CN101830964B - Triterpenoid compounds with anti-inflammatory activities - Google Patents
Triterpenoid compounds with anti-inflammatory activities Download PDFInfo
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- CN101830964B CN101830964B CN201010179553XA CN201010179553A CN101830964B CN 101830964 B CN101830964 B CN 101830964B CN 201010179553X A CN201010179553X A CN 201010179553XA CN 201010179553 A CN201010179553 A CN 201010179553A CN 101830964 B CN101830964 B CN 101830964B
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Abstract
The invention relates to the technical field of medicaments, and discloses seven new triterpenoid compounds with anti-inflammatory activities, which are extracted and separated from Chinese medicinal cherokee rose leaves. Through in vitro anti-inflammatory activity experiments, the compounds can remarkably inhibit luciferase expression caused by lipopolysaccharide (LPS) in 293-nfkb cells, present a dose dependency relationship and have obvious anti-inflammatory activities. Therefore, the compounds can be used for preparing new anti-inflammatory medicaments. The compounds provide new sources for preparing the anti-inflammatory medicaments.
Description
Technical field
The present invention relates to medical technical field, is 7 new triterpene compounds with anti-inflammatory activity that extraction separation obtains from the Chinese medicine Leaf of Cherokee Rose.
Background technology
Leaf of Cherokee Rose is the tender leaf of Rosaceae gul Fruit of Cherokee Rose (Rosa laevigata Michx.).It is very wide to distribute in China, mainly is distributed in areas such as East China, Central-South, southwest.Leaf of Cherokee Rose is a conventional Chinese medicine among the people, is one of staple of ancient prescription " children's indigestion powder in the army ", and it is hemorrhage to cure mainly metal-inflicted wound.From Leaf of Cherokee Rose, extracted triterpene compound urson, Jacaric acid, Rialic acid A, Oleanolic Acid, Crategolic acid, etc. triterpenic acid active component and Fruit of Cherokee Rose saponin A, Flos rosae multiflorae glycosides, Tormentic acid-28-O-β-triterpenoid saponin active components such as D-glucopyranoside, their chemical structure of general formula is following:
Wherein, R
1Expression hydrogen or hydroxyl, R
2Expression hydrogen or hydroxyl, R
3Expression hydrogen or glucopyranosyl, R
4Expression hydrogen or methyl, R
5Expression hydrogen or hydroxyl, R
6Expression hydrogen or methyl, R
7Expression methyl or methylol.But do not see the report that from Leaf of Cherokee Rose, separates 7 new triterpene compounds that obtain provided by the invention so far with anti-inflammatory activity.
Summary of the invention
The present invention provides a kind of new 7 triterpene compounds with anti-inflammatory activity that obtain that from the Chinese medicine Leaf of Cherokee Rose, separate, and their chemical structural formula is distinguished as follows:
Through the extracorporeal anti-inflammatory activity test, they all can significantly suppress luciferase expression in the 293-nfkb cell that LPS (LPS) causes, and present dose-dependence, show to have tangible anti-inflammatory activity.Therefore, can be used for preparing new anti-inflammatory drug.
The preparation method of The compounds of this invention is following:
1. prepare the Leaf of Cherokee Rose extract
With the Chinese medicine Leaf of Cherokee Rose by routine with 40～95% aqueous ethanolic solution soaked overnight, heating and refluxing extraction 2～3 times, each 1～3 hour, united extraction liquid, decompression and solvent recovery, extract medicinal extract;
2. extraction triterpene compound
Medicinal extract is dissolved in water, uses sherwood oil, ethyl acetate extraction successively, obtain sherwood oil and acetic acid ethyl acetate extract respectively, get acetic acid ethyl acetate extract, decompression and solvent recovery gets the ethyl acetate extract powder;
3. separation and purification
With the ethyl acetate extract powder by the decompression of silica gel conventionally column chromatography, with the methylene chloride-methanol system with 50: 1-20: 1-10: 1-5: 1-1: 1 carries out gradient elution, collects 20 parts of elutriants altogether; Every part of 2000ml merges 12-16 part, goes up silica gel decompression column chromatography behind the decompression and solvent recovery once more; With sherwood oil-acetone system with 20: 1-10: 1-5: 1-1: 1 carries out gradient elution, collects 10 parts of elutriants altogether, every part of 2000ml; 5-7 part is merged, carry out column chromatography with the open post of anti-phase ODS filler again behind the decompression and solvent recovery and separate, use the methanol-water gradient elution; Get 60% methanol-water wash-out part respectively, carry out the reversed phase high performance liquid phase behind the decompression and solvent recovery, with 70% methanol-water wash-out; Separate and obtain compound 1, compound 2, compound 3; Get 70% methanol-water wash-out part, carry out the reversed phase high performance liquid phase behind the decompression and solvent recovery,, separate obtaining compound 7 with 75% methanol-water wash-out; Get 80% methanol-water wash-out part, carry out the reversed phase high performance liquid phase behind the decompression and solvent recovery,, separate obtaining compound 4, compound 5, compound 6 with 85% methanol-water wash-out.
