CN113149840B - Wild pepper extract and preparation method and application thereof - Google Patents
Wild pepper extract and preparation method and application thereof Download PDFInfo
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- CN113149840B CN113149840B CN202110308494.XA CN202110308494A CN113149840B CN 113149840 B CN113149840 B CN 113149840B CN 202110308494 A CN202110308494 A CN 202110308494A CN 113149840 B CN113149840 B CN 113149840B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
- C07C69/66—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety
- C07C69/73—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of unsaturated acids
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
- C07C67/56—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
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Abstract
The invention discloses a wild pepper extract, a preparation method and application thereof, and relates to the technical field of medicinal compounds. The structural formula of the piper spicatum extract is shown as the formula I:
Description
Technical Field
The invention relates to the technical field of medical compounds, and particularly relates to a lindera glauca extract and a preparation method and application thereof.
Background
Inflammation is a complex defense reaction mainly for defending, wherein the body adopts self-tissue repair to eliminate damaged cells and foreign antigens under the action of inflammatory factors in living tissues with vascular systems. The inflammatory reaction is divided into acute inflammation and chronic inflammation, the acute inflammation takes red, swelling, heat and pain as main symptoms, is expressed by lymphocyte infiltration and activation, has the functions of immunity and defense, and can prevent and control the invasion and damage of pathogens. However, if inflammatory factors persist and damage tissues, chronic inflammation can be induced, leading to various diseases such as rheumatoid arthritis, diabetes, silicosis, and even cancer. The anti-inflammatory drugs commonly used in clinic at present mainly comprise non-steroidal anti-inflammatory drugs and steroidal anti-inflammatory drugs. They have good anti-inflammatory effect to a certain extent, but the body can generate a series of adverse reactions and tolerance after long-term or large-dose use, such as serious gastrointestinal tract injury, liver injury, cardiovascular toxicity risk, kidney injury and the like. In order to solve the adverse reaction and tolerance of the drugs, the search for new anti-inflammatory drug types and related drugs with novel framework types is always a hot spot in the research field of anti-inflammatory drugs.
Natural products are important sources for new drug discovery, and are important prodrug molecules for human prevention and treatment of diseases due to structural diversity and wide range of biological activities. About 62% of the drugs available worldwide are derived from natural products or from structural modifications of natural products. The plants are important components of natural medicines, plant resources on the earth are rich, metabolites are various, and finding the lead compound from the plants is an important way for obtaining new medicine candidate compounds and new medicines. The metabolite of the plant has wide physiological activities, such as anti-inflammatory, antibacterial, anti-tumor, immunoregulation, enzyme inhibition and other activities. The search for new drug source molecules from plants has been a hot spot in the field of drug research.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a wild pepper extract and a preparation method and application thereof.
The invention is realized in the following way:
in a first aspect, the present invention provides a piper spicatum extract, which has a structural formula shown in formula I:
in a second aspect, the present invention provides a method for preparing the piper spicatum extract according to the foregoing embodiment, which includes:
extracting the mountain pepper root powder with methanol, concentrating the extracting solution to obtain a concentrated extract, adding water into the concentrated extract for suspension, and extracting with petroleum ether to obtain a petroleum ether extract;
performing gradient elution separation on the petroleum ether extract by silica gel column chromatography, wherein the mobile phase is petroleum ether and acetone with the volume ratio of 100-25:1, and collecting petroleum ether: concentrating the eluate at acetone volume ratio of 35-25:1, performing gel Sephadex LH-20 chromatography, and performing gel chromatography with methanol-chloroform as eluent to obtain fructus Piperis extract with formula I.
In an alternative embodiment, the methanol extraction of the mountain pepper root powder comprises: cold soaking in methanol, percolating and extracting the powder of radix Linderae Strychnifoliae for 2-4 times, each time for 45-55 hr.
In an alternative embodiment, Sephadex LH-20 is chromatographed on Sephadex LH-20 gel using Sephadex LH-20 hydroxypropyl Sephadex.
In alternative embodiments, the methanol-chloroform volume ratio is 1-2: 1.
In an alternative embodiment, the mountain pepper root powder is obtained by pulverizing mountain pepper roots to 10-30 mesh.
In a third aspect, the present invention provides an anti-inflammatory composition comprising a novel compound produced by the extraction of piper spicatum according to the previous embodiments.
