CN101787068B - Steroid saponin extract of Smilax riparia A.DC. and preparation method and application thereof - Google Patents
Steroid saponin extract of Smilax riparia A.DC. and preparation method and application thereof Download PDFInfo
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- CN101787068B CN101787068B CN2010101150915A CN201010115091A CN101787068B CN 101787068 B CN101787068 B CN 101787068B CN 2010101150915 A CN2010101150915 A CN 2010101150915A CN 201010115091 A CN201010115091 A CN 201010115091A CN 101787068 B CN101787068 B CN 101787068B
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- DWYWIMZZFDUULS-ZEMUTCMASA-N CC(CCC(C1(C)C)OC(C2)C1[C@@](C)(CC1)C2C(CC2)C1[C@@](C)(CC1)[C@@H]2C[C@H]1O[C@@H](C(C1O)OC([C@H](C(C2)O)O)O[C@@H](C)C2O)OC(COC([C@H](C2O)O)O[C@@H](C)C2O)[C@H]1O)COC(OC(CO)[C@H](C1O)O)=C1O Chemical compound CC(CCC(C1(C)C)OC(C2)C1[C@@](C)(CC1)C2C(CC2)C1[C@@](C)(CC1)[C@@H]2C[C@H]1O[C@@H](C(C1O)OC([C@H](C(C2)O)O)O[C@@H](C)C2O)OC(COC([C@H](C2O)O)O[C@@H](C)C2O)[C@H]1O)COC(OC(CO)[C@H](C1O)O)=C1O DWYWIMZZFDUULS-ZEMUTCMASA-N 0.000 description 1
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Abstract
The invention provides a steroid saponin extract of Smilax riparia A.DC. and a preparation method and application thereof. Roots and rootstocks of Smilax riparia A.DC. are extracted by 0 to 100 percent aqueous solution of ethanol, and steroid saponin of the Smilax riparia A.DC. is separated by a macroporous adsorption resin method, wherein the steroid saponin accounts for 50 to 95 weight percent of the extract, and the extract contains one or more of four new compounds. The steroid saponin extract of the Smilax riparia A.DC. prepared is proved to have the effect of resisting rheumatoid arthritis and can be used for preparing products for preventing and/or treating the rheumatoid arthritis through pharmacological activity study.
Description
Technical field
The invention belongs to field of medicaments, relate to Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract and preparation method thereof, also relate to Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract and prevent and/or treat the purposes in the rheumatoid arthritis product in preparation.
Background technology
Rheumatoid arthritis be a kind of common serve as the autoimmune disorder of main performance with joint tissue chronic inflammation pathology.Its main pathological change is the synovium of joint cellular infiltration, and the synovial membrane screen forms, the erosion of cartilage and osseous tissue.The purpose of rheumatoid arthritis treatment is to stop course of disease development, promptly stops arthritis and destruction process, the prophylactic function forfeiture.Though the treatment measure is a lot, pharmacological agent is still the basis.Medicine comprises NSAIDs, steroidal anti-inflammatory medicine and disease adjusting antirheumatic at present, but all there is tangible untoward reaction in these medicines.Along with to inflammatory process, molecular genetics and biotech development, the understanding of etiology mechanism is deepened continuously, the investigator strives to find has specific medicine.Be target spot mainly, be target spot with the inflammatory mediator, be target spot, be target spot with the signal transduction pathway with the immunologically competent cell surface antigen with the inflammatory cytokine.
Root and the rhizome aboundresources of Liliaceae smilax plant Root and Rhizome of Ripqrian Greenbrier Smilax riparia A.DC have inrigorating qi and promoting blood circulation, the effect of channels sootheing and network vessel quickening; Cure mainly deficiency of vital energy edema, bones and muscles pain, hemiplegia; Dizziness headache, cough is spitted blood, bone tuberculosis; Leukorrhea [the new medical college in Jiangsu. Chinese medicine voluminous dictionary, Shanghai science tech publishing house, 1985:427.].Sashida Y etc. from Root and Rhizome of Ripqrian Greenbrier isolation identification 2 steroid saponin compound NSC 232021 3-O-α-L-rhamnopyranosyl-(1 → 6)-β-D-glucopyranosides; NSC 232021 3-O-β-D-glucopyranosyl-(1 → 4)-[α-L-rhamnopyranosyl-(1 → 6)]-β-D-glucopyranoside] [Sashida Y; Et al.Phytochemistry, 31 (7): 2439-2443.]; Li J etc. from Root and Rhizome of Ripqrian Greenbrier isolation identification 2 steroid saponin compound 3-O-α-L-rhamnopyranosyl-(1 → 2)-[α-L-rhamnopyranosyl-(1 → 6)]-β-D-Glucopyranose-3 β; 20 salmefamols-5 α-furan steroid-22 (23)-alkene-26-O-β-D-glucopyranoside; 3-O-α-L-rhamnopyranosyl-(1 → 2)-[α-L-rhamnopyranosyl-(1 → 6)]-β-D-Glucopyranose-3 β; 16 beta-diols-5 α-pregnant steroid-20-ketone-16-O-[5-hydroxy-4-methyl-valeric acid-5-O-β-D-glucopyranosyl ester] [Li J; Et al.Chem Pharm Bull, 54 (10): 1451-1454.].Do not see at present the product of high-content Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract; Do not see that relevant Root and Rhizome of Ripqrian Greenbrier steroidal saponin is at the research report that prevents and/or treats aspect the rheumatoid arthritis biological activity.
Summary of the invention
The objective of the invention is development and use and use still not Liliaceae smilax plant Root and Rhizome of Ripqrian Greenbrier Smilax riparia A.DC, especially its steroidal saponin constituents widely up to now.
This case contriver is through carrying out careful deep research to Root and Rhizome of Ripqrian Greenbrier; Invented Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract extraction separation purification process, further the extract chemical ingredients studied, isolation identification 4 kinds of compounds that content is high in the extract; Be new compound; The extract that makes proves to have the resisting rheumatoid arthritis effect through pharmacology activity research, thereby has accomplished the present invention.
The present invention provides a kind of Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract.
The steroidal saponin weight percentage is 50～95% in the Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract of the present invention.
The present invention provides Root and Rhizome of Ripqrian Greenbrier steroidal saponin preparation method of extract, and this method may further comprise the steps:
A) the Root and Rhizome of Ripqrian Greenbrier meal extracts with first solvent refluxing, filters, and extracting solution merges, the extracting solution concentrating under reduced pressure;
B) extract that step a) is obtained is dissolved in water, and through macroporous adsorbent resin column chromatography, it is closely colourless that water is eluted to elutriant, discards elutriant;
C) closely colourless with second solvent elution to elutriant, discard elutriant;
D) closely colourless with the 3rd solvent elution to elutriant, collect elutriant;
E) elutriant that step d) is obtained through decolorizing resin post D941 chromatogram, with the 4th solvent elution, is collected elutriant, concentrating under reduced pressure, the dry Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract that gets.
