CN113292427B - Split-ring-altane-type triterpenoid, preparation method and application thereof, and medicine - Google Patents

Split-ring-altane-type triterpenoid, preparation method and application thereof, and medicine Download PDF

Info

Publication number
CN113292427B
CN113292427B CN202010113708.3A CN202010113708A CN113292427B CN 113292427 B CN113292427 B CN 113292427B CN 202010113708 A CN202010113708 A CN 202010113708A CN 113292427 B CN113292427 B CN 113292427B
Authority
CN
China
Prior art keywords
compound
sub
fraction
fractions
mobile phase
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202010113708.3A
Other languages
Chinese (zh)
Other versions
CN113292427A (en
Inventor
于洋
姚新生
张维阳
鲁丹
李海波
胡宇慧
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jinan University
Original Assignee
Jinan University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jinan University filed Critical Jinan University
Priority to CN202010113708.3A priority Critical patent/CN113292427B/en
Publication of CN113292427A publication Critical patent/CN113292427A/en
Application granted granted Critical
Publication of CN113292427B publication Critical patent/CN113292427B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • A—HUMAN NECESSITIES
    • 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]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • 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
    • 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
    • C07C69/732—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 of unsaturated hydroxy carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/07—Optical isomers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2603/00—Systems containing at least three condensed rings
    • C07C2603/93—Spiro compounds
    • C07C2603/94—Spiro compounds containing "free" spiro atoms

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Public Health (AREA)
  • Pain & Pain Management (AREA)
  • Rheumatology (AREA)
  • Biochemistry (AREA)
  • Toxicology (AREA)
  • Medicines Containing Plant Substances (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

Abstract

The invention provides a split-ring-Argentine-type triterpenoid compound, a preparation method and application thereof, and a medicament, and belongs to the technical field of medicines. The split-ring altane triterpenoid is any one of a compound 1 to a compound 14, and is prepared by eluting a gardenia fruit extract subjected to alcohol extraction by using macroporous resin, and then performing multiple separation and HPLC (high performance liquid chromatography) to obtain the split-ring altane triterpenoid with high purity. The split-ring altane triterpenoids have certain anti-inflammatory activity and antioxidant activity, and can be used for preparing anti-inflammatory drugs and antioxidant drugs. The medicine containing the split-ring altane triterpenoid has good anti-inflammatory effect and antioxidant effect.