In an alternative embodiment, it further comprises a pharmaceutically acceptable pharmaceutical excipient.
In a fourth aspect, the present invention provides an application of the spicebush root extract described in the foregoing embodiments in preparing an anti-inflammatory drug.
In a fifth aspect, the present invention provides a use of the extract of piper spicatum according to the previous embodiment in the preparation of a medicament for inhibiting the production of NO induced by LPS.
The invention has the following beneficial effects:
the invention separates a long-chain sebutanolide derivative, namely, the sebobaolide F (namely, the piper amurensis extract with the structural formula I in the application) from the piper amurensis (Lindera glauca) plant, and the novel compound has the advantages of remarkably inhibiting the generation of NO induced by LPS and IC50Comprises the following steps: 1.73 +/-0.18 mu M, has the clinical application potential of anti-inflammatory treatment, can be used for preparing anti-inflammatory drugs, and has good application prospect.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The invention provides a lindera glauca extract, which has a structural formula shown as a formula I:
in the present application, the compound represented by formula I is named as secocosubamolide F, which is a long-chain secocobutanolide derivative. Experiments prove that the long-chain sebutanolide derivative sebosubamolide F derived from the plant of the Lindera glauca (Lindera glauca) has the obvious effect of inhibiting the generation of NO induced by LPS, has the clinical application potential of anti-inflammatory treatment, and can be used for preparing anti-inflammatory drugs.
Thus, also provided herein is an anti-inflammatory composition comprising the novel compounds produced as described above by extraction of piper spicatum. In alternative embodiments, the anti-inflammatory composition further comprises a pharmaceutically acceptable pharmaceutical excipient.
Further, the invention provides an application of the piper spicatum extract in preparing an anti-inflammatory drug.
Further, the present invention provides an application of the piper spicatum extract described in the foregoing embodiments in preparing a medicament capable of inhibiting the production of NO induced by LPS.
In addition, the present application also provides a preparation method of the above spicebush root extract, which is only exemplary, and comprises the following steps:
s1, the sources of the root medicinal materials of the wild pepper are as follows:
collected from the edge of the Linn (Xiaodi name) Lin (east longitude 110.4470 degrees; northern latitude 30.7057 degrees; altitude 1203m) of the Pistan village of the Yangshou Zhengzhen province, county, Ba Dong province, Hubei province, and identified as the root of mountain pepper (Lindera glauca) of the genus Lithocarpus of Lauraceae by Pogtian Philippines, university of traditional Chinese medicine, Guangzhou. Drying root of mountain pepper (Lindera glauca) in the sun, and pulverizing to 10-30 mesh.
S2, crude extraction:
cold soaking and percolating radix Linderae powder with methanol for 2-4 times, soaking for 45-55 hr each time, concentrating the extractive solution to obtain concentrated extract, suspending the concentrated extract with water and extracting with petroleum ether, specifically, suspending and dispersing the concentrated extract with distilled water, adding petroleum ether, extracting until colorless, and concentrating petroleum ether fraction to obtain petroleum ether extract.
S3, separation and purification:
performing gradient elution separation on the petroleum ether extract by silica gel column chromatography, wherein the mobile phase comprises petroleum ether and acetone in a volume ratio of 100-25:1, and collecting the petroleum ether: concentrating the eluate at acetone volume ratio of 35-25:1, performing gel Sephadex LH-20 chromatography, and performing gel chromatographic separation with methanol-chloroform (volume ratio of 1-2:1) as eluent to obtain fructus Piperis extract with formula I.
Preferably, when gradient elution is performed, the volume ratio of the mobile phase petroleum ether to acetone is from 100:1 → 55-45:1 → 35-25: 1.
In an alternative embodiment, Sephadex LH-20 is chromatographed on gel Sephadex LH-20 using Sephadex gel.
The invention separates a long-chain sebutanolide derivative sebosubamolide F from a Lindera glauca plant, and the new compound has the functions of obviously inhibiting the generation of NO induced by LPS and IC of the NO50Comprises the following steps: 1.73 +/-0.18 mu M, has the clinical application potential of anti-inflammatory treatment, can be used for preparing anti-inflammatory drugs, and has good application prospect.
The applicant can know that the piper spicatum extract provided by the application has the anti-inflammatory effect due to the general formula of the mother nucleus shown in formula II, and it is understood that the conventional substitutes for the piper spicatum extract provided by the application have the same or similar effect, and the substitutes for the piper spicatum extract include but are not limited to: secoisolanciolide (formula III), Secoiubaolide (formula IV), and Secokotomolide A (formula V).