In above step, wherein first solvent of step a) is 0～100% aqueous ethanolic solution; The step b) macroporous adsorbent resin comprises nonpolar or the low-pole macroporous adsorbent resin.Second solvent of step c) is 10～50% aqueous ethanolic solutions, and the 3rd solvent of step d) is 60～90% aqueous ethanolic solutions, and the 4th solvent of step e) is 90% aqueous ethanolic solution.
The present invention provides Root and Rhizome of Ripqrian Greenbrier steroidal saponin preparation method of extract, preferably may further comprise the steps:
A) the Root and Rhizome of Ripqrian Greenbrier meal adds 50～70% alcohol reflux, filters, and extracting solution merges, decompression recycling ethanol;
B) extract that step a) is obtained is dissolved in water, and through macroporous adsorptive resins D101 chromatogram, it is closely colourless that water is eluted to elutriant, discards elutriant;
C) closely colourless with 30% ethanol elution to elutriant, discard elutriant;
D) closely colourless with 70% ethanol elution to elutriant, collect elutriant;
E) elutriant that step d) is obtained, through decolorizing resin post D941 chromatogram, 6 times of column volume 90% ethanol elutions are collected elutriant, concentrating under reduced pressure, the dry Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract that gets.
Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract of the present invention extracts from Root and Rhizome of Ripqrian Greenbrier root and rhizome, therefrom separates obtaining 4 kinds of new compounds, and chemical structure is identified as follows:
Compound (1): 16-O-β-D-glucopyranosyl-(22S)-and courage steroid-5-alkene-3 β, 16 β, 22-triol-3-O-α-L-rhamnopyranosyl-(1 → 2)-[α-L-rhamnopyranosyl-(1 → 6)]-β-D-glucopyranoside
Amorphous solid, the Liebermann-Burchard reaction is blue-greenish colour, and the Molish reaction is purple,
1H-NMR with
13The C-NMR spectrum shows the characteristic of steroid saponin compound, infers that compound (1) is a steroid saponin compound.
HR-ESI MS shows quasi-molecular ion peak m/z 1033.5499 [M-H]
-, in conjunction with
13C-NMR and DEPT data-speculative molecular formula are C
51H
86O
21(calculated value C
51H
85O
21Relative molecular mass 1033.5583).
1The H-NMR spectrum is shown has methyl proton signal δ 0.90 (3H, s), 0.94 (3H, d, J=6.2Hz), 0.95 (3H; D, J=6.0Hz), 0.99 (3H, s), 1.21 (3H; D, J=7.2Hz), the methine protons signal δ 3.82 that links to each other with oxygen (1H, m), 4.31 (1H; M), 4.53 (1H, m), alkene hydrogen proton signal δ 5.28 (1H, br d).
13C-NMR shows has 51 carbon signals, and 27 steroidal parent nucleus carbon signals are wherein inferred in the structure in conjunction with DEPT and HMQC spectrum and to be contained 5 CH
3, 9 CH
2, 10 CH (wherein 3 δ 73.23,78.20 that link to each other with oxygen, 82.68,1 olefinic carbon δ 121.87), 3 quaternary carbons (wherein 1 olefinic carbon δ 140.96).The above data of integration analysis also contrast with document [Yin J, et al.J Nat Prod, 2003,66:646-650]; Infer that compound (1) aglycon is (22S)-courage steroid-5-alkene-3 β, 16 β, 22-triol; Comparison 16-O-β-D-Glucopyranose-(22S, 25S)-courage steroid-5-alkene-3 β, 16 β; 22,26-tetrol-3-O-α-L-rhamnopyranosyl-(1 → 2)-[α-L-rhamnopyranosyl-(1 → 4)]-β-D-glucopyranoside and 26-O-β-D-Glucopyranose-(22R, 25R)-courage steroid-5-alkene-3 β; 16 β, 22,26-tetrol-3-O-α-L-rhamnopyranosyl-(1 → 2)-β-D-glucopyranoside C-22 chemical shift further confirms 22S configuration [Sang S M; Et al.Food Chem, 2003: 499-506.; Hayes P Y, et al.Phytochemistry, 2008,68:623-630.], the NOESY spectrum shows the 16-H/22-H coherent signal, confirms that further 16-OH relative configuration is beta comfiguration.
1H-NMR shows has four sugared anomeric proton signal δ 4.74 (1H, d, J=7.8Hz), 4.89 (1H, d; J=7.2Hz), 5.37 (1H, br s) and 5.64 (1H, br s), two rhamnosyl characteristic methyl proton signal δ 1.65 (3H; D is J=6.2Hz) with 1.71 (3H, d, J 6.2Hz);
13C-NMR shows has 102.01,102.83,102.89 and 107.00, two rhamnosyl characteristic methyl of four sugared end group carbon signal δ carbon signal δ 18.51 and 18.66.Compound (1) HPLC method after acid hydrolysis is identified in the structure and is had D-glucose and L-rhamnosyl [Guo H Z, et al.Phytochemistry, 2004,65:481-484.];
1H-NMR shows that glucose anomeric proton even summation constant J=7.2 and J=7.8Hz infer that glucose is beta comfiguration,
13C-NMR shows that rhamnosyl C-5 position chemical shift is respectively δ 69.84 and 70.61 and infers that rhamnosyl is α configuration [Shao B, et al.Phytochemistry, 2007,68:623-630.].Integration analysis
1H-NMR,
13C-NMR, DEPT, HMQC, HMBC compose also and the document contrast, compound (1) sugar moieties data identical with Riparoside A [Li J, et al.Chem Pharm Bull, 2006,10:1451-1454.].The HMBC spectrum shows glucose anomeric proton δ 4.74 and 16 carbon δ of aglycon, 82.68 long-range coherent signals, infers in the structure to have 16-O-β-D-glucopyranosyl.The HMBC spectrum shows glucose anomeric proton δ 4.89 and 3 carbon δ 78.20 of aglycon; Rhamnosyl anomeric proton δ 5.64 and 2 carbon δ 79.50 of glucose; Rhamnosyl anomeric proton δ 5.37 and 6 carbon δ of glucose, 67.08 long-range coherent signals infer in the structure to have 3-O-α-L rhamnosyl-(1 → 2)-[α-L-rhamnosyl-(1 → 6)]-β-D-glucopyranosyl.The above data of analysis-by-synthesis; Authenticating compound (1) is 16-O-β-D-Glucopyranose-(22S)-courage steroid-5-alkene-3 β; 16 β, 22-triol-3-O-α-L-rhamnopyranosyl-(1 → 2)-[α-L-rhamnopyranosyl-(1 → 6)]-β-D-glucopyranoside.This compound is not seen bibliographical information.