Description

Split-ring-Argentine-type triterpenoid compound, preparation method and application thereof, and medicine
Technical Field
The invention relates to the technical field of medicines, and particularly relates to a split-ring altansane-type triterpene compound, a preparation method and application thereof, and a medicament.
Background
Gardenia (Gardenia jasminoides Ellis), namely, Plumbum preparatium, Gardenia jasminoides Ellis, Gardenia jasminoides Ellis, semen plantaginis, fructus fici Pumilae and the like, are dry mature fruits of Gardenia jasminoides belonging to the genus Gardenia (Gardenia) of the family Rubiaceae, and are mainly produced in Hunan, Jiangxi, Fujian, Zhejiang, Guangxi and the like of China. It is recorded in the pharmacopoeia of the Chinese edition, beginning from Shen nong Ben Cao Jing, listed as the "Zhongpin".
The fruit of gardenia is a common traditional Chinese medicine, and the gardenia yellow pigment is widely applied to food coloring and the like as a natural pigment, and is the first medical-edible dual-purpose resource issued by the ministry of health. Gardenia is cold in nature and bitter in taste, enters heart, lung and triple energizer meridians, has the functions of purging fire, relieving restlessness, clearing heat, promoting diuresis, cooling blood, removing toxicity and the like, and is mainly used for treating symptoms such as feverish diseases, vexation, jaundice due to damp-heat, conjunctival congestion, swelling and pain and the like.
Gardenia jasminoides ellis has various chemical components, and the chemical components of Gardenia jasminoides ellis are still to be studied deeply.
In view of this, the present application is specifically proposed.
Disclosure of Invention
The first purpose of the application comprises providing 14 brand-new seco-ring altane-type triterpenoids extracted from gardenia fruits for the first time.
A second object of the present invention is to provide a process for producing the above-mentioned seco-ring-ahltansane-type triterpene compound, which process enables efficient production of a high-purity seco-ring-ahltansane-type triterpene compound.
A third object of the present application comprises providing a use of the above-mentioned secocycloartane-type triterpene compound, for example, for the preparation of an anti-inflammatory agent or an antioxidant agent.
A fourth object of the present application includes providing a medicament containing the above-mentioned seco-cycloartane-type triterpenoid.
The technical problem solved by the application is realized by adopting the following technical scheme:
the application provides a split-ring altane-type triterpene compound, which is any one of a compound 1 to a compound 14, wherein the chemical structural formulas of the compound 1 to the compound 14 are as follows:
Figure BDA0002390841910000021
wherein,
r of Compound 1 1 ＝OH，R 2 ＝OH，R 3 ＝S 1 ，R 4 ＝H，R 5 ＝CH 2 OH and delta 4(28) ；
R of Compound 2 1 ＝OH，R 2 ＝OH，R 3 ＝S 3 ，R 4 ＝H，R 5 ＝CH 2 OH and delta 4(28) ；
R of Compound 3 1 ＝OH，R 2 ＝OH，R 3 ＝S 2 ，R 4 ＝H，R 5 ＝CH 2 (delta) and OH 4(28) ；
R of Compound 4 1 ＝OH，R 2 ＝OH，R 3 ＝S 4 ，R 4 ＝H，R 5 ＝CH 2 OH and delta 4(28) ；
R of Compound 5 1 ＝OH，R 2 ＝OH，R 3 ＝S 2 ，R 4 ＝OH，R 5 ＝CH 3 And (delta) 4(28) ；
R of Compound 6 1 ＝OH，R 2 ＝OH，R 3 ＝S 1 ，R 4 ＝OH，R 5 ＝CH 2 (delta) and OH 4(28) ；
R of Compound 7 1 ＝H，R 2 ＝OH，R 3 ＝S 2 ，R 4 ＝H，R 5 ＝CH 2 OH and delta 4(28) ；
R of Compound 8 1 ＝H，R 2 ＝OH，R 3 ＝S 2 ，R 4 ＝OH，R 5 ＝CH 2 (delta) and OH 4(28) ；
R of Compound 9 1 ＝OH，R 2 ＝OH，R 3 ＝S 1 ，R 4 ＝H，R 5 (ii) CHO and (delta) 4(28) ；
R of Compound 10 1 ＝OH，R 2 ＝OH，R 3 ＝S 2 ，R 4 ＝R 5 (ii) H and (delta) 4(28) ；
R of Compound 11 1 ＝OH，R 2 ＝OCH 3 ，R 3 ＝S 2 ，R 4 ＝H，R 5 ＝CH 2 OH and delta 4(28) ；
R of Compound 12 1 ＝OH，R 2 ＝OH，R 3 ＝S 2 ，R 4 ＝H，R 5 ＝CH 2 OH；
R of Compound 13 1 ＝OH，R 2 ＝OCH 3 ，R 3 ＝S 2 ，R 4 ＝H，R 5 ＝CH 2 OH；
R of Compound 14 1 ＝H，R 2 ＝OCH 3 ，R 3 ＝OH，R 4 ＝OH，R 5 ＝CH 2 (delta) and OH 4(28) ；
Wherein S is 1 To S 4 Respectively as follows:
Figure BDA0002390841910000031
in addition, the application also provides a preparation method of the schizocyclic altane triterpenoid, which comprises the following steps:
and (3) carrying out macroporous resin elution on the gardenia fruit extract subjected to alcohol extraction, wherein the conditions of the macroporous resin elution comprise that: eluting with low-alcohol-water as eluent at the volume concentration of 70-80% and 94-96%, and collecting 94-96% eluate to obtain target fraction GJ-5; chromatographic separation of the GJ-5 fraction yielded 13 total subcomponents 5A to 5M, which were subsequently prepared according to the desired compound in the corresponding manner as follows:
(a) carrying out chromatographic separation on the 5I component to obtain 5 sub-fractions in total from 5I1 to 5I 5; the 5I5 substream was subjected to HPLC separation to give compound 1 and compound 2.
(b) Carrying out chromatographic separation on the 5J component to obtain 14 sub-fractions in total from 5J1 to 5J 14; HPLC separation was performed on 5J5, 5J6, 5J8, 5J9 and 5J10 subflows to obtain compound 10, compound 5, compound 4, compound 3 and compound 13, respectively.
(c) Carrying out chromatographic separation on the 5H component to obtain 8 sub-fractions in total from 5H1 to 5H 8; HPLC separation was carried out on the 5H6 subflow and the 5H8 subflow, respectively, to give compound 9 and compound 7.
(d) Carrying out chromatographic separation on the 5K component to obtain 7 sub-fractions in total from 5K1 to 5K 7; HPLC separation was carried out on the 5K3 subflow and the 5K7 subflow, respectively, to obtain compound 8 and compound 6.
(e) Carrying out chromatographic separation on the 5L components to obtain 12 sub-fractions in total from 5L1 to 5L 12; HPLC separation was performed on the 5L11 subflow and the 5L12 subflow to obtain compound 11, compound 12 and compound 14.
In addition, the application also provides the application of the split-ring altane-type triterpenoid in preparing an anti-inflammatory drug or an antioxidant drug, for example, at least one of the compound 1, the compound 3, the compound 5, the compound 7, the compound 9 and the compound 12 can be used for preparing the anti-inflammatory drug, or at least one of the compound 4, the compound 5, the compound 7, the compound 10 and the compound 11 can be used for preparing the antioxidant drug.
In addition, the application also provides a medicament which contains the schizocyclic altane triterpenoid.
The split-ring-altane-type triterpene compound provided by the application, the preparation method and the application thereof, and the beneficial effects of a medicament comprise:
the secocycloartane triterpenoids are all brand-new compounds extracted from gardenia fruits and vegetables for the first time, and research on chemical components in gardenia is widened. The preparation method of the split-ring-Argentine triterpenoid is simple and easy to operate, and the compound with higher yield and purity can be prepared. The split-ring altane triterpenoids have certain anti-inflammatory activity and antioxidant activity, and can be used for preparing anti-inflammatory drugs or antioxidant drugs, especially compounds 1, 3, 5, 7, 9 and 12 have good anti-inflammatory (such as neuritis resisting) effect, and compounds 4, 5, 7, 10 and 11 have good antioxidant effect. The medicine containing the split-ring altane triterpenoid has better anti-inflammatory effect and antioxidant effect.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were carried out according to 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 split-ring aldinane triterpenoids provided by the embodiments of the present application, and the preparation method, application and a medicament thereof are specifically described below.
The split-ring altansane triterpenoid provided by the application is any one of a compound 1 to a compound 14, and the chemical structural formulas of the compound 1 to the compound 14 are as follows:
Figure BDA0002390841910000051
wherein,
r of Compound 1 1 ＝OH，R 2 ＝OH，R 3 ＝S 1 ，R 4 ＝H，R 5 ＝CH 2 OH and delta 4(28) ；
R of Compound 2 1 ＝OH，R 2 ＝OH，R 3 ＝S 3 ，R 4 ＝H，R 5 ＝CH 2 OH and delta 4(28) ；
R of Compound 3 1 ＝OH，R 2 ＝OH，R 3 ＝S 2 ，R 4 ＝H，R 5 ＝CH 2 (delta) and OH 4(28) ；
R of Compound 4 1 ＝OH，R 2 ＝OH，R 3 ＝S 4 ，R 4 ＝H，R 5 ＝CH 2 OH and delta 4(28) ；
R of Compound 5 1 ＝OH，R 2 ＝OH，R 3 ＝S 2 ，R 4 ＝OH，R 5 ＝CH 3 And (delta) 4(28) ；
R of Compound 6 1 ＝OH，R 2 ＝OH，R 3 ＝S 1 ，R 4 ＝OH，R 5 ＝CH 2 OH and delta 4(28) ；
R of Compound 7 1 ＝H，R 2 ＝OH，R 3 ＝S 2 ，R 4 ＝H，R 5 ＝CH 2 (delta) and OH 4(28) ；
R of Compound 8 1 ＝H，R 2 ＝OH，R 3 ＝S 2 ，R 4 ＝OH，R 5 ＝CH 2 (delta) and OH 4(28) ；
R of Compound 9 1 ＝OH，R 2 ＝OH，R 3 ＝S 1 ，R 4 ＝H，R 5 (ii) CHO and (delta) 4(28) ；
R of Compound 10 1 ＝OH，R 2 ＝OH，R 3 ＝S 2 ，R 4 ＝R 5 H and Δ 4(28) ；
R of Compound 11 1 ＝OH，R 2 ＝OCH 3 ，R 3 ＝S 2 ，R 4 ＝H，R 5 ＝CH 2 (delta) and OH 4(28) ；
R of Compound 12 1 ＝OH，R 2 ＝OH，R 3 ＝S 2 ，R 4 ＝H，R 5 ＝CH 2 OH；
R of Compound 13 1 ＝OH，R 2 ＝OCH 3 ，R 3 ＝S 2 ，R 4 ＝H，R 5 ＝CH 2 OH；
R of Compound 14 1 ＝H，R 2 ＝OCH 3 ，R 3 ＝OH，R 4 ＝OH，R 5 ＝CH 2 (delta) and OH 4(28) ；
Wherein S is 1 To S 4 Respectively as follows:
Figure BDA0002390841910000061
Figure BDA0002390841910000062
it is worth noting that Δ 4(28) represents a double bond midway between positions 4-28.