Wherein R1 and R2 are independently selected from OCH3OH, substituted or unsubstituted alkyl, substituted or unsubstituted alkynyl.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The present example provides an extract of piper spicatum, which has a structural formula shown in formula I:
the preparation method comprises the following steps:
(1) the mountain pepper root medicinal material source:
the root of mountain pepper is obtained from the edge of the Linn (Xiaodi name) forest (east longitude 110.4470 degrees; northern latitude 30.7057 degrees; altitude 1203m) of the Yangtze Cuishatan Piper in Shandong county of Ba county of Hubei province, and is identified as the root of mountain pepper (Lindera glauca) of the genus Lithocarpus of Lauraceae by Potentilla Pzetta, university of traditional Chinese medicine of Guangzhou. Sun drying root of fructus Piperis, and pulverizing to 20 mesh;
(2) crude extraction:
cold soaking and percolating radix Linderae powder with methanol for 3 times, soaking for 48 hr each time, concentrating the extractive solution to obtain concentrated extract, suspending and dispersing the concentrated extract with distilled water, adding petroleum ether, extracting to colorless, and concentrating petroleum ether fraction to obtain petroleum ether extract.
(3) Separation and purification:
performing gradient elution on the petroleum ether extract by silica gel column chromatography, eluting from 100:1 → 50:1 → 30:1 by using a petroleum ether/acetone solvent according to the volume ratio, collecting petroleum ether/acetone (30:1) eluent, concentrating, performing chromatography by using Sephadex LH-20 hydroxypropyl Sephadex, and performing gel chromatographic separation by using methanol-chloroform with the volume ratio of 1:1 as an eluent to obtain a compound secocosubamolide F shown as the formula I.
Experimental example 1
The new compound secocosubamolide F is subjected to structural test and analysis, and the following experimental data are obtained:
the new compound secocosubamolide F: c19H30O4，(+)-HRESIMSm/z323.2220[M+H]+(calculated 323.2222).
The NMR data for compound secocosubamolide F are shown in Table 1.
TABLE 1 NMR data (CDCl) of the compound secoubamolide F3，400MHz/100MHz,ppm)
Experimental example 2: anti-inflammatory cell screening model of compound secocosubamolide F
1. Culture and treatment of cells
RAW264.7 cells were cultured in vitro and cultured and passaged routinely at 37 ℃ and 5% carbon dioxide concentration in a DMEM high-glucose medium containing 10% FBS.
2. Compound intervention
(1) Experimental methods
The new compound, secosubalide F or indomethacin, was dissolved in DMSO and formulated with media to different drug concentrations, 3 duplicate wells in each concentration, and model control wells (LPS only) and blank control wells (cells and media) were set.
RAW264.7 cells in logarithmic growth phase were selected at 2X 104cells/well density plate, adding drugs with different concentrations into corresponding 96-well plates after co-culturing for 12h, and adding LPS with the final concentration of 1 mug/mL after 2h to induce macrophage differentiation into an inflammatory state. After 48h of culture, 50 mu L of cell supernatant is taken, 50 mu L of Griess reagent I and 50 mu L of Griess reagent II of the nitric oxide detection kit are added respectively, and the content of NO is measured by utilizing a Griess method.
Inhibition rate ═ 1- (NO)treat-NOcontrol)/(NOmodel-NOcontrol)]×100％
Experimental example 3: effect of Compounds on cell viability
MTT is reduced to water-insoluble blue crystals by succinate dehydrogenase in mitochondria of living cells and deposited in living cells, while dead cells do not function.
Experimental methods
Compounds of secosulamolide F or indomethacin were dissolved in DMSO and the media used to prepare different drug concentrations of 100. mu.M, 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.125. mu.M, 1.563. mu.M, 3 replicates per concentration, and blank wells (cells and media) were set.
RAW264.7 cells in logarithmic growth phase were selected at 2X 104cells/well density plate, after 12h of co-culture, different concentrations of drug were added to the respective 96-well plates, after 48h of culture, 10. mu.L of 5mg/mL MTT solution was added to each well, after 4 h of culture, the medium was aspirated, and 150. mu.L DMSO was added to each well. Shaking up by shaking. The absorbance value (A) at a wavelength of 570nm was measured with a microplate reader. The inhibition of cell growth by drugs is expressed in terms of survival, with higher survival indicating lower drug toxicity.