1H-NMR with
13The C-NMR digital data is seen table 1.
Table 1 compound (1)
1H-NMR with
13C-NMR data (C
5D
5N)
Compound (2): 26-O-β-D-glucopyranosyl-(25S)-and furan steroid-5,20 (22)-diene-3 β, 26-glycol-3-O-α-L-rhamnopyranosyl-(1 → 2)-[α-L-rhamnopyranosyl-(1 → 6)]-β-D-glucopyranoside
Amorphous solid, the Liebermann-Burchard reaction is blue-greenish colour, and the Molish reaction is purple, and the Ehrlich reagent react takes on a red color,
1H-NMR with
13The C-NMR spectrum shows the characteristic of steroid saponin compound, infers that compound (2) is the furostanol saponin compounds.
HR-ESI MS shows quasi-molecular ion peak m/z 1029.5331 [M-H]
-, in conjunction with
13C-NMR data-speculative molecular formula is C
51H
82O
21(calculated value C
51H
81O
21Relative molecular mass 1029.5270).
1The H-NMR spectrum is shown has methyl proton signal δ 0.72 (3H, s), 0.92 (3H, s), 1.05 (3H; D, J=6.6Hz), 1.64 (3H, s), the methine protons signal δ 3.97 (1H that link to each other with oxygen; M), 4.81 (1H, m) link to each other with oxygen together with proton signal δ 3.50 (1H, dd, J=9.4; 7.0Hz) and 4.07 (1H, dd, J=9.0,6.0Hz).
13C-NMR shows has 51 carbon signals, and 27 steroidal parent nucleus carbon signals are wherein inferred in the structure in conjunction with the HMQC spectrum and to be contained 4 CH
3, 10 CH
2(wherein 1 link to each other with oxygen δ 75.25), 8 CH (wherein 2 δ 78.49,84.53 that link to each other with oxygen, 1 olefinic carbon δ 121.67), 5 quaternary carbons (wherein 3 olefinic carbon δ 103.54,141.08,152.52).The NMR spectrum is shown has 5,20 (22)-diene-first characteristic feature δ 1.64 (3H, s, H-21) proton signal of furostanol saponin; δ 103.54 (C-20), 121.67 (C-6), 141.08 (C-5), 152.52 (C-22) carbon signal [Yokosuka A; Et al.J Nat Prod, 2002,65:1293-1298.].H
2-26 humorous proton chemical shifts value of delta a-δ b=0.57 infer the 25S configuration, and 25R configuration chemical shift difference δ a-δ b＜0.48 [Agrawal P K.Steroids, 2005,70:715-724.],
13The C-NMR spectrum shows that δ 33.77 (C-25) is arranged, and 75.25 (C-26) and 17.21 (C-27) carbon signal further confirm 25S configuration [Yokosuka A, et al.J Nat Prod, 2002,65:1293-1298.; Dong M, et al.Tetrahedron, 2001,74:501-506.].The above data of integration analysis also contrast [Yokosuka A, et al.J Nat Prod, 2002,65:1293-1298.] with document, and authenticating compound (2) aglycon is (25S)-furan steroid-5,20 (22)-diene-3 β, the 26-glycol.
1H-NMR shows has four sugared anomeric proton signal δ 4.84 (1H, d, J=7.8Hz), 4.91 (1H, d; J=7.8Hz), 5.37 (1H, br s) and 5.65 (1H, br s), two rhamnosyl characteristic methyl proton signal δ 1.67 (3H; D, and J=6.2Hz) with 1.71 (3H, d, J=6.2Hz);
13C-NMR shows has 101.99,102.80,102.88 and 105.19, two rhamnosyl characteristic methyl of four sugared end group carbon signal δ carbon signal δ 18.50 and 18.67.Compound (2) HPLC method after acid hydrolysis is identified in the structure and is had D-glucose and L-rhamnosyl [Guo H Z, et al.Phytochemistry, 2004,65:481-484.];
1H-NMR shows that glucose anomeric proton even summation constant J=7.8Hz infers that glucose is beta comfiguration,
13C-NMR shows that rhamnosyl C-5 position chemical shift is respectively δ 69.83 and 70.61 and infers that rhamnosyl is α configuration [Shao B, et al.Phytochemistry, 2007,68:623-630.].Integration analysis
1H-NMR,
13C-NMR, HMQC, HMBC compose also and the document contrast, compound (2) sugar moieties data identical with Riparoside A [Li J, et al.Chem Pharm Bull, 2006,10:1451-1454.].The HMBC spectrum shows glucose anomeric proton δ 4.84 and 26 carbon δ of aglycon, 75.25 long-range coherent signals, infers in the structure to have 26-O-β-D-glucopyranosyl.The HMBC spectrum shows glucose anomeric proton δ 4.91 and 3 carbon δ 78.49 of aglycon; Rhamnosyl anomeric proton δ 5.37 and 6 carbon δ 67.08 of glucose; Rhamnosyl anomeric proton δ 5.65 and 2 carbon δ of glucose, 79.50 long-range coherent signals infer in the structure to have 3-O-α-L rhamnosyl-(1 → 2)-[α-L-rhamnosyl-(1 → 6)]-β-D-glucopyranosyl.The above data of analysis-by-synthesis; Authenticating compound (2) is 26-O-β-D-Glucopyranose-(25S)-furan steroid-5; 20 (22)-diene-3 β, 26-glycol-3-O-α-L-rhamnopyranosyl-(1 → 2)-[α-L-rhamnopyranosyl-(1 → 6)]-β-D-glucopyranoside.This compound is not seen bibliographical information.
1H-NMR with
13The C-NMR digital data is seen table 2.
Table 2 compound (2)
1H-NMR with
13C-NMR data (C
5D
5N)
Compound (3): 26-O-β-D-glucopyranosyl-(25S)-and 5 α-furan steroid-20 (22)-alkene-3 β, 26-glycol-3-O-α-L-rhamnopyranosyl-(1 → 2)-[α-L-rhamnopyranosyl-(1 → 6)]-β-D-glucopyranoside
Amorphous solid, the Liebermann-Burchard reaction is blue-greenish colour, and the Molish reaction is purple, and the Ehrlich reagent react takes on a red color,
1H-NMR with
13The C-NMR spectrum shows the characteristic of steroid saponin compound, infers that compound (3) is the furostanol saponin compounds.