In the application, the seco-ring-Argentine triterpenoids are all separated from gardenia fruits. Wherein the Gardenia fruit can be mature Gardenia (Gardenia jasminoides Ellis) fruit collected from Cinnamomum kanehirae Hayata. Before preparation, the harvested mature fruits may be dried.
The preparation method of the split-ring altane triterpenoid compound comprises the following steps:
eluting fructus Gardeniae fruit extract with macroporous resin under the following conditions: eluting with lower alcohol-water as eluent at 70-80% and 94-96% volume concentration of lower alcohol in the eluent, and collecting 94-96% of the eluate to obtain target fraction GJ-5. Wherein the lower alcohol comprises methanol or ethanol. Chromatographic separation of the GJ-5 fraction into a total of 13 subcomponents 5A to 5M, followed by preparation according to the desired compound in the corresponding manner as follows (it is to be noted that "corresponding" herein is understood that when the desired compound is only any one of the compounds 1 to 14, it is possible to perform in a certain manner from a to e, and when the desired compound is two or more of the compounds 1 to 14, it is possible to perform in a different manner from a to e in combination):
(a) carrying out chromatographic separation on the 5I component to obtain 5 sub-fractions in total from 5I1 to 5I 5; the 5I5 substream was subjected to HPLC separation to give compound 1 and compound 2.
(b) Carrying out chromatographic separation on the 5J component to obtain 14 sub-fractions in total from 5J1 to 5J 14; HPLC separation was performed on 5J5, 5J6, 5J8, 5J9 and 5J10 subflows to obtain compound 10, compound 5, compound 4, compound 3 and compound 13, respectively.
(c) Carrying out chromatographic separation on the 5H component to obtain 8 sub-fractions in total from 5H1 to 5H 8; HPLC separation was carried out on the 5H6 subflow and the 5H8 subflow, respectively, to obtain compound 9 and compound 7.
(d) Carrying out chromatographic separation on the 5K component to obtain 7 sub-fractions in total from 5K1 to 5K 7; HPLC separation was carried out on the 5K3 subflow and the 5K7 subflow, respectively, to obtain compound 8 and compound 6.
(e) Carrying out chromatographic separation on the 5L components to obtain 12 sub-fractions in total from 5L1 to 5L 12; HPLC separation was performed on the 5L11 sub-stream and the 5L12 sub-stream, respectively, to give compound 12 and compound 14.
In reference, the extraction of the gardenia fruit extract may include the following steps: the gardenia fruit is extracted by refluxing with an alcohol reagent.
The alcohol reagent may be, but not limited to, ethanol-water solution, and may be other common alcohol reagents. When an ethanol-water solution is used as the extractant, the concentration of ethanol in the ethanol-water solution is preferably 55 to 75vt%, for example 55 vt%, 60 vt%, 65vt%, 70% or 75%, etc., more preferably 60 vt%.
In some embodiments, the feed-to-liquid ratio of the alcoholic reagent to the gardenia fruit may be 1g: 4-20mL, such as 1g: 4mL, 1g: 6mL, 1g: 8mL or 1g: 10mL or 1g:20mL, etc., preferably 1g: 4 mL.
In some embodiments, the number of reflux extractions may be 1, 2, or more than 2. In some preferred embodiments, the number of reflux extractions is 1-5 (e.g., may be 2 or 3). When the reflux extraction time is only 1 time, the reflux extraction time can be 5-7 h; when the reflux extraction is carried out for 2 times or more than 2 times, the time of each reflux extraction can be 0.5-2.5h, and after the extraction is carried out according to the conditions, the extracting solutions obtained after the multiple extractions are combined.
Alternatively, the macroporous resin elution described above may be performed using HP-20 macroporous resin.
In reference, the ratio of the volume of the eluent used for each elution component to the medicinal materials in the elution process of the macroporous resin can be 6-10 mL: 1 g.
In some preferred embodiments, the elution is performed in a ratio of 70% and 95% by volume of ethanol in the eluent, i.e. the volume ratio of ethanol to water is 70:30 and 95: 5.
By reference, the chromatographic separation of the GJ-5 component may include, for example:
using the mixed solution of cyclohexane-ethyl acetate-formic acid as an eluent, and respectively eluting the components according to the volume concentration of 99:1:0-100:0, 98.5:1.5:0-99.5:0.5:0, 96.5:3.5:0-97.5:2.5:0, 94.5:5.5:0-95.5:4.5:0, 92.5:7.5:0-93.5:6.5:0, 89.5:10.5:0-90.5:9.5:0, 84.5:15.5:0-85.5:14.5:0, 79.5:20.5:0-80.5:19.5:0, 69.5:30.5:0-70.5:29.5:0, 59.5:40.5:0-60.5:39.5:0, 49.5:50.5: 0.5:0, 49.5: 0.5:0, 99.5: 0-99.5: 0:0.5: 0, 98.5: 0:0: 0-98.5: 0.5:0, 98.5: 0: 0-98.5: 0:0:0, 100.5: 0:0: 0:0.5: 0, 1.5: 0:0: 0.5:0, 1.5: 0:0: 0.5:0, 100.5: 0:0: 0.5: 0:0: 0.5:0, 100.5: 0:0.5: 0, 100.5: 0:0.5: 0:0, 98.5: 0:0.5: 0, 1.5: 0.5: 0:0: 0.5: 0:0:0, and 100.5: 0.5: 0:0: 0.5: 0:0: 0:0.5: 0:0: 0:0.5: 0, and 100.5: 0.5: 0:0: 0.5: 0:0: 0.5:0, and 100.5: 0:0: 0.5: 0:0: 0.5:0, and 100.5: 0:0.5: 0:0: 0.5: 0:0: 0:0.5: 0:0, elution fractions with consistent results were detected and pooled to give 5A to 5M.
In some embodiments, the GJ-5 component is eluted at a concentration of 100:0:0, 99:1:0, 97:3:0, 95:5:0, 93:7:0, 90:10:0, 85:15:0, 80:20:0, 70:30:0, 60:40:0, 50:50:0, 0:100:0, 0:99:1, 0:98:2, and 0:0:100 by volume. Wherein, the detection can be carried out by high performance liquid chromatography, and the 'consistent result' indicates that the peak number and the time are approximately the same, as follows.
Alternatively, the volume of eluent used for each eluted component during the chromatographic separation of the GJ-5 component may be 4 to 8L.
By reference, in step (a), the chromatographic separation of the 5I component comprises: eluting with methanol as mobile phase, collecting fractions eluted from the mobile phase of 50-80mL as one sub-fraction, and sequentially collecting 5 sub-fractions to obtain 5I 1-5I 5.
In some embodiments, the 5I component can be subjected to exclusion chromatography using a Sephadex LH-20 gel chromatography column.
Further, the HPLC separation (preparation) conditions for the 5I5 subfractions included:
a chromatographic column: phenomenex Gemini C-18column, the specification of the chromatographic column can be phi 10 × 25 mm; flow rate: 2.5-3.5 mL/min; mobile phase: 58-62vt% acetonitrile.
In some preferred embodiments, the flow rate is 3mL/min and the mobile phase is 60 vt% acetonitrile.
After separation (preparation) according to the HPLC separation conditions described above, the retention time t is collected R The substance 23.5min is compound 1, and the retention time t is collected R Compound 2 was obtained after 26.5 min. It is noted that the above retention times may deviate somewhat within certain acceptable limits, as follows.
By reference, in step (b), the chromatographic separation of the 5J fraction may comprise: and (3) eluting the 5J component by taking methanol-water as a mobile phase according to the volume concentration of 74:26-76:24 and 99:1-100: 0. When elution is carried out at a volume concentration of 74:26-76:24, the fraction eluted from 750mL of mobile phase per 500-. When elution is carried out at a volume concentration of 99:1-100:0, the fraction eluted from the mobile phase of 750mL each 500-750mL is taken as one sub-fraction, and 2 sub-fractions are collected in turn. The fractions eluted with consistent results were detected and combined to give a sub-stream of 5J1 to a sub-stream of 5J 14.
In some preferred embodiments, the 5J fraction is eluted at a concentration of 75:25 and 100:0 by volume of methanol-water as described above.
Alternatively, in the present application, the 5J fraction can be chromatographically separated using an ODS column.
Further, HPLC separation (preparation) conditions for the 5J5, 5J6, 5J8, 5J9 and 5J10 sub-fractions may include:
and (3) chromatographic column: phenomenex Gemini C-18 column; flow rate: 2.5-3.5 mL/min; the flows corresponded to 52-58vt% acetonitrile, 58-62vt% acetonitrile and 52-58vt% acetonitrile, respectively.
In some preferred embodiments, the flow rate is 3mL/min, and the flows correspond to 55 vt% acetonitrile, 60 vt% acetonitrile and 55 vt% acetonitrile, respectively.
After separation (preparation) according to the HPLC separation conditions described above, the retention time t is collected R The substance with the retention time of 26min is the compound 10, and the retention time t is collected R Compound 5 is obtained after 45min, and the retention time t is collected R Compound 4 is obtained after 27min, and the retention time t is collected R The substance which is 23min is the compound 3, and the retention time t is collected R Compound 13 was obtained when 28.5 min.
In reference, the conditions under which the 5H fraction is chromatographically separated in step (c) may include: the 5H fraction was eluted at 50:50 and 100:0 volume concentrations with methanol-water as the mobile phase. When the elution is carried out with the volume concentration of 50:50, taking the fraction eluted by each 300-mL mobile phase as a sub-fraction, and sequentially collecting 10 sub-fractions; when elution is performed with the volume concentration of 100:0, every 600mL of eluted fractions of the mobile phase are taken as one sub-fraction, and 2 sub-fractions are sequentially collected. The fractions eluted with consistent results were detected and combined to give a sub-stream of 5H1 to a sub-stream of 5H 8.