Embodiment
Combine embodiment at present, the present invention is described in detail.
Embodiment 1. preparations triterpene compound 1～7 of the present invention
1. prepare the Leaf of Cherokee Rose extract
Get 2 kilograms of Chinese medicine Leaf of Cherokee Rose, with 70% aqueous ethanolic solution soaked overnight of 10 times of volumes, heating and refluxing extraction 3 times, each 2 hours, united extraction liquid, decompression and solvent recovery gets Leaf of Cherokee Rose extract medicinal extract;
2. extraction triterpene compound
Medicinal extract is dissolved in water, uses sherwood oil, ethyl acetate extraction successively, obtain sherwood oil and acetic acid ethyl acetate extract respectively, get acetic acid ethyl acetate extract, decompression and solvent recovery gets the ethyl acetate extract powder;
3. separation and purification
With the ethyl acetate extract powder by the decompression of silica gel conventionally column chromatography, with the methylene chloride-methanol system with 50: 1-20: 1-10: 1-5: 1-1: 1 carries out gradient elution, collects 20 parts of elutriants altogether; Every part of 2000ml merges 12-16 part, goes up silica gel decompression column chromatography behind the decompression and solvent recovery once more; With sherwood oil-acetone system with 20: 1-10: 1-5: 1-1: 1 carries out gradient elution, collects 10 parts of elutriants altogether, every part of 2000ml; 5-7 part is merged, carry out column chromatography with the open post of anti-phase ODS filler again behind the decompression and solvent recovery and separate, use the methanol-water gradient elution; Get 60% methanol-water wash-out part respectively, carry out the reversed phase high performance liquid phase behind the decompression and solvent recovery, with 65% methanol-water wash-out; Separate and obtain compound 1 20mg, compound 2 15mg, compound 3 15mg; Get 70% methanol-water wash-out part, carry out the reversed phase high performance liquid phase behind the decompression and solvent recovery, with 75% methanol-water wash-out; Separate obtaining compound 7 30mg, get 80% methanol-water wash-out part, carry out the reversed phase high performance liquid phase behind the decompression and solvent recovery; With 85% methanol-water wash-out; Separate and obtain compound 4 13mg, compound 5 25mg, compound 6 20mg.
Through the magnetic resonance detection analysis, confirm the chemical structure of each compound of the present invention, their physico-chemical property and spectral data are respectively:
Compound 1: white unformed powder, 142～146 ℃ of mp.[α]
D 20+25°(c＝0.16，MeOH)。UV (MeOH) λ
Max(log) nm:243 (4.26); IR (KBr)
Max: 3420 (OH), 2937,2837,1712 (C=O), 1634,1384cm
-1HRESIMS, m/z 481.3291 [M+Na]
+(calcd forC
29H
46O
4Na, 481.3294);
13C and
1H NMR data are seen table 1 and table 2.
Compound 2: white unformed powder, 220～225 ℃ of mp.[α]
D 20-13°(c＝0.173，MeOH)。UV (MeOH) λ
Max(log) nm:240 (4.26); IR (KBr)
Max: 3446 (OH), 2925,2873,2832,1636cm
-1HRESIMS, m/z 465.3343 [M+Na]
+(calcd for C
29H
46O
3Na, 465.3345);
13C and
1H NMR data are seen table 1 and table 2.
Compound 3: white unformed powder, 259～263 ℃ of mp.[α]
D 20+10°(c＝0.07，MeOH)。UV (MeOH) λ
Max(log) nm:240 (4.26); IR (KBr)
Max: 3406 (OH), 2935,2868,1701 (C=O), 1458,1384cm
-1HRESIMS, m/z 525.3188 [M+Na]
+(calcd forC
30H
46O
6Na, 525.3192);
13C and
1H NMR data are seen table 1 and table 2.
Compound 4: white unformed powder, mp 248～252C.[α]
D 18+0°(c＝0.08，MeOH)。IR (KBr)
Max: 3421 (OH), 2941,1698 (C=O), 1384cm
-1HRESIMS, m/z 495.3451 [M+Na]
+(cacld for C
30H
48O
4Na, 495.3450);
13C and
1H NMR data are seen table 1 and table 2.
Compound 5: white unformed powder, 216～220 ℃ of mp.[α]
D 20+24°(c＝0.22，MeOH)。IR (KBr)
Max: 3420 (OH), 2940,1695 (C=O), 1384cm
-1HRESIMS, m/z 527.3344 [M+Na]
+(calcd for C
30H
48O
6Na, 527.3349);
13C and
1H NMR data are seen table 1 and table 2.
Compound 6: white unformed powder, mp 292～295C.[α]
D 20+63°(c＝0.085，MeOH)。IR (KBr)
Max: 3420 (OH), 2946,1700 (C=O), 1384cm
-1HRESIMS, m/z 511.3396 [M+Na]
+(calcd for C
30H
48O
5Na, 511.3399);
13C and
1H NMR data are seen table 1 and table 2.