HR-ESI MS shows quasi-molecular ion peak m/z 1031.5412 [M-H]
-, in conjunction with
13C-NMR and DEPT data-speculative molecular formula are C
51H
84O
21(calculated value C
51H
83O
21Relative molecular mass 1031.5427).
1The H-NMR spectrum is shown has methyl proton signal δ 0.70 (3H, s), 0.71 (3H, s), 1.06 (3H; D, J=6.7Hz), 1.65 (3H, s), the methine protons signal δ 3.97 (1H that link to each other with oxygen; M), 4.81 (1H, m), link to each other with oxygen together with proton signal δ 3.52 (1H, dd; J=9.5, and 6.9Hz) with 4.09 (1H, dd, J=9.4,5.8Hz).
13C-NMR shows has 51 carbon signals, and 27 steroidal parent nucleus carbon signals are wherein inferred in the structure in conjunction with DEPT and HMQC spectrum and to be contained 4 CH
3, 11 CH
2(wherein 1 link to each other with oxygen δ 75.37), 8 CH (wherein 2 link to each other with oxygen δ 78.03,84.67), 4 quaternary carbons (2 olefinic carbon δ 103.77,152.53 wherein.The NMR spectrum is shown has 20 (22)-alkene-first characteristic feature δ 1.65 (3H, s, H-21) proton signal, δ 103.77 (C-20), 152.53 (C-22) carbon signal [Yokosuka A, et al.J Nat Prod, 2002,65:1293-1298.] of furostanol saponin.H
2-26 humorous proton chemical shifts value of delta a-δ b=0.57 infer the 25S configuration, and 25R configuration chemical shift difference δ a-δ b＜0.48 [Agrawal P K.Steroids, 2005,70:715-724.],
13The C-NMR spectrum shows that δ 33.83 (C-25) is arranged, and 75.37 (C-26) and 17.32 (C-27) carbon signal further confirm 25S configuration [OleszekW, et al.JAgric Food Chem, 2001,49:4392-4396.; BedirE, et al.J Nat Prod, 2000,63:1699-1701.].
13The C-NMR spectrum shows that δ 44.79 (C-5) is arranged, and 54.47 (C-9) and 12.44 (C-19) carbon signal are inferred A/B ring trans configuration, 5 α-furostanol saponin [Woo M H, et al.J Nat Prod, 1992,55:1129-1135.; Sautour M, et al.J Nat Prod, 2005,68:1489-1493.].The NOESY spectrum shows 5-H/9-H, 5-H/14-H, 9-H/14-H, 11-H/19-H
3, 11-H/18-H
3, the 16-H/17-H coherent signal is inferred A/B ring trans, B/C and C/D ring trans, D/E ring cis configuration.The above data of integration analysis also contrast with document [Yokosuka A, et al.J Nat Prod, 2002,65:1293-1298.], and authenticating compound (3) aglycon is 25 (S)-5 α-furan steroid-20 (22)-alkene-3 β, the 26-glycol.
1H-NMR shows has four sugared anomeric proton signal δ 4.84 (1H, d, J=7.7Hz), 4.94 (1H, d; J=7.8Hz), 5.40 (1H, d is J=0.8Hz) with 5.64 (1H, br s); Two rhamnosyl characteristic methyl proton signal δ 1.67 (3H, d, J=6.2Hz) with 1.71 (3H, d, J=6.2Hz);
13C-NMR shows has 102.19,102.32,102.94 and 105.21, two rhamnosyl characteristic methyl of four sugared end group carbon signal δ carbon signal δ 18.58 and 18.76.Compound (3) HPLC method after acid hydrolysis is identified in the structure and is had D-glucose and L-rhamnosyl [Guo H Z, et al.Phytochemistry, 2004,65:481-484.];
1H-NMR shows that glucose anomeric proton even summation constant J=7.7 and J=7.8Hz infer that glucose is beta comfiguration,
13C-NMR shows that rhamnosyl C-5 position chemical shift is respectively δ 69.92 and 70.72 and infers that rhamnosyl is α configuration [Shao B, et al.Phytochemistry, 2007,68:623-630.].Integration analysis
1H-NMR,
13C-NMR, DEPT, HMQC, HMBC compose also and the document contrast, compound (3) sugar moieties data identical with Riparoside A [Li J, et al.Chem Pharm Bull, 2006,10:1451-1454.].The HMBC spectrum shows glucose anomeric proton δ 4.84 and 26 carbon δ of aglycon, 75.37 long-range coherent signals, infers in the structure to have 26-O-β-D-glucopyranosyl.The HMBC spectrum shows glucose anomeric proton δ 4.94 and 3 carbon δ 78.03 of aglycon; Rhamnosyl anomeric proton δ 5.40 and 6 carbon δ 67.42 of glucose; Rhamnosyl anomeric proton δ 5.64 and 2 carbon δ of glucose, 79.71 long-range coherent signals infer in the structure to have 3-O-α-L rhamnosyl-(1 → 2)-[α-L-rhamnosyl-(1 → 6)]-β-D-glucopyranosyl.The above data of analysis-by-synthesis; Authenticating compound (3) is 26-O-β-D-Glucopyranose-(25S)-5 α-furan steroid-20 (22)-alkene-3 β, 26-glycol-3-O-α-L-rhamnopyranosyl-(1 → 2)-[α-L-rhamnopyranosyl-(1 → 6)]-β-D-glucopyranoside.This compound is not seen bibliographical information.
1H-NMR with
13C-NMR digital data such as table 3.
Table 3 compound (3)
1H-NMR with
13The C-NMR data
Compound (4): NSC 232021 3-O-α-L-rhamnopyranosyl-(1 → 2)-[α-L-rhamnopyranosyl-(1 → 6)]-β-D-glucopyranoside
White powder, the Liebermann-Burchard reaction is blue-greenish colour, and the Molish reaction is purple, and the Ehrlich reagent react is negative,
1H-NMR with
13The C-NMR spectrum shows the characteristic of steroid saponin compound, infers that compound (4) is the spirostanol saponin compounds.
HR-ESI MS shows quasi-molecular ion peak m/z 869.4877 [M-H]
-, in conjunction with
13C-NMR data-speculative molecular formula is C
45H
74O
16(calculated value C
45H
73O
16Relative molecular mass 869.4899).
1The H-NMR spectrum is shown has methyl proton signal δ 0.67 (3H, s), 0.82 (3H, s), 1.09 (3H, d, J=7.2Hz), 1.20 (3H, d, J=7.1Hz).
13C-NMR spectrum shows that 45 carbon signals are arranged, and wherein 27 steroidal parent nucleus carbon signals combine to contain 4 CH in the HMQC spectrum supposition structure
3, 11 CH
2, 9 CH (wherein 2 link to each other with oxygen δ 77.87,81.27), 3 quaternary carbons.