In some preferred embodiments, the 5H fraction is eluted at a concentration of 50:50 and 100:0 by volume of methanol-water as described above.
Alternatively, in the present application, the 5H fraction can be chromatographically separated using an ODS column.
Further, HPLC separation (preparation) conditions for the 5H6 and 5H8 sub-fractions may include:
a chromatographic column: phenomenex Gemini C-18 column; flow rate: 2.5-3.5 mL/min; the flows corresponded to 62-68vt% acetonitrile and 76-80vt% acetonitrile, respectively. Preferably, the mobile phases each contain 0.08 to 0.12vt% (e.g. 0.1 vt%) of acetic acid or formic acid or trifluoroacetic acid to avoid peak tailing.
In some preferred embodiments, the above flow rate is 3mL/min, corresponding to a flow of 65vt% acetonitrile (containing 0.1 vt% acetic acid or formic acid or trifluoroacetic acid) and 78 vt% acetonitrile (containing 0.1 vt% acetic acid or formic acid or trifluoroacetic acid), respectively.
After separation according to the HPLC separation conditions, the retention time t is collected R The substance with 21min is the compound 9, and the retention time t is collected R Compound 7 was obtained after 15 min.
By reference, in step (d), the chromatographic separation of the 5K fraction may comprise: and (3) eluting the 5K component by using methanol-water as a mobile phase according to the volume concentration of 59:41-61:39 and 99:1-100: 0. When elution is carried out with the volume concentration of 59:41-61:59, taking the fraction eluted by every 300-600mL mobile phase as a sub-fraction, and sequentially collecting 7 sub-fractions; when elution is carried out with the volume concentration of 99:1-100:0, every 600mL of eluted fractions of the mobile phase are taken as one sub-fraction, and 2 sub-fractions are collected in turn. The fractions eluted with consistent results were detected and combined to give a sub-stream of 5K1 to a sub-stream of 5K 7.
In some preferred embodiments, the 5K fraction is eluted at a concentration of 60:40 and 100:0 by volume of methanol-water as described above.
Alternatively, in the present application, the 5K fraction can be chromatographically separated using an ODS column.
Further, the HPLC separation (preparation) conditions for the 5K3 sub-fraction and the 5K7 sub-fraction may include:
a chromatographic column: phenomenex Gemini C-18 column; flow rate: 2.5-3.5 mL/min; the flows corresponded to 60-65vt% acetonitrile and 52-58vt% acetonitrile, respectively. Preferably, the mobile phases each contain from 0.08 to 0.12vt% (e.g. 0.1 vt%) of acetic acid or formic acid or trifluoroacetic acid.
In some preferred embodiments, the flow rate is 3mL/min, with flows corresponding to 63 vt% acetonitrile (containing 0.1 vt% acetic acid or formic acid or trifluoroacetic acid) and 55 vt% acetonitrile (containing 0.1 vt% acetic acid or formic acid or trifluoroacetic acid), respectively.
After separation according to the HPLC separation conditions, the retention time t is collected R The substance with the retention time t of 40min is the compound 8 R The substance which lasts for 20min is the compound 6.
By reference, in step (e), the chromatographic separation of the 5L fraction may comprise: and (3) eluting 5L of components by taking methanol-water as a mobile phase according to the volume concentration of 74:26-76:24 and 99:1-100: 0. When elution is carried out at a volume concentration of 74:26-76:24, the fraction eluted from every 250-500mL of mobile phase is taken as one sub-fraction, and 12 sub-fractions are collected in turn; when elution is carried out at a volume concentration of 99:1-100:0, every 500mL of the mobile phase eluted from the column is taken as one sub-fraction, and 2 sub-fractions are sequentially collected. The fractions eluted with consistent results were detected and combined to give a 5L1 subflow to a 5L12 subflow.
In some preferred embodiments, the 5K fraction is eluted at a concentration of 75:25 and 100:0 by volume of methanol-water as described above.
Alternatively, in the present application, the 5L fraction can be chromatographically separated using an ODS column.
Further, the HPLC separation (preparation) conditions for the 5L11 sub-fraction and the 5L12 sub-fraction may include:
and (3) chromatographic column: phenomenex Gemini C-18 column; flow rate: 2.5-3.5 mL/min; mobile phase: 58-62vt% acetonitrile. Preferably, the mobile phase contains 0.08 to 0.12vt% of acetic acid or formic acid or trifluoroacetic acid.
In some preferred embodiments, the flow rate is 3mL/min and the mobile phase is 60 vt% acetonitrile (containing 0.1 vt% acetic acid or formic acid or trifluoroacetic acid).
After separation according to the HPLC separation conditions, the retention time t is collected R 33min substance is compound 11, and the retention time t is collected R The 39min substance was compound 14.
The structures of the above compounds 1 to 14 are shown in table 1 by nuclear magnetic and mass spectrometric identification.
Identification results of the Compounds of Table 1
Figure BDA0002390841910000121
Figure BDA0002390841910000131
Figure BDA0002390841910000141
Figure BDA0002390841910000151
In addition, the application also provides the application of the schizocyclic altane triterpenoid, such as anti-inflammatory drugs or anti-oxidation drugs.
Preferably, at least one of compound 1, compound 3, compound 5, compound 7, compound 9 and compound 12 can be used for the preparation of an anti-inflammatory drug. In reference, at least one of compound 1, compound 3, compound 5, compound 7, compound 9, and compound 12 can be used for the preparation of an anti-inflammatory agent that inhibits NO production, such as an anti-neuritis agent. Wherein the anti-neuritis agent comprises an anti-neurodegenerative disease agent, such as anti-Alzheimer's disease or anti-Parkinson's disease agent.
Preferably, at least one of compound 4, compound 5, compound 7, compound 10 and compound 11 can be used for preparing an antioxidant drug.
In addition, the application also provides a medicament which contains the schizocyclic altane triterpenoid, preferably at least one of the compound 1, the compound 3, the compound 4, the compound 5, the compound 7, the compound 9, the compound 10, the compound 11 and the compound 12 so as to have better anti-inflammatory effect or antioxidant effect.
Example 1
40kg of dried gardenia fruit medicinal material collected from Jiangxi is taken, and is extracted by refluxing for 3 times (2 hours each time) of 4 times (the material-liquid ratio is 1g: 4mL) of ethanol-water solution with the ethanol concentration of 60 vt%, the extracting solution is combined, and the gardenia extract is obtained by decompression and concentration, wherein 6.2kg of gardenia extract is obtained. Gradient eluting with ethanol-water as eluent at the volume ratio of ethanol to water of 70:30 and 95:5 through HP-20 macroporous resin, collecting 95: and 5, eluting the part to obtain a target fraction GJ-5. The ratio of the volume of the eluent used for each eluted component to the medicinal materials is 6 mL: 1g of the total weight of the composition.
GJ-5(80g) was subjected to silica gel column chromatography using a cyclohexane-ethyl acetate-methanol gradient elution at 100:0:0, 99:1:0, 97:3:0, 95:5:0, 93:7:0, 90:10:0, 85:15:0, 80:20:0, 70:30:0, 60:40:0, 50:50:0, 0:100:0, 0:99:1, 0:98:2, and 0:0:100 concentrations by volume, and the volume of eluent used for each eluted fraction was 6L, yielding 13 sub-fractions (5A to 5M).
Performing SephadexLH-20 gel exclusion chromatography separation on 5I component by taking methanol as mobile phase, performing isocratic elution in the separation process, taking the fraction eluted from each 60mL mobile phase as one sub-fraction, sequentially collecting 5 sub-fractions to obtain 5I 1-5I 5, and preparing the sub-fraction 5I5 by semi-preparative HPLC to obtain a compound 1 (t-type HPLC) R 23.5min) and compound 2 (t) R 26.5 min). Sub-fraction 5I5 the column used in the preparation was phenomenex GeminiC-18column (. phi.10X 25mm), the flow rate was 3mL/min, and the mobile phase was 60 vt% acetonitrile.
ODS column chromatography separation was performed on 5J column using methanol-water as mobile phase at 75:25 and 100:0 by volume concentration. When elution is carried out with the volume concentration of 75:25, the fraction eluted from each 600mL of mobile phase is taken as one sub-fraction, and 14 sub-fractions are sequentially collected; when the elution is carried out with the volume concentration of 100:0, the fraction eluted from each 600mL of mobile phase is taken as a sub-fraction, and 2 sub-fractions are sequentially collected; fractions with consistent results were detected and pooled to give 14 sub-fractions (5J1 to 5J 14).
In which subflow 5J5 was subjected to HPLC to afford compound 10 (t) R 26min), subfraction 5J6 was prepared by HPLC to afford compound 5 (t) R (45 min), subfraction 5J8 by HPLC to afford compound 4 (t) R 27min), subfraction 5J9 was prepared by HPLC to afford compound 3 (t) R (23 min), subflow 5J10 by HPLC to afford compound 13 (t, t) R 28.5 min). Sub-fraction 5J5, sub-fraction 5J6, sub-fraction 5J8, sub-fraction 5J9 and sub-fraction 5J10 all the chromatographic columns used for the preparation were Phenomenex GeminiC-18column (. phi.10X 25mm), the flow rate was 3mL/min, and the mobile phases were 55 vt% acetonitrile, 60 vt% acetonitrile and 55 vt% acetonitrile, respectively.