Survival (%) - (a)1–A0)/(A2–A0)]X 100% where A0Absorbance value of blank, A1Is the absorbance value of the sample, A2Absorbance values for positive control wells.
Five samples at 200. mu.M, 100. mu.M, 50. mu.M, 25. mu.M, 12.5. mu.M concentrations were assayed, and a dose-inhibition curve was plotted to obtain the CC thereof50The value is obtained. Each sample was assayed in triplicate.
The results of experimental examples 2 and 3 are shown in table 2:
TABLE 2 inhibition of inflammatory factor NO by the compound secoubamolide F
The results show that: secosubamolide F compound shows strongerThe anti-NO production activity of (1.73. + -. 0.18. mu.M) in IC50 is stronger than that of Indomethacin (IC) as a positive control5024.0 ± 0.36). The compound secocosubamolide F does not show cytotoxic activity at the concentration of 100 mu M, which indicates that the compound has good potential for further preclinical research.
In summary, the invention separates a long-chain sebutanolide derivative, namely, the sebobaolide F (the extract of the Lindera glauca with the structural formula I in the application) from the plant of the Lindera glauca, and the novel compound has the advantages of remarkably inhibiting the generation of NO induced by LPS and IC of the NO50Comprises the following steps: 1.73 +/-0.18 mu M, has the clinical application potential of anti-inflammatory treatment, can be used for preparing anti-inflammatory drugs, and has good application prospect.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. The piper spicatum extract is characterized by having a structural formula shown as a formula I:
2. A method for preparing the piper spicatum extract as claimed in claim 1, which comprises:
extracting the mountain pepper root powder with methanol, concentrating the extracting solution to obtain a concentrated extract, adding water into the concentrated extract for suspension, and extracting with petroleum ether to obtain a petroleum ether extract;
performing gradient elution separation on the petroleum ether extract by silica gel column chromatography, wherein the mobile phase is petroleum ether and acetone with the volume ratio of 100-25:1, and collecting the petroleum ether: concentrating the eluate at acetone volume ratio of 35-25:1, performing gel Sephadex LH-20 chromatography, and performing gel chromatography with methanol-chloroform as eluent to obtain fructus Piperis extract with formula I.
3. The method for preparing the mountain pepper extract as claimed in claim 2, wherein the extracting the mountain pepper root powder with methanol comprises: cold soaking in methanol, percolating and extracting the powder of radix Linderae Strychnifoliae for 2-4 times, each time for 45-55 hr.
4. The method for preparing the piper spicatum extract as claimed in claim 2, wherein the volume ratio of methanol to chloroform is 1-2: 1.
5. The method for preparing the mountain pepper extract as claimed in claim 2, wherein the mountain pepper root powder is obtained by pulverizing mountain pepper roots to 10-30 mesh.
6. An anti-inflammatory composition comprising the piper spicatum extract of claim 1.
7. An anti-inflammatory composition according to claim 6, further comprising a pharmaceutically acceptable pharmaceutical excipient.
8. Use of the piper spicatum extract of claim 1 for the preparation of an anti-inflammatory agent.
9. Use of the extract of piper spicatum of claim 1 for the manufacture of a medicament capable of inhibiting the production of NO induced by LPS.
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Non-Patent Citations (4)
| Title |
|---|
| 三种山胡椒属植物的化学成分及生物活性研究;雷洁萍;《中国优秀博硕士学位论文全文数据库(硕士)工程科技Ⅰ辑》;20170715(第07期);第B014-277页 * |
| 四川山胡椒和尖叶蓝花楹化学成分及生物活性的研究;袁娟娟;《中国优秀博硕士学位论文全文数据库(硕士) 医药卫生科技辑》;20181015(第10期);第E057-44页 * |
| 四川山胡椒的化学成分及三种山胡椒属植物成分的活性筛选;张嘉穗;《中国优秀博硕士学位论文全文数据库(硕士)医药卫生科技辑》;20170215(第02期);第E057-337页 * |
| 绒毛山胡椒的化学成分及其生物活性研究;魏国清;《中国优秀博硕士学位论文全文数据库(硕士) 医药卫生科技辑》;20160125(第01期);第E057-44页 * |
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