13The C-NMR spectrum shows that δ 16.36 (C-27) carbon signal is arranged, and infers the 25S configuration, and 25R configuration δ 17.06 (C-27) [Sautour M, et al.J Nat Prod, 2005,68:1489-1493.; Sautour M, et al.Planta Med, 2006,72:667-670.].
13The C-NMR spectrum shows that δ 44.68 (C-5) is arranged, and 54.44 (C-9) and 12.35 (C-19) carbon signal are inferred A/B ring trans configuration, 5 α-spirostanol saponin [Roboson V P, et al.Phytochemistry, 1996,43:465-469.].The NOESY spectrum shows 5-H/9-H, 5-H/14-H, 9-H/14-H, 11-H/19-H
3, 11-H/18-H
3, the 16-H/17-H coherent signal is inferred A/B ring trans, B/C and C/D ring trans, D/E ring cis configuration.Comprehensive above data also contrast [Roboson V P, et al.Phytochemistry, 1996,43:465-469.] with document, and authenticating compound (4) aglycon is a NSC 232021.
1H-NMR shows has three sugared anomeric proton signal δ 4.92 (1H, d, J=7.8Hz), 5.38 (1H, br s) and 5.63 (1H, br s), two rhamnosyl characteristic methyl proton signal δ 1.65 (3H, d, J=6.2Hz) with 1.70 (3H, d, J=6.2Hz);
13C-NMR shows has three sugared end group carbon signal δ 102.12,102.28 and 102.90, two rhamnosyl characteristic methyl carbon signal δ 18.51 and 18.69.Compound (4) HPLC method after acid hydrolysis is identified in the structure and is had D-glucose and L-rhamnosyl [Guo H Z, et al.Phytochemistry, 2004,65:481-484.];
1H-NMR shows that glucose anomeric proton even summation constant J=7.8Hz infers that glucose is beta comfiguration,
13C-NMR shows that rhamnosyl C-5 position chemical shift is respectively δ 69.84 and 70.64 and infers that rhamnosyl is α configuration [Shao B, et al.Phytochemistry, 2007,68:623-630.].Integration analysis
1H-NMR,
13C-NMR, HMQC, HMBC compose also and the document contrast, compound (4) sugar moieties data identical with Riparoside A [Li J, et al.Chem Pharm Bull, 2006,10:1451-1454.].The HMBC spectrum shows glucose anomeric proton δ 4.92 and 3 carbon δ 77.87 of aglycon; Rhamnosyl anomeric proton δ 5.38 and 6 carbon δ 67.34 of glucose; Rhamnosyl anomeric proton δ 5.64 and 2 carbon δ of glucose, 79.68 long-range coherent signals infer in the structure to have 3-O-α-L rhamnosyl-(1 → 2)-[α-L-rhamnosyl-(1 → 6)]-β-D-glucopyranosyl.The above data of analysis-by-synthesis, authenticating compound (4) are NSC 232021 3-O-α-L-rhamnopyranosyl-(1 → 2)-[α-L-rhamnopyranosyl-(1 → 6)]-β-D-glucopyranoside.This compound is not seen bibliographical information.
1H-NMR with
13C-NMR digital data such as table 4.
Table 4 compound (4)
1H-NMR with
13C-NMR data (C
5D
5N)
Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract of the present invention prevents and/or treats the purposes in the rheumatoid arthritis product in preparation.
The present invention adopts the injection Freund's complete adjuvant to set up experimental rheumatoid arthritis rat model; This model is to estimate the classical model of resisting rheumatoid disease arthritis drug to the immune inflammation effect; After injection Freund's complete adjuvant sensitization in the rat toes; Its pathology is divided into two kinds of primary and Secondary cases: the primary pathology mainly shows as and causes scorching partial inflammatory reaction in early days, causes the right foot swelling peaking of scorching back 18h, continues to alleviate gradually behind the 3d; The Secondary cases pathology comes across and causes about scorching back 15d, shows as the swelling of offside (left hind) and forelimb pin, and " sacroiliitis " brief summary, allergic pannus and weight loss etc. appear in ear and afterbody.Cytokine is one group of synthetic by immune cell and non-immunocyte, that secretion produces small-molecule substance, has the various biological function.With the closely-related cytokine major part of rheumatoid arthritis by monocyte, scavenger cell, inoblast and T emiocytosis, wherein TNF-α, IL-1, IL-6 are virulence factor, IL-10, IL-4 are the protection factor.Adopt the effect of adjuvant arthritis rats model research Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract to secondary inflammation, the result shows, Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract 800mgkg
-1And 400mgkg
-1Dose groups has obvious restraining effect to adjuvant arthritis rat secondary inflammation, in institute's dosage scope obvious dose-effect relationship is arranged, and can obviously reduce TNF-α in the serum, IL-1 β and IL-6 content.Chondrus ocellatus Holmes colloidality rat paw edema model is an acute inflammation model that is usually used in estimating the classics of medicine anti-inflammatory action, can make in giving rat paw that the local trichangiectasia of rat, vascular permeability increase, ooze out during the injection X 5189, oedema etc. is a series of is similar to the acutely inflamed reaction of human body.Adopt chondrus ocellatus Holmes colloidality rat paw edema model research Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract to acutely inflamed effect, the result shows, Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract 800mgkg
-1And 400mgkg
-1Dose groups on Carrageenan property foot swelling rat acute inflammation has obvious restraining effect, in institute's dosage scope obvious dose-effect relationship is arranged.Acetic acid twisting method mouse model and hot plate method mouse model are the anodyne screening models of using always, easy and simple to handle, be convenient to observe, effect is obvious, its advantage is that stimulation location is stronger with the specificity that stimulates character.Licking the back foot reflex is a kind of reaction that mouse is taken place when coming to harm sexual stimulus with turning round body reflection, shows as the stimulation that receives heat and causes pain, and mouse can show the reaction of licking metapedes.The stimulation that the abdominal cavity receives acetic acid causes pain, and mouse can show the contraction belly, stretches hind leg, raises writhing responses such as buttocks, and the elementary maincenter of this type of reflection is positioned at spinal cord, and reflex arc is simpler, and the correspondence between stimulation and the reaction is good.Therefore, there is the objective indicator of nociception in Chang Zuowei reflection rat limbs, adopt acetic acid twisting method mouse model and hot plate method mouse model research Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract analgesic activity, and the result shows, Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract 1000mgkg
-1And 500mgkg
-1Dose groups Dichlorodiphenyl Acetate induced mice pain reaction has obvious restraining effect, wherein 1000mgkg
-1The dose groups effect is better than Frosst) group 100mgkg
-1, in institute's dosage scope obvious dose-effect relationship is arranged, hot plate induced mice pain reaction there is obvious restraining effect equally.The Superlysoform rat model is a chronic pain model preferably; When causing the spontaneous activity of C fiber immediately, subcutaneous injection Superlysoform increases severely; The single fast outstanding mediator of warp is delivered to the spinal cord projection neuron; And continue and import outburst mutually into second, be reflected as wide power dorsal horn neuronal activation, cause pain.The C fiber stimulation that continues in addition makes wide power cell import into the C fiber and showed strong discharge, and brought out nervus centralis pain sensation facilitation state.Its pain characteristics are pain phases when dividing two; I be mutually since directly stimulate the peripheral nerve end slightly due to; The generation release that II then is mainly inflammatory mediator mutually causes; Adopt Superlysoform rat model research Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract analgesic activity, the result shows, Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract 800mgkg
-1, 400mgkg
-1And 200mgkg
-1Dose groups to first o'clock of rat pain reaction due to the Superlysoform mutually with obvious restraining effect was all arranged in second o'clock mutually.Pain and inflammation are two big cardinal symptoms of rheumatoid arthritis, and experimental result shows that Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract has the obvious suppression effect to rational inflammation of immunological disease and nonspecific inflammation, has significant analgesia role simultaneously.The above results shows that Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract can be used for preparation and prevents and/or treats the rheumatoid arthritis product, and the preparation of pharmaceutical compositions that also can contain Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract prevents and/or treats in the rheumatoid arthritis product.