Compound 7: white unformed powder, 290～294 ℃ of mp.[α]
D 20+3°(c＝0.173，CH
2Cl
2)。IR (KBr)
Max: 3425 (OH), 2939,1740 (C=O) cm
-1HRESIMS, m/z 611.3925 [M+Na]
+(calcd for C
35H
56O
7Na, 611.3924);
13C and
1H NMR data are seen table 1 and table 2.
Table 1. compound 1-7's
13C-NMR spectrum data (pyridine-d
5, 125MHz)
aIn?CDCl
3
Table 2. compound 1-7's
1H-NMR spectrum data (pyridine-d
5, 500MHz)
aIn?CDCl
3
The test of The compounds of this invention anti-inflammatory activity
Adopt the influence of luciferase intensity in 1～7 pair of LPS of compound (LPS) inductive NF-κ B-luc, the 293 Cell Line cells to prove its anti-inflammatory activity.
1. the preparation of medicament solution:
1) using DMSO to be mixed with final concentration respectively compound 1～7 is 20mg/ml solution, places-4 ℃ of preservations;
2) LPS:L2654 (sigma) uses dH
2It is 100 μ g/ml solution that O is mixed with concentration ,-20 ℃ of preservations.
2. cell strain: use anti-inflammatory drug screening implement cell strain NF-κ B-luc 293 Cell Line, provide by pharmacology teaching and research room of pharmaceutical college of The 2nd Army Medical College.
3. TP:
By the conventional recovery cell and the cultivation of going down to posterity, use the perfect medium of DMEM+10%FBS; Get P2 for the logarithmic phase cell, use the method for trysinization to prepare cell suspension, inoculating cell is to 96 porocyte culture plates, and every porocyte suspension is 100 μ l, and cell count is 1 * 10
4Individual; Each compound is divided into 6 groups by every hole different pharmaceutical concentration, and promptly 0 μ g/ml, 12.5 μ g/ml, 25 μ g/ml, 50 μ g/ml, 100 μ g/m and 200 μ g/ml organize, and 5%CO is put in 3 every group multiple holes
2Incubator adds LPS (final concentration is 1 μ g/ml) in 37 ℃ of culturing cells 4 hours, continues to cultivate 48 hours, collects respectively by routine and respectively organizes cell sample, carries out uciferase activity and detects.Whether the detection data to luciferase are carried out statistical study, inhibited for the inflammatory reaction of LPS inductive to confirm the medicine pre-treatment.The result sees table 3.
Table 3. compound 1-7 is to luciferase intensity detection (RLU) (MV ± variance) in LPS (1ug/ml) the inductive NF-κ B-luc 293 Cell Line cells
Annotate: * P＜0.05, * * P＜0.01; Medicine pretreated group vs model group
Visible by table 3, in 293 cells, compound 1-7 can obviously suppress the NF-κ B activation that LPS causes, and presents dose-dependence, shows that The compounds of this invention has significant anti-inflammatory activity, therefore, can be used for preparing anti-inflammatory drug.
Claims (2)
1. triterpene compound with anti-inflammatory activity is characterized in that chemical structural formula is respectively:
Compound 5
Compound 7.
2. the application of the described triterpene compound of claim 1 in the preparation anti-inflammatory drug.
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| CN102584929A (en) * | 2011-12-29 | 2012-07-18 | 广西壮族自治区药用植物园 | Extracting and purifying method of cherokee rose root saponin D and E in cherokee rose root |
| CN110577562B (en) * | 2019-05-13 | 2022-06-07 | 株洲千金药业股份有限公司 | Triterpenoid and pharmaceutically acceptable salt thereof, and preparation method and application thereof |
| CN111303237B (en) * | 2020-03-06 | 2022-05-03 | 上海市闵行区中心医院 | Polyhydroxy-substituted oleanane pentacyclic triterpenoid, preparation method thereof and application thereof in preparing antifungal medicines |
| CN114642677A (en) * | 2020-12-21 | 2022-06-21 | 上海凯屹医药科技有限公司 | Stable liquid medicine composition containing ilexoside compound |
| CN112773809A (en) * | 2021-03-22 | 2021-05-11 | 苏州大学 | Application of multinoside in preparing medicine for treating inflammatory bowel disease |
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| CN101590134A (en) * | 2009-06-30 | 2009-12-02 | 沈阳药科大学 | Has total triterpene of garden burnet root of anti-inflammatory and analgesic effect and preparation method thereof |
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| CN101590134A (en) * | 2009-06-30 | 2009-12-02 | 沈阳药科大学 | Has total triterpene of garden burnet root of anti-inflammatory and analgesic effect and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| Sosa S. 等.Topical anti-inflammatory activity of Bauhinia tarapotensis leaves.《Phytomedicine》.2002,第9卷(第7期),646－653. * |
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