ODS column chromatography separation was performed on 5H with methanol-water as the mobile phase at 50:50 and 100:0 by volume. When elution is carried out with the volume concentration of 50:50, the fraction eluted by every 400mL of eluent is taken as one sub-fraction, and 10 sub-fractions are sequentially collected; when the elution is carried out with the volume concentration of 50:50, the fraction eluted by every 400mL of eluent is taken as one sub-fraction, and 1 sub-fraction is collected in sequence; fractions with consistent results were detected and pooled, yielding 8 sub-fractions (5H1 to 5H 8).
Fractionation of 5H6 by HPLC gave compound 9 (t) R 21min), subflow 5H8 was separated by HPLC to give compound 7 (t) R 15 min). Sub-fraction 5H6 and sub-fraction 5H8 the chromatographic columns used for the preparation were PhenomenexGeminiC-18column (. phi.10X 25mm) with a flow rate of 3mL/min and mobile phases of 65vt% acetonitrile and 78 vt% acetonitrile, respectively, containing 0.1 vt% acetic acid.
Performing ODS column chromatography separation on 5K by taking methanol-water as a mobile phase according to the volume concentration of 60:40 and 100: 0. When elution is carried out with the volume concentration of 60:40, the fraction eluted by every 400mL of eluent is used as one sub-fraction, and 7 sub-fractions are sequentially collected; when the volume concentration is 100:0 for elution, the fraction eluted by every 400mL of eluent is taken as one sub-fraction, and 2 sub-fractions are sequentially collected; the fractions with consistent results were detected and pooled to give 7 sub-fractions (5K to 5K 7).
Fractionation of 5K3 by HPLC gave compound 8 (t) R 40min), subfraction 5K7 was separated by HPLC to give compound 6 (t) R 20 min). Sub-fraction 5K3 and sub-fraction 5K7 the chromatographic columns used for the preparation were all PhenomenexGeminiC-18column (. phi.10X 25mm) with a flow rate of 3mL/min and mobile phases of 63 vt% acetonitrile and 55 vt% acetonitrile, respectively, containing 0.1 vt% acetic acid.
ODS column chromatography separation was performed on 5L with methanol-water as a mobile phase at a volume concentration of 75:25 and 100: 0. When elution is carried out with the volume concentration of 75:25, the fraction eluted by every 400mL of eluent is taken as one sub-fraction, and 12 sub-fractions are sequentially collected; when the volume concentration is 100:0 for elution, the fraction eluted by every 400mL of eluent is taken as one sub-fraction, and 2 sub-fractions are sequentially collected; the fractions with consistent results were detected and pooled to give 12 sub-fractions (5L1 to 5L 12).
Sub-fraction 5L11 prepared by HPLC to give compound 11 (t) R 33 min). The column used in the preparation of sub-fraction 5L11 was phenomenex gemini C-18column (phi 10X 25mm), the flow rate in the preparation was 3mL/min, the mobile phase was 60 vt% acetonitrile, and the mobile phase contained 0.1 vt% acetic acid. Fraction 5L12 Using the same procedure and experimental conditions Compound 12 (t) R 42min) and compound 14 (t) R ＝39min)。
Example 2
Taking dried gardenia fruit medicinal materials collected from the west of Yangxi, carrying out reflux extraction for 2 times (2.5 hours each time) by 6 times (the material-liquid ratio is 1g: 6mL) of ethanol-water solution with the ethanol concentration of 55 vt%, combining extracting solutions, and carrying out reduced pressure concentration to obtain gardenia extract. And carrying out gradient elution by using ethanol-water as an eluent through HP-20 macroporous resin according to the volume ratio of the ethanol to the water of 75:25 and 94:6 to obtain GJ-5. The ratio of the volume of the eluent used for each eluted component to the medicinal materials is 8 mL: 1 g.
Silica gel column chromatography was performed using a cyclohexane-ethyl acetate-methanol gradient elution at concentrations of 98.5:1.5:0, 96.5:3.5:0, 94.5:5.5:0, 92.5:7.5:0, 89.5:10.5:0, 84.5:15.5:0, 79.5:20.5:0, 69.5:30.5:0, 59.5:40.5:0, 49.5:50.5:0, 0:99.8:0.2, 0:98.8:1.2, 0:97.5:2.5 and 0:0.5:99.5 eluents, 4L in volume, per eluting component, to give 13 sub-components (5A to 5M).
Methanol is used as a mobile phase to perform SephadexLH-20 gel exclusion chromatographic separation on the 5I component, isocratic elution is adopted in the separation process, each 50mL of fraction eluted from the mobile phase is used as one sub-fraction, 5 sub-fractions are sequentially collected to obtain 5I 1-5I 5, and the sub-fraction 5I5 is prepared into the compound 1 and the compound 2 through semi-preparative HPLC. Sub-fraction 5I5 the column used in the preparation was Phenomenex GeminiC-18column (. phi.10X 25mm), the flow rate was 2.5mL/min, and the mobile phase was 58vt% acetonitrile.
ODS column chromatography separation was performed on 5J with methanol-water as a mobile phase at 74:26 and 99:1 volume concentrations. When the elution is carried out with the volume concentration of 74:26, the fraction eluted from every 500mL of mobile phase is taken as one sub-fraction, and 14 sub-fractions are sequentially collected; when the elution is carried out with the volume concentration of 99:1, the fraction eluted from every 500mL of mobile phase is taken as a sub-fraction, and 2 sub-fractions are sequentially collected; the fractions with consistent results were detected and pooled to give 14 sub-fractions (5J1 to 5J 14).
Wherein, sub-fraction 5J5 is prepared by HPLC to obtain compound 10, sub-fraction 5J6 is prepared by HPLC to obtain compound 5, sub-fraction 5J8 is prepared by HPLC to obtain compound 4, sub-fraction 5J9 is prepared by HPLC to obtain compound 3, and sub-fraction 5J10 is prepared by HPLC to obtain compound 13. Sub-fraction 5J5, sub-fraction 5J6, sub-fraction 5J8, sub-fraction 5J9 and sub-fraction 5J10 all the chromatographic columns used for the preparation were Phenomenex Gemini C-18column (. phi.10X 25mm), the flow rate was 2.5mL/min, and the mobile phases were 52 vt% acetonitrile, 58vt% acetonitrile and 52 vt% acetonitrile, respectively.
And (3) performing ODS column chromatography separation on 5H by taking methanol-water as a mobile phase according to the volume concentration of 50:50 and 100: 0. When elution is carried out with the volume concentration of 50:50, the fraction eluted by eluent of every 300mL is taken as one sub-fraction, and 7 sub-fractions are sequentially collected; when the volume concentration is 100:0 for elution, the fraction eluted by eluent of every 300mL is taken as one sub-fraction, and 2 sub-fractions are collected in sequence; fractions with consistent results were detected and pooled, yielding 8 sub-fractions (5H1 to 5H 8).
Fractionation of 5H6 gave compound 9 by HPLC and fractionation of 5H8 gave compound 7 by HPLC. Subfraction 5H6 and subfraction 5H8 both Phenomenex GeminiC-18column (. phi.10X 25mm) were prepared using 2.5mL/min flow rate mobile phase of 62vt% acetonitrile and 76 vt% acetonitrile respectively containing 0.08 vt% acetic acid.
ODS column chromatography separation was performed on 5K at 59:41 and 99:1 volume concentrations using methanol-water as a mobile phase. When the elution is carried out with the volume concentration of 59:41, the fraction eluted by each 300mL of eluent is taken as one sub-fraction, and 7 sub-fractions are sequentially collected; when the elution is carried out with the volume concentration of 99:1, the fraction eluted by eluent of every 300mL is taken as one sub-fraction, and 2 sub-fractions are collected in sequence; the fractions with consistent results were detected and pooled to give 7 sub-fractions (5K to 5K 7).
Fractionation 5K3 gave compound 8 by HPLC, and fractionation 5K7 gave compound 6 by HPLC. Sub-fraction 5K3 and sub-fraction 5K7 the chromatographic columns used for the preparation were all PhenomenexGeminiC-18column (. phi.10X 25mm) with a flow rate of 2.5mL/min and mobile phases of 60 vt% acetonitrile and 52 vt% acetonitrile, respectively, containing 0.08 vt% acetic acid.
ODS column chromatography separation was performed on 5L at 74:26 and 99:1 volume concentrations using methanol-water as a mobile phase. When the elution is carried out by using the volume concentration of 74:26, the fraction eluted by every 250mL of eluent is taken as one sub-fraction, and 12 sub-fractions are sequentially collected; when the elution is carried out with the volume concentration of 99:1, the fraction eluted by every 250mL of eluent is taken as one sub-fraction, and 2 sub-fractions are sequentially collected; the fractions with consistent results were detected and pooled to give 12 sub-fractions (5L1 to 5L 12).
The sub-fraction 5L11 was prepared by HPLC to give compound 11, the column used in the preparation of sub-fraction 5L11 was phenomenex GeminiC-18column (. phi.10X 25mm), the flow rate during the preparation was 2.5mL/min, the mobile phase was 58vt% acetonitrile, and the mobile phase contained 0.08 vt% acetic acid. Fraction 5L12 using the same method and experimental conditions gave compound 12 and compound 14.
Example 3