Root and Rhizome of Ripqrian Greenbrier steroidal saponin preparation method of extract technology of the present invention is simple, easy to operate, and technology is prone to grasp, and energy consumption is little, and solvent is recyclable to be recycled, and production cost is low.
Root and Rhizome of Ripqrian Greenbrier steroidal saponin preparation method of extract advantage of the present invention is 50～95% for steroidal saponin weight percentage wherein; Contain one or more of 4 kinds of new compounds; This is that technology was reported as yet in the past; Because content is high, thereby has improved curative effect, satisfied preparation prevents and/or treats the needs of rheumatoid arthritis product.
The present invention also provides the pharmaceutical prepn with Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract of the present invention and pharmaceutically acceptable carrier or vehicle preparation.These pharmaceutical prepns are selected from following formulation: tablet, sugar coated tablet, film coated tablet, enteric coated tablet, effervescent tablet, sublingual tablet, capsule, hard capsule, soft capsule, microcapsule, microspheres agent, granule, pill, pill, powder, paste, oral liquid, suspensoid, solution, aerosol, injection; Injectable emulsion, freeze-dried powder can also be prepared into slowly-releasing or controlled release preparation as required.
The pharmaceutical prepn that contains Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract of the present invention; When pharmaceutical formulations, can add the medicine acceptable carrier; Said medicine acceptable carrier is from oxidation inhibitor, a flat iron plate for making cakes mixture, tensio-active agent, weighting agent, disintegrating agent, wetting agent, solvent, slow-release material, enteric material, pH regulator agent, correctives, pigment etc., and common carrier is like mannitol, DEXTRAN 500.000, lactose, glucose, sorbyl alcohol, Xylitol, water for injection, injection ethanol, sodium-chlor, silicon derivative, Mierocrystalline cellulose, derivatived cellulose, gelatin, Vinylpyrrolidone polymer, glycerine, tween 80, agar, lime carbonate, polyoxyethylene glycol, Schardinger dextrins, phospholipids material, talcum powder, Magnesium Stearate, calcium stearate etc.
Below through specific embodiment the present invention is further described, be not to qualification of the present invention, according to prior art well known in the art, embodiment of the present invention is not limited to specific embodiment.
Embodiment
Embodiment 1
Get Root and Rhizome of Ripqrian Greenbrier root and rhizome meal 20Kg, add 8 times of water gaging refluxing extraction 3 times, each 2 hours, filter, extracting solution merges, and decompression recycling ethanol gets extract.Get extract and be dissolved in water, be added on the HPD100 macroporous adsorptive resins, it is closely colourless that water is eluted to elutriant, discards elutriant; Closely colourless with 10% ethanol elution to elutriant, discard elutriant, closely colourless with 90% ethanol elution to elutriant; Collect elutriant, 90% ethanol eluate is added on the D941 decolorizing resin post, with 6 times of column volume 90% ethanol elutions; Collect elutriant, concentrating under reduced pressure, the dry Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract 215g that gets, wherein contents of steroid saponin 52%.
Embodiment 2
Get Root and Rhizome of Ripqrian Greenbrier root and rhizome meal 20Kg, add 8 times of amount 30% alcohol reflux 3 times, each 2 hours, filter, extracting solution merges, and decompression recycling ethanol gets extract.Get extract and be dissolved in water, be added on the HPD300 macroporous adsorptive resins, it is closely colourless that water is eluted to elutriant, discards elutriant; Closely colourless with 50% ethanol elution to elutriant, discard elutriant, closely colourless with 90% ethanol elution to elutriant; Collect elutriant, 90% ethanol eluate is added on the D941 decolorizing resin post, with 6 times of column volume 90% ethanol elutions; Collect elutriant, concentrating under reduced pressure, the dry Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract 72g that gets, wherein contents of steroid saponin 95%.
Embodiment 3
Get Root and Rhizome of Ripqrian Greenbrier root and rhizome meal 20Kg, add 8 times of amount 50% alcohol reflux 3 times, each 2 hours, filter, extracting solution merges, and decompression recycling ethanol gets extract.Get extract and be dissolved in water, be added on the HPD400 macroporous adsorptive resins, it is closely colourless that water is eluted to elutriant, discards elutriant; Closely colourless with 30% ethanol elution to elutriant, discard elutriant, closely colourless with 70% ethanol elution to elutriant; Collect elutriant, 70% ethanol eluate is added on the D941 decolorizing resin post, with 6 times of column volume 90% ethanol elutions; Collect elutriant, concentrating under reduced pressure, the dry Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract 136g that gets, wherein contents of steroid saponin 88%.
Embodiment 4
Get Root and Rhizome of Ripqrian Greenbrier root and rhizome meal 20Kg, add 8 times of amount 70% alcohol reflux 3 times, each 2 hours, filter, extracting solution merges, and decompression recycling ethanol gets extract.Get extract and be dissolved in water, be added on the D101 macroporous adsorptive resins, it is closely colourless that water is eluted to elutriant, discards elutriant; Closely colourless with 30% ethanol elution to elutriant, discard elutriant, closely colourless with 70% ethanol elution to elutriant; Collect elutriant, 70% ethanol eluate is added on the D941 decolorizing resin post, with 6 times of column volume 90% ethanol elutions; Collect elutriant, concentrating under reduced pressure, the dry Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract 124g that gets, wherein contents of steroid saponin 84%.
