSO (content is lower than 5%) is used for dissolving the sample, and then the sample is diluted by PBS buffer solution, so that the concentration of sanggenon D and the extract in the reaction is shown in Table 1.
5) Taking allopurinol as a positive control, sequentially adding 200 mu L of a sample solution to be tested, 200 mu L of a positive control solution and 500 mu L, PBS 2800 mu L of a blank solution and 500 mu L, PBS mu L of an enzyme solution into a test tube, incubating at 37 ℃ for 15min, adding 500 mu L of a substrate to start reaction, reading 1 time at 295nm every 30s, recording absorbance A, and counting for 5 min.
4. Results of the experiment
The inhibition (%) was calculated from the change in the A values of the sample group and blank group using the following formula: the inhibition (%) (1-a sample/a blank) × 100%, the results of inhibition are shown in the following table.
TABLE 1 inhibition of xanthine oxidase by test substances
The result shows that the allopurinol has good dose-effect relationship on the inhibition effect of the xanthine oxidase activity by changing the concentration of the allopurinol. The sanggenon D has good dose-effect relationship on the inhibition of xanthine oxidase activity under the concentration of 2.0, 1.0, 0.5, 0.2, 0.1 and 0.05mg/ml, and has strong inhibition. And has stronger activity than allopurinol, stronger activity than mulberry twig extract and IC50About 20 times or so.
Detailed Description
The present invention is illustrated in detail by the following examples, which are not intended to limit the invention thereto, and the following specific embodiments are provided:
examples 1,Extraction and preparation of sanggenon D
Crushing 10kg of cortex mori into coarse powder, performing reflux extraction for 3 times by using 40% ethanol solution in an amount which is 8 times that of the coarse powder, performing filtration, directly passing an extracting solution through a macroporous resin column, eluting 1 column volume by using 40% ethanol, discarding an eluent, eluting 4 column volumes by using 70% ethanol solution, collecting ethanol eluent, recovering ethanol, adding acid into a concentrated solution to adjust the pH value to 2, and standing and precipitating; refluxing the precipitate with 6 times of acetone, filtering while hot, adding silica gel column, gradient eluting with acetone water solution, collecting effective components, recovering acetone, filtering to obtain coarse crystal, separating with methanol-water (70-30) as mobile phase, separating with C18 normal pressure column, collecting effective components, and mixing to obtain sanggenon D.
Examples 2,Preparation of ramulus Mori extract
Taking dry ramulus Mori, adding ethanol, refluxing for three times with 2 hr each time, mixing extractive solutions, filtering, recovering ethanol from filtrate under reduced pressure, concentrating into soft extract, and drying under reduced pressure to obtain ramulus Mori extract.
Claims (6)
1. Application of sanggenon D in preparing medicine for treating hyperuricemia is disclosed.
2. The use according to claim 1, wherein sanggenon D is prepared by the following steps: crushing cortex mori radicis or mulberry twigs or mulberry leaves into coarse powder, performing reflux extraction for 2-3 times by using 30-40% ethanol solution in an amount which is 6-10 times that of the coarse powder, directly passing an extracting solution through a macroporous resin column, eluting 1-3 column volumes by using 30-40% ethanol, discarding an eluent, eluting 2-5 column volumes by using 50-80% ethanol solution, collecting ethanol eluent, recovering ethanol, adding acid into a concentrated solution to adjust the pH value to 1-4, standing for precipitation, performing reflux on a precipitate by using organic solvent in an amount which is 5-10 times that of the precipitate, filtering while hot, adding a silica gel column, performing gradient elution by using acetone aqueous solution, collecting effective components, recovering acetone, and standing for crystallization; filtering out the crude crystal, passing through C18 column with methanol-water (70-30) as mobile phase, collecting the effective components, and mixing to obtain sanggenon D.
3. The use according to claim 1 or 2, wherein the sanggenon D has a purity of 95-98%.
4. The use according to claim 1, wherein the medicament is in a dosage form selected from the group consisting of a suspension, granules, capsules, powders, tablets, emulsions, solutions, drops, injections, suppositories, enemas, aerosols, patches and drops.
5. The use of claim 4, wherein the medicament further comprises a pharmaceutically acceptable excipient.
6. The use according to claim 5, wherein the pharmaceutically acceptable excipient comprises any one or a combination of at least two of diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, tonicity adjusting agents, surfactants, coating materials, pH adjusting agents, antioxidants, bacteriostats or buffers.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103156869A (en) * | 2013-03-26 | 2013-06-19 | 赵全成 | Sanggenone C and sanggenone D extracted from morus plants and new medicine application of composition |
| CN105452269A (en) * | 2013-06-17 | 2016-03-30 | 尤妮金公司 | Compositions and methods for joint health |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103156869A (en) * | 2013-03-26 | 2013-06-19 | 赵全成 | Sanggenone C and sanggenone D extracted from morus plants and new medicine application of composition |
| CN105452269A (en) * | 2013-06-17 | 2016-03-30 | 尤妮金公司 | Compositions and methods for joint health |
Non-Patent Citations (2)
| Title |
|---|
| 夏道宗.青梅有效部位防治高尿酸血症和痛风的作用及机制研究.《中国博士学位论文全文数据库》.2011,E057-11. * |
| 青梅有效部位防治高尿酸血症和痛风的作用及机制研究;夏道宗;《中国博士学位论文全文数据库》;20111231;第15-16页 * |
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