Taking dried gardenia fruit medicinal materials collected from Jiangxi, carrying out reflux extraction for 1 time and 6 hours by 4.5 times (the material-liquid ratio is 1g: 10mL) of ethanol-water solution with the ethanol concentration of 65vt percent, and carrying out reduced pressure concentration to obtain gardenia extract. And carrying out gradient elution by using ethanol-water as an eluent according to the volume ratio of the ethanol to the water of 0:1, 32:68, 52:48, 72:28 and 96:4 through HP-20 macroporous resin to obtain 5 elution components GJ-1 to GJ-5. The ratio of the volume of the eluent used for each elution component to the medicinal materials is 10 mL: 1g of the total weight of the composition.
Silica gel column chromatography was performed using a cyclohexane-ethyl acetate-methanol gradient elution at 99.5:0.5:0, 97.5:2.5:0, 95.5:4.5:0, 93.5:6.5:0, 90.5:9.5:0, 85.5:14.5:0, 80.5:19.5:0, 70.5:29.5:0, 60.5:39.5:0, 50.5:49.5:0, 0:100:0, 0:99.5:0.5, 0:98.5:1.5, and 0:0:100 by volume, using 8L of eluent for each eluted fraction, to give 13 sub-fractions (5A to 5M).
Methanol is used as a mobile phase to perform SephadexLH-20 gel exclusion chromatographic separation on the 5I component, isocratic elution is adopted in the separation process, the fraction eluted from each 80mL of mobile phase is used as one sub-fraction, 5 sub-fractions are sequentially collected to obtain 5I 1-5I 5, and the sub-fraction 5I5 is prepared by semi-preparative HPLC to obtain a compound 1 and a compound 2. Sub-fraction 5I5 the column used in the preparation was Phenomenex GeminiC-18column (. phi.10X 25mm), the flow rate was 3.5mL/min, and the mobile phase was 62vt% acetonitrile.
ODS column chromatography separation was performed on 5J column with methanol-water as mobile phase at 76:24 and 100:0 by volume concentration. When elution is carried out with the volume concentration of 76:24, the fraction eluted from every 750mL of mobile phase is taken as one sub-fraction, and 15 sub-fractions are sequentially collected; when the volume concentration is 100:0 for elution, the fraction eluted from each 750mL of mobile phase is taken as one sub-fraction, and 2 sub-fractions are sequentially collected; the fractions with consistent results were detected and pooled to give 14 sub-fractions (5J1 to 5J 14).
Wherein, subflow 5J5 is prepared by HPLC to obtain compound 10, subflow 5J6 is prepared by HPLC to obtain compound 5, subflow 5J8 is prepared by HPLC to obtain compound 4, subflow 5J9 is prepared by HPLC to obtain compound 3, and subflow 5J10 is prepared by HPLC to obtain compound 13. Sub-fraction 5J5, sub-fraction 5J6, sub-fraction 5J8, sub-fraction 5J9 and sub-fraction 5J10 all the chromatographic columns used for the preparation were Phenomenex GeminiC-18column (. phi.10X 25mm), the flow rate was 3.5mL/min, and the mobile phases were 58vt% acetonitrile, 62vt% acetonitrile and 58vt% acetonitrile, respectively.
ODS column chromatography separation was performed on 5H with methanol-water as the mobile phase at 50:50 and 100:0 by volume. When elution is carried out with the volume concentration of 50:50, the fraction eluted by eluent of every 600mL is taken as one sub-fraction, and 10 sub-fractions are sequentially collected; when the elution is carried out with the volume concentration of 100:0, the fraction eluted by every 600mL of eluent is taken as one sub-fraction, and 2 sub-fractions are sequentially collected; the fractions with consistent results were detected and pooled to give 8 sub-fractions (5H1 to 5H 8).
Fractionation of 5H6 gave compound 9 by HPLC and fractionation of 5H8 gave compound 7 by HPLC. Sub-fraction 5H6 and sub-fraction 5H8 the chromatographic columns used for the preparation were Phenomenex GeminiC-18column (. phi.10X 25mm) with a flow rate of 3.5mL/min and mobile phases of 68vt% acetonitrile and 80vt% acetonitrile, respectively, containing 0.12vt% acetic acid.
ODS column chromatography separation was performed on 5K with methanol-water as the mobile phase at 61:39 and 100:0 by volume. When elution is carried out with the volume concentration of 61:39, the fraction eluted by eluent of every 600mL is taken as one sub-fraction, and 7 sub-fractions are sequentially collected; when the elution is carried out with the volume concentration of 100:0, the fraction eluted by eluent of every 600mL is taken as one sub-fraction, and 2 sub-fractions are collected in sequence; fractions with consistent results were detected and pooled to give 7 sub-fractions (5K to 5K 7).
Fractionation with 5K3 gave compound 8 and fractionation with 5K7 gave compound 6. Both the column used for the preparation of subfraction 5K3 and subfraction 5K7 were phenomenex GeminiC-18column (. phi.10X 25mm) with a flow rate of 3.5mL/min and mobile phases 63 vt% acetonitrile and 55 vt% acetonitrile, respectively, containing 0.12vt% acetic acid.
ODS column chromatography separation was performed on 5L with methanol-water as a mobile phase at 76:24 and 100:0 by volume concentration. When elution is carried out with the volume concentration of 76:24, the fraction eluted by every 500mL of eluent is taken as one sub-fraction, and 12 sub-fractions are sequentially collected; when the elution is carried out with the volume concentration of 100:0, the fraction eluted by every 500mL of eluent is taken as one sub-fraction, and 2 sub-fractions are sequentially collected; the fractions with consistent results were detected and pooled to give 12 sub-fractions (5L1 to 5L 12).
The sub-fraction 5L11 was prepared by HPLC to give compound 11, the column used in the preparation of sub-fraction 5L11 was PhenomenexGeminiC-18column (. phi.10X 25mm), the flow rate during the preparation was 3.5mL/min, the mobile phase was 62vt% acetonitrile, and the mobile phase contained 0.12vt% acetic acid. Fraction 5L12 using the same procedure and experimental conditions gave compound 12 and compound 14.
Test example 1
The anti-inflammatory activity of the 14 secocycloartane-type triterpenoids obtained was tested in example 1.
Specifically, a BV-2 microglia cell inflammation model induced by LPS is established aiming at the intracerebral neuroinflammation, and the anti-neuritis activity of the compound is researched. The cultured BV-2 cell strain is taken out for experiment, and the cell density is adjusted to 2.5 multiplied by 10 4 Adding cell suspensions into a 96-well plate according to the number/mL of cells in groups, and incubating for 24h and then carrying out an experiment; grouped according to experimental requirements, each group having 3 parallel wells. After 24 hours of culture, corresponding liquid medicine, culture medium and LPS (the final concentration is 0.5 mu g/mL) are added according to groups, and after further 24 hours of culture, cell supernatant is collected, and the NO concentration is detected by the Griess method. Dexamethasone was used as a positive control.
The results are shown in table 2, and it is worth noting that table 2 shows only a few compounds having significant effects and corresponding results, and that compounds not shown also have anti-neuritic effects, but only compounds not shown have significant effects.
TABLE 2 results of anti-inflammatory Activity
Figure BDA0002390841910000221
Figure BDA0002390841910000231
As can be seen from table 2, compounds 1, 3, 5, 7, 9 and 12 significantly inhibited NO production, indicating that they have potential anti-neuritic effects.
Test example 2
The 14 types of split-ring-cycloartane-type triterpenoids obtained in example 1 were subjected to an anti-oxidation-radical scavenging test.
Specifically, DPPH: adding 1014.4 μ L ethanol into DPPH8.0mg to obtain a solution with a concentration of 2 × 10 -2 M mother liquor; rutin (rutin): rutin 12.2mg, adding 1000 μ L DMSO, and making into 2 × 10 -2 M mother liquor as positive control; the test compound (1-14) was dissolved in DMSO to prepare 2X 10 -2 M mother liquor. Mixing 100 μ L of 2X 10 -4 M sample solution and 100. mu.L of 2X 10 -4 The M DPPH solution was added to a 96-well plate, placed on a shaker to shake for 30min, and the absorbance value (OD517) was measured at 517 nm.
Radical scavenging rate (OD) Solvent(s) -OD Sample (I) /OD Solvent(s) ×100％。
Rutin is used as positive control.
The results are shown in table 3, and it is worth explaining that table 3 shows only a few compounds having significant effects and corresponding results, and that compounds not shown also have antioxidant effects, but compounds not shown have significant effects.
TABLE 3 antioxidant results
Compound number EC 50 (μM)
4 17.31±4.08
5 5.08±0.41
7 6.72±0.96
10 7.36±1.34
11 8.98±1.13
Rutin (rutin) 8.00±1.01
As can be seen from table 3, compounds 4, 5, 7, 10, and 11 have potential antioxidant activity.
In summary, the split-ring altane triterpenoids provided by the application are all brand-new compounds extracted from gardenia fruits and vegetables for the first time, and the research on chemical components in gardenia is widened. The preparation method of the split-ring-Argentine triterpenoid is simple and easy to operate, and the compound with higher yield and purity can be prepared. The split-ring altane triterpenoids have certain anti-inflammatory activity and antioxidant activity, and can be used for preparing anti-inflammatory drugs or antioxidant drugs, especially the compound 1, the compound 3, the compound 5, the compound 7, the compound 9 and the compound 12 have good anti-inflammatory (such as neuritis) effects, and the compound 4, the compound 5, the compound 7, the compound 10 and the compound 11 have good antioxidant effects. The medicine containing the split-ring altane triterpenoid has better anti-inflammatory effect and antioxidant effect.
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 (14)