Embodiment 5
Get Root and Rhizome of Ripqrian Greenbrier root and rhizome meal 20Kg, add 8 times of amount 90% alcohol reflux 3 times, each 2 hours, filter, extracting solution merges, and decompression recycling ethanol gets extract.Get extract and be dissolved in water, be added on the D101 macroporous adsorptive resins, it is closely colourless that water is eluted to elutriant, discards elutriant; Closely colourless with 10% ethanol elution to elutriant, discard elutriant, closely colourless with 60% ethanol elution to elutriant; Collect elutriant, 60% ethanol eluate is added on the D941 decolorizing resin post, with 6 times of column volume 90% ethanol elutions; Collect elutriant, concentrating under reduced pressure, the dry Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract 166g that gets, wherein contents of steroid saponin 73%.
Embodiment 6
Get Root and Rhizome of Ripqrian Greenbrier root and rhizome meal 20Kg, add 8 times of amount 90% alcohol reflux 3 times, each 2 hours, filter, extracting solution merges, and decompression recycling ethanol gets extract.Get extract and be dissolved in water, be added on the D101 macroporous adsorptive resins, it is closely colourless that water is eluted to elutriant, discards elutriant; Closely colourless with 30% ethanol elution to elutriant, discard elutriant, closely colourless with 80% ethanol elution to elutriant; Collect elutriant, 80% ethanol eluate is added on the D941 decolorizing resin post, with 6 times of column volume 90% ethanol elutions; Collect elutriant, concentrating under reduced pressure, the dry Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract 159g that gets, wherein contents of steroid saponin 69%.
Embodiment 7
Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract is with the pharmaceutical prepn of acceptable carrier pharmaceutically or vehicle preparation.These pharmaceutical prepns are selected from following formulation: tablet, sugar coated tablet, film coated tablet, enteric coated tablet, effervescent tablet, sublingual tablet, capsule, hard capsule, soft capsule, microcapsule, microspheres agent, granule, pill, pill, powder, paste, oral liquid, suspensoid, solution, aerosol, injection; Injectable emulsion, freeze-dried powder; Can also be prepared into slowly-releasing or controlled release preparation as required; When pharmaceutical formulations, can add the medicine acceptable carrier; Said medicine acceptable carrier is from oxidation inhibitor, a flat iron plate for making cakes mixture, tensio-active agent, weighting agent, disintegrating agent, wetting agent, solvent, slow-release material, enteric material, pH regulator agent, correctives, pigment etc., and common carrier is like mannitol, DEXTRAN 500.000, lactose, glucose, sorbyl alcohol, Xylitol, water for injection, injection ethanol, sodium-chlor, silicon derivative, Mierocrystalline cellulose, derivatived cellulose, gelatin, Vinylpyrrolidone polymer, glycerine, tween 80, agar, lime carbonate, polyoxyethylene glycol, Schardinger dextrins, phospholipids material, talcum powder, Magnesium Stearate, calcium stearate.Above-mentioned preparation technology is this area routine operation, does not add at this and gives unnecessary details.
Embodiment 8
Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract is the pharmaceutical prepn that pharmaceutical prepn that raw material makes not only comprises independent use Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract, also comprises adding the pharmaceutical prepn of Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract as activeconstituents.
Embodiment 9
Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract is to the influence of adjuvant arthritis rat paw edema
Get 48 of male rats, wherein (every milliliters of liquid paraffin contains 8mgH to 40 right back sufficient plantar subcutaneous injection Freund's complete adjuvants of rat
37The RV mycobacterium hominis) 100 μ L sensitization, 8 of normal control groups give 100 μ L whiterusss, and the sensitization rat is divided into model control group at random behind the 13d, tripterygium glycosides group (25mgkg
-1), Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract high dose group (800mgkg
-1), middle dose groups (400mgkg
-1) and low dose group (200mgkg
-1).13d-24d ig every day administration is 1 time after each treated animal sensitization, and control group gives with volume zero(ppm) water (10mLkg
-1).Measure the right back sufficient sole of the foot Zhou Jing of portion of rat respectively at 18d after the sensitization, 21d and 24d, calculate the swelling degree of the paw of the right back sufficient sole of the foot of rat in different time, the scorching metapedes sole of the foot Zhou Jing of swelling degree of the paw=cause-cause scorching front foot sole of the foot Zhou Jing, the result sees table 5.
Table 5 Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract is to the influence of adjuvant arthritis rat paw edema
Compare with model control group:
*P＜0.05
*P＜0.01
The result shows, Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract 800mgkg
-1And 400mgkg
-1Dose groups has obvious restraining effect to adjuvant arthritis rat secondary inflammation, in institute's dosage scope obvious dose-effect relationship is arranged.
Embodiment 10
Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract is to the influence of adjuvant arthritis rat inflammatory cytokine
24d abdominal injection 3% Chloral Hydrate 300mgkg after the sensitization
-1Anesthesia, aorta abdominalis blood sampling, centrifugal 5min (3000rpm) separation of serum.Adopt the ELISA test kit to measure TNF-α, IL-1 β and IL-6 content respectively, the result sees table 6.
Table 6 Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract is to the influence of adjuvant arthritis rat blood serum TNF-α, IL-1 β and IL-6
Compare with model control group:
*P＜0.05
*P＜0.01; Compare with the normal control group:
#P＜0.05
The result shows, Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract 800mgkg
-1And 400mgkg
-1Dose groups can obviously reduce TNF-α in the adjuvant arthritis rat blood serum, IL-1 β and IL-6 content.
Embodiment 11
The influence of Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract on Carrageenan property rat paw edema
Get 50 of male rats, be divided into model control group at random, Ibuprofen BP/EP group (80mgkg
-1), Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract high dose group (800mgkg
-1), middle dose groups (400mgkg
-1) and low dose group (200mgkg
-1).Every day, the ig administration was 1 time, continuous 6d, and control group gives with volume zero(ppm) water (10mLkg
-1).Behind each treated animal last administration 1h, aseptic 1% X 5189, the 100 μ L of right back sufficient plantar subcutaneous injection cause inflammation.Measure the right back sufficient sole of the foot Zhou Jing of portion of rat respectively at causing scorching back 2h, 3h and 4h, calculate the swelling degree of the right back sufficient sole of the foot of rat in different time, the scorching metapedes sole of the foot Zhou Jing of swelling degree of the paw=cause-cause scorching front foot sole of the foot Zhou Jing, the result sees table 7.
The influence of table 7 Root and Rhizome of Ripqrian Greenbrier ethanol extraction on Carrageenan property rat paw edema
Compare with model control group:
*P＜0.05
*P＜0.01
The result shows, Root and Rhizome of Ripqrian Greenbrier ethanol extraction 800mgkg
-1And 400mgkg
-1Dose groups on Carrageenan property foot swelling rat acute inflammation has obvious restraining effect, in institute's dosage scope obvious dose-effect relationship is arranged.
Embodiment 12
The influence of Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract Dichlorodiphenyl Acetate induced mice pain reaction
Get 70 of male mices, be divided into model control group at random, Frosst) group (100mgkg
-1), Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract high dose group (1000mgkg
-1), middle dose groups (500mgkg
-1) and low dose group (250mgkg
-1), every day, the ig administration was 1 time, continuous 6d, and control group gives with volume zero(ppm) water (10mLkg
-1).1h after each treated animal last administration, the glacial acetic acid solution 0.3mL of abdominal injection 0.8%, behind the injection acetic acid in the 5min opening entry 10min mouse turn round the body number of times, shrink, stretch that hind leg raises with trunk, buttocks is the writhing response positive with belly, the result sees table 8.
The influence of table 8 Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract Dichlorodiphenyl Acetate induced mice pain reaction
Compare with model control group:
*P＜0.05
*P＜0.01.
The result shows, Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract 1000mgkg
-1And 500mgkg
-1Dose groups Dichlorodiphenyl Acetate induced mice pain reaction has obvious restraining effect, wherein 1000mgkg
-1The dose groups effect is better than Frosst) 100mgkg
-1, in institute's dosage scope obvious dose-effect relationship is arranged.
Embodiment 13
Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract is to the influence of hot plate induced mice pain reaction
Get female mice, carry out the threshold of pain screening in advance, get threshold of pain, take into account body weight and threshold of pain and be divided into model control group at random, Srm-Rhotaard group (5mgkg 70 of 5-30s mouse
-1), Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract high dose group (1000mgkg
-1), middle dose groups (500mgkg
-1) and low dose group (250mgkg
-1).Every day, the ig administration was 1 time, continuous 6d, and control group gives with volume zero(ppm) water (10mLkg
-1).1h hot-plate instrument after the last administration (50 ± 0.5 ℃) is measured the mouse threshold of pain, and is still reactionless behind the mouse 60s, takes out mouse, calculates with 60s.2 threshold of pain of every mouse assay are got its average as the preceding threshold of pain of dispensing, and the result sees table 9.
Table 9 Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract is to the influence of hot plate induced mice pain reaction
Compare with model control group:
*P＜0.05
*P＜0.01
The result shows, Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract 1000mgkg
-1And 500mgkg
-1Dose groups has obvious restraining effect to hot plate induced mice pain reaction.
Embodiment 14
Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract is to the influence of rat pain reaction due to the Superlysoform
Get 48 of male rats, be divided into model control group at random, Srm-Rhotaard group (5mgkg
-1), Frosst) group (100mgkg
-1), Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract high dose group (800mgkg
-1), middle dose groups (400mgkg
-1) and low dose group (200mgkg
-1), each treated animal ig every day administration 1 time, continuous 6d, control group gives with volume zero(ppm) water (10mLkg
-1).1h after each treated animal last administration, the formalin solution 100 μ L of right back toes subcutaneous injection 2.0%, pain reaction (lick and the cause scorching foot) cumulative time of observing injection back 0-5min (first o'clock phase) and 15-30min (mutually) rat at second o'clock, the result sees table 10.
Table 10 Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract is to the influence of rat pain reaction due to the Superlysoform
Compare with model control group:
*P＜0.05
*P＜0.01
The result shows, Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract 800mgkg
-1, 400mgkg
-1And 200mgkg
-1With second o'clock obvious restraining effect was arranged all mutually mutually at first o'clock of the rat pain reaction due to each dose groups PARA FORMALDEHYDE PRILLS(91,95), explain that it all has obvious restraining effect to maincenter and peripheral pain.
Claims (4)
1. Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract is characterized in that from Root and Rhizome of Ripqrian Greenbrier root and rhizome, extracting, and wherein the steroidal saponin weight percentage is 50～95%, and the preparation method may further comprise the steps:
A) the Root and Rhizome of Ripqrian Greenbrier meal extracts with first solvent refluxing, filters, and extracting solution merges, the extracting solution concentrating under reduced pressure;
B) extract that step a) is obtained is dissolved in water, and through macroporous adsorbent resin column chromatography, it is closely colourless that water is eluted to elutriant, discards elutriant;
C) closely colourless with second solvent elution to elutriant, discard elutriant;
D) closely colourless with the 3rd solvent elution to elutriant, collect elutriant;
E) elutriant that step d) is obtained through decolorizing resin post D941 chromatogram, with the 4th solvent elution, is collected elutriant, concentrating under reduced pressure, the dry Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract that gets;
In above step; Wherein first solvent of step a) is 0～100% aqueous ethanolic solution; The step b) macroporous adsorbent resin comprises nonpolar or the low-pole macroporous adsorbent resin; Second solvent of step c) is 10～50% aqueous ethanolic solutions, and the 3rd solvent of step d) is 60～90% aqueous ethanolic solutions, and the 4th solvent of step e) is 90% aqueous ethanolic solution.
2. Root and Rhizome of Ripqrian Greenbrier steroidal saponin preparation method of extract according to claim 1 is characterized in that, may further comprise the steps:
A) the Root and Rhizome of Ripqrian Greenbrier meal adds 50～70% alcohol reflux, filters, and extracting solution merges, decompression recycling ethanol;
B) extract that step a) is obtained is dissolved in water, and through macroporous adsorptive resins D101 chromatogram, it is closely colourless that water is eluted to elutriant, discards elutriant;
C) closely colourless with 30% ethanol elution to elutriant, discard elutriant;
D) closely colourless with 70% ethanol elution to elutriant, collect elutriant;
E) elutriant that step d) is obtained, through decolorizing resin post D941 chromatogram, 6 times of column volume 90% ethanol elutions are collected elutriant, concentrating under reduced pressure, the dry Root and Rhizome of Ripqrian Greenbrier steroidal saponin extract that gets.
3. the described Root and Rhizome of Ripqrian Greenbrier steroidal saponin of claim 1 extract prevents and/or treats the purposes in the rheumatoid arthritis product in preparation.
4. the pharmaceutical composition that contains the described Root and Rhizome of Ripqrian Greenbrier steroidal saponin of claim 1 extract prevents and/or treats the purposes in the rheumatoid arthritis product in preparation.
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