1. A split-ring altane-type triterpene compound is characterized in that the split-ring altane-type triterpene compound is any one of a compound 1 to a compound 13, and the chemical structural formulas of the compound 1 to the compound 13 are as follows:
Figure 618429DEST_PATH_IMAGE001
wherein
r of the Compound 1 1 =OH，R 2 =OH，R 3 =S 1 ，R 4 =H，R 5 =CH 2 OH and delta 4(28) ；
R of the Compound 2 1 =OH，R 2 =OH，R 3 =S 3 ，R 4 =H，R 5 =CH 2 (delta) and OH 4(28) ；
R of said Compound 3 1 =OH，R 2 =OH，R 3 =S 2 ，R 4 =H，R 5 =CH 2 (delta) and OH 4(28) ；
R of said Compound 4 1 =OH，R 2 =OH，R 3 =S 4 ，R 4 =H，R 5 =CH 2 (delta) and OH 4(28) ；
R of said Compound 5 1 =OH，R 2 =OH，R 3 =S 2 ，R 4 =OH，R 5 =CH 3 And (delta) 4(28) ；
R of said Compound 6 1 =OH，R 2 =OH，R 3 =S 1 ，R 4 =OH，R 5 =CH 2 OH and delta 4(28) ；
R of said Compound 7 1 =H，R 2 =OH，R 3 =S 2 ，R 4 =H，R 5 =CH 2 OH and delta 4(28) ；
R of said Compound 8 1 =H，R 2 =OH，R 3 =S 2 ，R 4 =OH，R 5 =CH 2 (delta) and OH 4(28) ；
R of said Compound 9 1 =OH，R 2 =OH，R 3 =S 1 ，R 4 =H，R 5 = CHO and delta 4(28) ；
R of said Compound 10 1 =OH，R 2 =OH，R 3 =S 2 ，R 4 = R 5 H and delta 4(28) ；
R of said Compound 11 1 =OH，R 2 =OCH 3 ，R 3 =S 2 ，R 4 =H，R 5 =CH 2 OH and delta 4(28) ；
R of said Compound 12 1 =OH，R 2 =OH，R 3 =S 2 ，R 4 =H，R 5 =CH 2 OH；
R of said Compound 13 1 =OH，R 2 =OCH 3 ，R 3 =S 2 ，R 4 =H，R 5 =CH 2 OH；
Said Δ 4(28) represents a double bond intermediate the 4-28 positions;
wherein S is 1 To S 4 Respectively as follows:
Figure 78884DEST_PATH_IMAGE002
，
Figure 893257DEST_PATH_IMAGE003
，
Figure 715719DEST_PATH_IMAGE004
and
Figure 45069DEST_PATH_IMAGE005
。
2. a process for the preparation of a split-ring alditol-type triterpene compound according to claim 1, comprising the steps of:
and (3) carrying out macroporous resin elution on the gardenia fruit extract subjected to alcohol extraction, wherein the conditions of the macroporous resin elution comprise that: eluting with ethanol-water as eluent at the volume concentration of 70-80% and 94-96%, and collecting 94-96% eluate to obtain target fraction GJ-5; chromatographic separation of the GJ-5 fraction yielded 13 total subcomponents 5A to 5M, which were subsequently prepared according to the desired compound in the corresponding manner as follows:
(a) carrying out chromatographic separation on the 5I component to obtain 5 sub-fractions in total from 5I1 to 5I 5; separating the 5I5 sub-stream by HPLC to obtain compound 1 and compound 2;
(b) carrying out chromatographic separation on the 5J component to obtain 14 sub-fractions in total from 5J1 to 5J 14; respectively carrying out HPLC separation on the 5J5 sub-stream, the 5J6 sub-stream, the 5J8 sub-stream, the 5J9 sub-stream and the 5J10 sub-stream to correspondingly obtain a compound 10, a compound 5, a compound 4, a compound 3 and a compound 13;
(c) carrying out chromatographic separation on the 5H component to obtain 8 sub-fractions in total from 5H1 to 5H 8; respectively carrying out HPLC separation on the 5H6 sub-flow and the 5H8 sub-flow to correspondingly obtain a compound 9 and a compound 7;
(d) carrying out chromatographic separation on the 5K component to obtain 7 sub-fractions in total from 5K1 to 5K 7; respectively carrying out HPLC separation on the 5K3 sub-flow and the 5K7 sub-flow to correspondingly obtain a compound 8 and a compound 6;
(e) carrying out chromatographic separation on the 5L components to obtain 12 sub-fractions in total from 5L1 to 5L 12; HPLC separation is respectively carried out on the 5L11 sub-flow and the 5L12 sub-flow, and a compound 11 and a compound 12 are correspondingly obtained;
eluting with HP-20 macroporous resin;
the extraction of the gardenia fruit extract comprises the following steps: reflux extracting fructus Gardeniae with alcohol reagent;
the alcohol reagent comprises an ethanol-water solution; the concentration of ethanol in the ethanol-water solution is 55-75 vt%;
the ratio of the alcohol reagent to the gardenia fruit is 1g: 4-20 mL;
the reflux extraction times are 1-5 times, and the reflux extraction time is 0.5-2.5h each time;
the chromatographic separation of the GJ-5 component comprises the following steps:
using cyclohexane-ethyl acetate-formic acid mixed solution as eluent, collecting elution components according to the volume concentration of 99:1:0-100:0, 98.5:1.5:0-99.5:0.5:0, 96.5:3.5:0-97.5:2.5:0, 94.5:5.5:0-95.5:4.5:0, 92.5:7.5:0-93.5:6.5:0, 89.5:10.5:0-90.5:9.5:0, 84.5:15.5:0-85.5:14.5:0, 79.5:20.5:0-80.5:19.5:0, 69.5:30.5:0-70.5:29.5:0, 59.5:40.5:0-60.5:39.5:0, 49.5:50.5: 0.5:0-50.5: 0.5:0, 99.5: 0-99: 0.5: 0:0.5: 0, 99: 0:0.5: 0: 1: 0-99.5:0.5:0, and 100:0: 0: 5:0.5: 0:0: 0:0.5: 0:0.5: 0.0.5: 0.0.0.0: 0.0.5: 0.5: 0.0.0: 0.5: 0.0.5: 0.5: 0.0.0.0.0.5: 0.5: 0.0.0.0.0.0.0.5: 0.0.0.0.0.0.0.0.0.0.0.0.5: 0.5: 0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.5: 0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.1: 0.0.1 to 100 to, detecting and combining elution components with consistent results to obtain the 5A to the 5M;
the volume of eluent used for each elution component in the chromatographic separation process of the GJ-5 component is 4-8L.
3. The process for the preparation of a secocycloartane-type triterpene compound according to claim 2, wherein the chromatographic separation of the 5I component comprises: eluting with methanol as mobile phase, collecting 5 sub-fractions per 50-80mL of the fraction eluted from the mobile phase as one sub-fraction, and obtaining 5I 1-5I 5;
carrying out exclusion chromatographic separation on the 5I component by adopting a Sephadex LH-20 gel chromatographic column;
the HPLC separation conditions for the 5I5 subfractions included:
and (3) chromatographic column: phenomenex Gemini C-18 column;
flow rate: 2.5-3.5 mL/min;
mobile phase: 58-62vt% acetonitrile.
4. The process for the preparation of a secocycloartane-type triterpene compound according to claim 2, wherein the chromatographic separation of the 5J component comprises: eluting the 5J component by using methanol-water as a mobile phase according to the volume concentration of 74:26-76:24 and 99:1-100: 0;
when the elution is carried out with the volume concentration of 74:26-76:24, every 500-750mL of fraction eluted from the mobile phase is taken as one sub-fraction, and 15 sub-fractions are sequentially collected;
when the elution is carried out with the volume concentration of 99:1-100:0, every 500-750mL of the fraction eluted from the mobile phase is taken as a sub-fraction, and 2 sub-fractions are sequentially collected;
detecting and combining elution components with consistent results to obtain the 5J1 subflow and the 5J14 subflow;
performing chromatographic separation on the 5J component by adopting an ODS (oxide dispersion strengthened) column;
the conditions for HPLC separation of the 5J5 subfraction, the 5J6 subfraction, the 5J8 subfraction, the 5J9 subfraction and the 5J10 subfraction comprise:
a chromatographic column: phenomenex Gemini C-18 column;
flow rate: 2.5-3.5 mL/min;
the flows corresponded 52-58vt% acetonitrile, 58-62vt% acetonitrile and 52-58vt% acetonitrile, respectively.
5. The process for the preparation of a seco-ring aldane-type triterpene compound of claim 2 wherein the conditions for chromatographic separation of the 5H component comprise: eluting the 5H component by taking methanol-water as a mobile phase according to the volume concentration of 50:50 and 100: 0;
when the elution is carried out with the volume concentration of 50:50, taking the fraction eluted from the mobile phase of every 300-600mL as a sub-fraction, and sequentially collecting 10 sub-fractions;
when the elution is carried out with the volume concentration of 100:0, taking each 600mL fraction eluted from the mobile phase as a sub-fraction, and sequentially collecting 2 sub-fractions;
detecting and combining elution components with consistent results to obtain the 5H1 subfractions to the 5H8 subfractions;
carrying out chromatographic separation on the 5H component by adopting an ODS (ozone depleting substance) column;
HPLC separation conditions for the 5H6 sub-fraction and the 5H8 sub-fraction include:
and (3) chromatographic column: phenomenex Gemini C-18 column;
flow rate: 2.5-3.5 mL/min;
flowing acetonitrile at 62-68vt% and acetonitrile at 76-80 vt%;
the mobile phase during HPLC separation of the 5H6 subflow and the 5H8 subflow both contained 0.08-0.12vt% of acetic acid or formic acid or trifluoroacetic acid.
6. The process for the preparation of a secocycloartane-type triterpene compound according to claim 2, wherein the chromatographic separation of the 5K component comprises: eluting the 5K component by taking methanol-water as a mobile phase according to the volume concentration of 59:41-61:39 and 99:1-100: 0;
when the elution is carried out with the volume concentration of 59:41-61:39, taking the fraction eluted from the mobile phase of every 300-600mL as a sub-fraction, and sequentially collecting 7 sub-fractions;
when the volume concentration is 99:1-100:0 for elution, every 300-600mL of the fraction eluted from the mobile phase is taken as a sub-fraction, and 2 sub-fractions are sequentially collected;
detecting and combining the elution components with consistent results to obtain the 5K1 sub-fraction to the 5K7 sub-fraction;
performing chromatographic separation on the 5K component by adopting an ODS (oxide dispersion strengthened) column;
the conditions for HPLC separation of the 5K3 sub-fraction and the 5K7 sub-fraction comprise:
a chromatographic column: phenomenex Gemini C-18 column;
flow rate: 2.5-3.5 mL/min;
the flow corresponds to 60-65vt% acetonitrile and 52-58vt% acetonitrile respectively;
the mobile phase during HPLC separation of the 5K3 sub-fraction and the 5K7 sub-fraction both contained 0.08-0.12vt% of acetic acid or formic acid or trifluoroacetic acid.
7. The process for the preparation of a seco-cycloartane-type triterpene compound according to claim 2, wherein the chromatographic separation of the 5L component comprises: eluting the 5L component by taking methanol-water as a mobile phase according to the volume concentration of 74:26-76:24 and 99:1-100: 0;
when the elution is carried out with the volume concentration of 74:26-76:24, every 500mL of fractions eluted from the mobile phase are taken as one sub-fraction, and 12 sub-fractions are sequentially collected;
when the elution is carried out with the volume concentration of 99:1-100:0, taking the fraction eluted from the mobile phase of every 500mL as one sub-fraction, and sequentially collecting 2 sub-fractions;
detecting and combining the elution components with consistent results to obtain the 5L1 subfractions to the 5L12 subfractions;
performing chromatographic separation on the 5L components by adopting an ODS (oxide dispersion strengthened) column;
HPLC separation conditions for the 5L11 sub-fraction and the 5L12 sub-fraction include:
a chromatographic column: phenomenex Gemini C-18 column;
flow rate: 2.5-3.5 mL/min;
mobile phase: 58-62vt% acetonitrile;
the mobile phase during the HPLC separation of the 5L11 sub-fraction and the 5L12 sub-fraction contained 0.08-0.12vt% of acetic acid or formic acid or trifluoroacetic acid.
8. Use of the split-ring aldrin-type triterpenoid of claim 1 in the preparation of an anti-inflammatory agent.
9. The use according to claim 8, wherein at least one of said compound 1, said compound 3, said compound 5, said compound 7, said compound 9 and said compound 12 is used for the manufacture of an anti-inflammatory medicament for the inhibition of NO production.
10. The use according to claim 8, wherein at least one of said compound 1, said compound 3, said compound 5, said compound 7, said compound 9 and said compound 12 is used for the manufacture of a medicament for the treatment of neuritis.
11. The use of claim 10, wherein the anti-neuritis agent comprises an anti-neurodegenerative disease agent.
12. The use according to claim 11, wherein the anti-neurodegenerative disease agent comprises an anti-alzheimer's disease or anti-parkinson's disease agent.
13. Use of the split-ring aldinane triterpenoid of claim 1 in the preparation of an antioxidant medicament.
14. A pharmaceutical agent comprising the split-ring cycloartane-type triterpene compound of claim 1.
CN202010113708.3A 2020-02-24 2020-02-24 Split-ring-altane-type triterpenoid, preparation method and application thereof, and medicine Active CN113292427B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202010113708.3A CN113292427B (en) 2020-02-24 2020-02-24 Split-ring-altane-type triterpenoid, preparation method and application thereof, and medicine

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010113708.3A CN113292427B (en) 2020-02-24 2020-02-24 Split-ring-altane-type triterpenoid, preparation method and application thereof, and medicine

Publications (2)

Publication Number Publication Date
CN113292427A CN113292427A (en) 2021-08-24
CN113292427B true CN113292427B (en) 2022-07-26

Family

ID=77318521

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010113708.3A Active CN113292427B (en) 2020-02-24 2020-02-24 Split-ring-altane-type triterpenoid, preparation method and application thereof, and medicine

Country Status (1)

Country Link
CN (1) CN113292427B (en)

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101824068B (en) * 2010-05-06 2015-07-08 沈阳药科大学 Cyclic-ahltin type triterpenoid saponin compound and preparation method and application thereof
CN109180623A (en) * 2018-09-21 2019-01-11 河南中医药大学 The preparation method and applications of the new terpene A of 3,4- driffractive ring cycloartane type cape jasmine and the new terpene F of cape jasmine in water cape jasmine

Also Published As

Publication number Publication date
CN113292427A (en) 2021-08-24

Similar Documents

Publication Publication Date Title
EP1800685B1 (en) Steroidal saponin pharmaceutical composition, its preparation method and use
US7811997B2 (en) Composition containing total triterpenoid sapogenins extracted from bamboo, and the preparation method and use thereof
AU2004200624B2 (en) Medicinal preparation containing phenylethanoid glycosides extracted from herbaceous plant, cistanche tubulosa (schenk.) wight, process of making the same, and uses of the same
BRPI0616319A2 (en) Composition and method for extracting a species of panax
US7150887B2 (en) Herbal pharmaceutical compositions for prophylaxis and/or treatment of cardiovascular diseases and the method of preparing the same
CN101810659A (en) Total saponins of salvia chinensis benth, and medical application and preparation method thereof
KR101928767B1 (en) Novel gypenoside compound isolated from gynostemma pentaphyllum
Zhang et al. Discovery of anti-inflammatory terpenoids from Mallotus conspurcatus croizat
CN105085534B (en) A kind of alkaloid compound and its extraction separation method
CN113292427B (en) Split-ring-altane-type triterpenoid, preparation method and application thereof, and medicine
CN104820029B (en) Content detection method for compound houttuynia cordata mixture
CN114891058B (en) Picrorhiza cucurbitacina alkyl type saponin extract and application thereof in preparation of medicine for treating constipation
TWI438001B (en) Plant extract for treating diabetes and process for making same
CN106822071B (en) Chinese medicinal effective component for treating coronary heart disease and hyperlipidemia, its preparation method and method for separating effective component from the same
CN113925948B (en) Use of rhizoma Zingiberis recens total extract or its active component, pharmaceutical composition and preparation method
CN111329866B (en) Application of pentacyclic triterpenoid in preparation of anti-migraine medicine
CN104402960B (en) The luxuriant and rich with fragrance ester A of furancarboxylic acid pentamethylene many hydrogen and extracting method thereof and pharmaceutical use
CN114288331A (en) Method for simultaneously extracting volatile oil from effective parts of total flavonoids and total saponins of Chinese medicinal fenugreek, extract and application thereof
CN107669751A (en) A kind of clarification process and method of quality control of the pharmaceutical composition that there is treatment red blood trace on face to act on
CN101612184A (en) Multiradiate fleabane extract, the compositions that contains this extract and preparation method and purposes
CN101284030B (en) Quality control methods of hairy holly root medicinal materials, extract or hairy holly root preparation
CN107158113A (en) A kind of Chinese medicine composition, Its Preparation Method And Use
WO2017220051A2 (en) Benserazide hydrochloride pharmaceutical composition and medical use thereof for lowering blood sugar
CN102552359A (en) Preparation method of Erigeron multiradiatus extractive
CN107260816B (en) A kind of pharmaceutical composition with antidepression effect and preparation method thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant