CN103058970A - Inula wissmannian extracts, preparation for same, and application of extracts in preparation of anti-inflammatory medicine - Google Patents
Inula wissmannian extracts, preparation for same, and application of extracts in preparation of anti-inflammatory medicine Download PDFInfo
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Abstract
The invention provides a preparation for inula wissmannian extracts and an application of the extracts in preparation of an anti-inflammatory medicine. The extracts are germacrane sesquiterpene lactone inula wissmannian lactones A, B, C, D and E and inula cappa lactone with the following chemical structural formulas and absolute configurations shown in the specification, wherein the inula wissmannian lactones A, B, C, D and E and the inula cappa lactone generate an inhibition effect on the NO of the RAW264.7 macrophage induced by LPS (lipopolysaccharide); and in an xylene-induced ear swelling mouse model and a Freund complete adjuvant arthritis rat model, the inula wissmannian lactones A, B, C, D and E and the inula cappa lactone are found to be able to obviously inhibit the ear swelling and foot swelling of experimental animals, and the result indicates that the inula wissmannian lactones A, B, C, D and E and the inula cappa lactone are good in anti-inflammatory activity, capable of being used for preparing an anti-inflammatory medicine, and great in application value. The invention provides an extraction and separation method for the inula wissmannian lactones A, B, C, D and E and the inula cappa lactone.
Description
Technical field
The present invention relates to natural drug, be specifically related to extraction and separation method, the purposes of the southern regions of the Yunnan Province Sheepear Inula Herb effective constituent; Relate in particular to extraction and separation method and the application in the preparation anti-inflammatory drug of the southern regions of the Yunnan Province Sheepear Inula Herb extract Germacrane Sesquiterpenoids lactone.
Background technology
Composite family (Compositae) Inula (Inula L.) plant has 100 kinds approximately, mainly is distributed in Mediterranean Zone, in other area in Europe, Africa and Asia distribution is arranged also.This platymiscium in state-owned more than 20 plant and most mutation, wherein more than half plants have medicinal record (Chinese Plants will editorial board of the Chinese Academy of Sciences, Chinese Plants will [M], Beijing: Science Press, 1979,75:248-281).Chinese scholars is successively separated compound (the Zhao YM that obtains the structure types such as flavones, sesquiterpene lactones, terpene from this platymiscium, Zhang ML, Shi QW, Kiyota H.Chemical constituents of plants from the genus Inula.Chemistry ﹠amp; Biodiversity2006; 3:371-383).Sesquiterpene lactones is the characteristic chemical constituent of this platymiscium, also is main activeconstituents.Have the multiple effects such as anti-inflammatory, antitumor, antiviral, expelling parasite desinsection, immunosuppression.Sesquiterpene lactones is also simple in structure because of it, and has multiple mother nucleus structure type, can be used as the lead compound of research and development medicine.Plant the southern regions of the Yunnan Province Sheepear Inula Herb (Inula wissmanniana Hand.-Mazz.) is Inulaplants, and the Chinese yunnan area is commonly called as " torch stalk ", is undershrub, produces south of Yunnan (Yuanjiang River, screen limit).Be born in low mountain and open spacious hillside fields, 1200 ~ 1650 meters of height above sea level.The inventor has carried out the deep chemical constitution study of system to the southern regions of the Yunnan Province Sheepear Inula Herb, therefrom separate obtaining 8 Germacrane Sesquiterpenoids constituents, find the southern regions of the Yunnan Province Sheepear Inula Herb extract and the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the Sheepear Inula Herb lactone have significant anti-inflammatory activity.At present, there is no the bibliographical information of the southern regions of the Yunnan Province Sheepear Inula Herb chemical ingredients and pharmacologically active thereof, also have no the report relevant with the southern regions of the Yunnan Province Sheepear Inula Herb method for preparing extractive, purposes among the present invention.
Summary of the invention
The purpose of technical problem to be solved by this invention is research and design the southern regions of the Yunnan Province Sheepear Inula Herb extract and extraction and separation method and purposes.
The invention provides a kind of the southern regions of the Yunnan Province Sheepear Inula Herb extract, described extract is mainly Germacrane Sesquiterpenoids the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the Sheepear Inula Herb lactone, and their structural formula and absolute configuration are as follows respectively:
The southern regions of the Yunnan Province Sheepear Inula Herb lactone first:
The southern regions of the Yunnan Province Sheepear Inula Herb lactone second:
The southern regions of the Yunnan Province Sheepear Inula Herb lactone third:
The southern regions of the Yunnan Province Sheepear Inula Herb lactone fourth:
The southern regions of the Yunnan Province Sheepear Inula Herb lactone penta:
The Sheepear Inula Herb lactone:
The southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the Sheepear Inula Herb lactone be colourless block crystallization, be soluble in chloroform, acetone, methyl alcohol, the ethanol, molecular formula is followed successively by C
22H
30O
6, C
20H
28O
5, C
20H
28O
4, C
19H
26O
4, C
15H
22O
3, C
22H
30O
7Its chemical structure and absolute configuration by high resolution mass spectrum in conjunction with one dimension, two dimensional NMR wave spectrum and X-ray single crystal diffraction Analysis deterrmination.
Another object of the present invention has provided the preparation method of described the southern regions of the Yunnan Province Sheepear Inula Herb extract.
Preparation of the present invention comprises the preparation of extracting separation and monomeric compound.
Preparation method of the present invention comprises the following steps:
(1) the southern regions of the Yunnan Province Sheepear Inula Herb herb is dry, pulverize, extract 3～4 times with 1 ~ 10 times of amount 80～95% ethanol soaking at room temperature, each 12～24 hours, merge to get extracting solution;
(2) the concentrating under reduced pressure extracting solution gets water suspendible fluid extract to without the alcohol flavor, adds 1～3 times of water dilution, uses successively 2～6 times of sherwood oils, methylene dichloride, ethyl acetate extraction, and reclaim under reduced pressure respectively extracts part, gets without aqueous extract;
(3) silica gel column chromatography is used at above-mentioned methylene dichloride position, methylene chloride/methanol system gradient elution take volume ratio as 100:0～2:1, thin-layer chromatography detects, collect respectively and contain the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the flow point of Sheepear Inula Herb lactone, again through dextran gel column chromatography, methylene chloride/methanol wash-out take volume ratio as 1:1 finally by liquid chromatography preparation, gets the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the Sheepear Inula Herb lactone.
Another purpose of the present invention has provided the application of described the southern regions of the Yunnan Province Sheepear Inula Herb extract in the preparation anti-inflammatory drug.
The inventor by the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the Sheepear Inula Herb lactone to cell and animal experiment, the result proves, the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the Sheepear Inula Herb lactone suppress the IC that scavenger cell RAW264.7 produces NO
50Value is respectively 4.90,2.17, the positive medicine IC of 5.09,1.09,1.75,10.28 μ M(aminoguanidine
500.37 μ M); In caused by dimethylbenzene xylene ear swelling mouse and Freund's complete adjuvant rat model of arthritis, find the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the Sheepear Inula Herb lactone can obviously suppress ear swelling and the foot swelling of laboratory animal.Therefore, the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the Sheepear Inula Herb lactone have preferably anti-inflammatory activity, can be used for preparing anti-inflammatory drug.
Medicine of the present invention is the pharmaceutical composition that is comprised of as activeconstituents and pharmaceutical excipient the southern regions of the Yunnan Province Sheepear Inula Herb extract.
Medicine of the present invention is the pharmaceutical composition that is comprised of as activeconstituents and pharmaceutical excipient the southern regions of the Yunnan Province Sheepear Inula Herb lactone.
Medicine of the present invention is the pharmaceutical composition that is comprised of as activeconstituents and pharmaceutical excipient the southern regions of the Yunnan Province Sheepear Inula Herb lactone first.
Medicine of the present invention is the pharmaceutical composition that is comprised of as activeconstituents and pharmaceutical excipient the southern regions of the Yunnan Province Sheepear Inula Herb lactone second.
Medicine of the present invention is the pharmaceutical composition that is comprised of as activeconstituents and pharmaceutical excipient the southern regions of the Yunnan Province Sheepear Inula Herb lactone third.
Medicine of the present invention is the pharmaceutical composition that is comprised of as activeconstituents and pharmaceutical excipient the southern regions of the Yunnan Province Sheepear Inula Herb lactone fourth.
Medicine of the present invention is the pharmaceutical composition that is comprised of as activeconstituents and pharmaceutical excipient the southern regions of the Yunnan Province Sheepear Inula Herb lactone penta.
The present invention discloses the southern regions of the Yunnan Province Sheepear Inula Herb extract first, the southern regions of the Yunnan Province Sheepear Inula Herb lactone, the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth and penta; And their extraction and separation method and the application in pharmacy be provided, and provide new antiradiation drug for clinical, bring glad tidings to the patient, larger clinical value and social benefit are arranged.
Description of drawings
Fig. 1 the southern regions of the Yunnan Province Sheepear Inula Herb lactone first X diffraction structure
Fig. 2 the southern regions of the Yunnan Province Sheepear Inula Herb lactone the third X diffraction structure
Embodiment
Present embodiment is implemented under take technical solution of the present invention as prerequisite, has provided detailed embodiment and process, but protection scope of the present invention is not limited to following embodiment.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
Embodiment 1 the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the preparation of Sheepear Inula Herb lactone
After the southern regions of the Yunnan Province Sheepear Inula Herb herb (10.0kg) drying and crushing, 25 ℃ of room temperatures lower with 95% ethanol (30L) cold soaking extract 4 times (24,12,12,12h), reclaim solvent, concentrate dryly without aqueous extract (631.4g); Total medicinal extract is suspended in the 1.5L water and extracts respectively 3 times with 5L sherwood oil, methylene dichloride, ethyl acetate successively, decompression and solvent recovery obtains respectively petroleum ether part (133.2g), methylene dichloride position (179.6g) and ethyl acetate extract (28.1g).
With the upper normal phase silicagel column of the methylene dichloride position 120g of the southern regions of the Yunnan Province Sheepear Inula Herb, use successively methylene chloride-methanol (100% methylene dichloride, 100:1,50:1,20:1,10:1,5:1 and 100% methyl alcohol) gradient elution, elutriant detects through thin-layer chromatography, merges the elutriant of similar composition, and decompression and solvent recovery gets 8 wash-out parts.Get wherein the 1st wash-out part, upper silica gel column chromatography is with petroleum ether-ethyl acetate (20:1) wash-out, and through the elutriant of the similar composition of thin-layer chromatography combining data detection, decompression and solvent recovery gets 6 wash-out flow points.The 2nd wash-out flow point (2.9g) wherein with the least possible methylene dichloride dissolving, left standstill under 25 ℃ of the room temperatures, separate out block crystallization, be accredited as the southern regions of the Yunnan Province Sheepear Inula Herb lactone third (1.52g); The 3rd flow point (3.3g) leaves standstill under 25 ℃ of the room temperatures in the least possible dichloromethane solution, separates out block crystallization the southern regions of the Yunnan Province Sheepear Inula Herb lactone first (1.23g); After dissolving, filter with Chromatographic Pure Methanol (3mL), the 4th flow point (0.9g) through the preparative chromatography method, with 70% methanol-eluted fractions, obtain compound the southern regions of the Yunnan Province Sheepear Inula Herb lactone fourth (t
R40.0min, 12.5mg).In addition, again with the 2nd wash-out part (12.4g), behind methyl alcohol (30mL) dissolution filter, upper MCI column chromatography with 80% methanol-eluted fractions, obtains 2 flow points.The 1st flow point (2.6g) further gone up Sephadex LH-20 gel column chromatography, with methylene chloride-methanol 1:1 wash-out, elutriant detects through thin-layer chromatography, 8 elutriants that merge similar composition, reclaim solvent and get 8 wash-out flow points, again wherein the 3rd (0.7g) and the 4th flow point (0.9g) are gone up respectively the preparative chromatography post, with 55% methyl alcohol (10L) wash-out, obtain the southern regions of the Yunnan Province Sheepear Inula Herb lactone penta (t
R32.0min, 25.6mg), Sheepear Inula Herb lactone (t
R58.2min, 31.1mg), and the southern regions of the Yunnan Province Sheepear Inula Herb lactone second (t
R75.0min, 30.0mg).
The southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the Sheepear Inula Herb lactone
1H-NMR and
13The C-NMR data see Table respectively 1 and table 2, and optical rotational activity spectrum, infrared spectra, mass spectrum, high resolution mass spectrum and X-ray single crystal diffraction data are as follows respectively:
The southern regions of the Yunnan Province Sheepear Inula Herb lactone first: brilliant without color lump; C
22H
30O
6;
(c0.10, CH
2Cl
2); IR (KBr) ν
Max2931,1768,1726,1661,1456,1382,1314,1245,1162,1134,1071,1019,975cm
-1; ESIMS m/z413.2[M+Na]
+, m/z389.2[M-H]
-; HRESIMS m/z413.1943[M+Na]
+(calcd for C
22H
30O
6Na, 413.1940); Crystallization data: C
22H
30O
6, CH
2Cl
2, M=390.46, orthorhombic, space group P2 (1) 2 (1) 2 (1), a=6.7751 (3)
α=90 °; B=8.9058 (4)
β=90 °; C=35.0890 (14)
γ=90 °; V=2117.19 (16)
Z=4, D
Calcd=1.225mg/m
3, crystallite size 0.20 * 0.15 * 0.10mm
3.Cu K α (λ=1.54178
), F (000)=840, T=133 (2) K.R value is respectively R
1=0.0317, and wR
2=0.0873, for22899 observed reflections[I〉2 σ (I)] the .Flack parameter is 0.05 (15).Relevant crystallization data trnascription can be in (the storage number: CCDC835957) of Cambridge crystallization Data centre Free Acquisition, address: CCDC, 12Union Road, Cambridge CB21EZ, UK (fax:+441223336033or e-mail:data_request@ccdc.cam.ac.uk).
The southern regions of the Yunnan Province Sheepear Inula Herb lactone second: brilliant without color lump; C
20H
28O
5;
(c0.10, CH
2Cl
2); IR (KBr) ν
Max3430,2928,2861,1763,1707,1650,1453,1381,1245,1150,1021,975cm
-1; ESIMS m/z371.1[M+Na]
+, m/z347.1[M-H]
-; HRESIMS m/z371.1836[M+Na]
+(calcd for C
20H
28O
5Na, 371.1834).
The southern regions of the Yunnan Province Sheepear Inula Herb lactone third: brilliant without color lump; C
20H
28O
4;
(c0.10, CH
2Cl
2); IR (KBr) ν
Max2937,2923,2856,1766,1705,1643,1450,1379,1234,1153,1144,1037,973cm
-1; ESIMS m/z355.2[M+Na]
+, m/z331.2[M-H]
-; HRESIMS m/z355.1856[M+Na]
+(calcd for C
20H
28O
4Na, 355.1855); Crystallization data: C
20H
28O
4, CH
2Cl
2, M=332.42, orthorhombic, space group P2 (1) 2 (1) 2 (1), a=11.9746 (5)
α=90 °; B=12.3428 (5)
β=90 °; C=12.4853 (5)
γ=90 °; V=1845.33 (13)
Z=4, D
Calcd=1.197mg/m
3, crystallite size 0.20 * 0.15 * 0.10mm
3.Cu K α (λ=1.54178
), F (000)=720, T=273 (2) K.R value is R respectively
1=0.0276, and wR
2=0.0767, for20005 observed reflections[I〉2 σ (I)] the .Flack parameter is 0.02 (15).Relevant crystallization data trnascription can be in (the storage number: CCDC835958) of Cambridge crystallization Data centre Free Acquisition, address: CCDC, 12Union Road, Cambridge CB21EZ, UK (fax:+441223336033or e-mail:data_request@ccdc.cam.ac.uk).
The southern regions of the Yunnan Province Sheepear Inula Herb lactone fourth: brilliant without color lump; C
22H
30O
6;
(c0.05, CH
2Cl
2); IR (KBr) ν
Max2930,2863,1770,1713,1633,1571,1454,1383,1317,1281,1250,1141,1072,1010,976cm
-1; ESIMS m/z341.2[M+Na]
+, m/z317.2[M-H]
-; HRESIMS:m/z341.1733[M+Na]
+(calcd for C
19H
26O
4Na, 341.1729).
The southern regions of the Yunnan Province Sheepear Inula Herb lactone penta: brilliant without color lump; C
15H
22O
3;
(c0.10, CH
2Cl
2); IR (KBr) ν
Max3441,2936,2865,1765,1663,1457,1382,1274,1249,1144,1079,1012,973cm
-1; ESIMS m/z273.1[M+Na]
+, m/z249.1[M-H]
-; HRESIMS:m/z273.1466[M+Na]
+(calcd for C
15H
22O
3Na, 273.1467).
Sheepear Inula Herb lactone: brilliant without color lump; C
22H
30O
7; ESIMS m/z429.2[M+Na]
+, m/z405.2[M-H]
-
Table 1 the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the Sheepear Inula Herb lactone
1H-NMR data (CDCl
3, 400MHz)
Continued 1
Compound atom numbering is seen structural formula of compound figure; Nuclear magnetisation displacement study unit is ppm; Be the coupling constant value in the bracket, unit is Hz.
Table 2 the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the Sheepear Inula Herb lactone
13C-NMR data (CDCl
3, 400MHz)
Compound atom numbering is seen structural formula of compound figure; Nuclear magnetisation displacement study unit is ppm.
Embodiment 2 the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the Sheepear Inula Herb lactone suppress the NO that LPS induces RAW264.7 and generate active
(1) sample configuration
The southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta that embodiment 1 makes, Sheepear Inula Herb lactone and positive control drug aminoguanidine (Sigma-Aldrich, purity〉98.0%)) use the DMSO(methyl-sulphoxide) (Merck) after the dissolving, adding PBS (-) (phosphate buffered saline buffer) is made into the solution of 10mM, further dilution is 0,0.1,0.5, the sample of 5,20 μ M gradient concentrations.LPS (Lipopolysaccharides, lipopolysaccharides, sigma, Cat.L-2880) with 10 μ g/mL is inductor.
(2) experimental technique
Mouse macrophage RAW264.7(is purchased from Shanghai cell resource center of Sheng Ke institute) at 37 ° of C, 5%CO
2Cellar culture is in the DMEM nutrient solution in the incubator.During experiment with 1 μ L/mL LPS(lipopolysaccharides) (being dissolved in the distilled water) to add 100mL concentration be 2 * 10
6In the cell suspension of μ g/mL, indirectly reflect the NO growing amount with the Griess method by the content of measuring cell conditioned medium liquid nitrite behind the 18h: get the 100mL cell culture fluid, add in the equivalent Griess(lattice this) reagent, measure light absorption value.
(3) judgement criteria and statistical method
Survey optical density in 570nm wavelength place, with NaNO
2Standardized solution drawing standard curve, the concentration of calculating nitrite.Each is organized experimental result and carries out statistical study with SPSS software ONE WAY ANNOVA method (Chicago,U.S SPSS Inc.).
(4) experimental result
Experimental result show the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the Sheepear Inula Herb lactone induce the NO of RAW264.7 scavenger cell to generate to LPS obvious inhibitions activity (the results are shown in Table 3) to be arranged, illustrate that it has anti-inflammatory activity.
Table 3 the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the Sheepear Inula Herb lactone suppress the NO that LPS induces RAW264.7 and generate active
(1) sample configuration
The southern regions of the Yunnan Province Sheepear Inula Herb total extract that embodiment 1 makes, petroleum ether part, methylene dichloride position, ethyl acetate extract and the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta, Sheepear Inula Herb lactone, use respectively the 0.5%Tween-80(tween-80) solution preparation becomes the suspension of desired concn, and is for subsequent use.Before use aspirin tablet (Sigma-Aldrich company) is mixed with the suspension of 200mg/kg as positive control.The administration volume of each group is 20mL/kg, oral administration.
(2) experimental technique
220 of Kunming mouses, male, 18～22g is divided into 22 groups at random, 10 every group.If group model contrast, one group of positive control and 20 groups of administration groups.After mouse is bought the week that conforms, the beginning administration.Medication is: positive drug acetylsalicylic acid successive administration 5 days, and once a day, 0.5mL/ time, dosage is 200mg/kg; Each treatment group successive administration 5 days, once a day, 0.5mL/ inferior, the southern regions of the Yunnan Province Sheepear Inula Herb total extract group dosage is 400mg/kg, 800mg/kg, sherwood oil, methylene dichloride, ethyl acetate extract dosage are 200mg/kg, 400mg/kg, the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and Sheepear Inula Herb lactone dosage be 200mg/kg, 100mg/kg.Model group successive administration 5 days, once a day, 0.5mL/ time.30min after the administration of mouse last, the auris dextra two sides is coated with dimethylbenzene 0.05mL/ and only causes inflammation, and left ear is not coated with, and puts to death behind the 60min, gets the same position of left and right sides ear disk with the 6mm punch tool, precision weighing two ear weight.
(3) judgement criteria and statistical method
Difference with auris dextra and left ear weight represents the swelling degree.Each is organized experimental result and carries out statistical study with SPSS software ONE WAY ANNOVA method.
(4) experimental result
Positive drug (acetylsalicylic acid), the southern regions of the Yunnan Province Sheepear Inula Herb total extract, low, the high dose group of petroleum ether part, methylene dichloride position, ethyl acetate extract and the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta, Sheepear Inula Herb lactone, the ear swelling that p-Xylol causes has obvious restraining effect (P＜0.05), wherein, the most obvious with methylene dichloride position and the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta, Sheepear Inula Herb lactone high dose group effect.This experiment show the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the Sheepear Inula Herb lactone have obvious anti-inflammatory activity (seeing Table 4).
The impact that table 4. the southern regions of the Yunnan Province Sheepear Inula Herb total extract, each extract part and the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta, Sheepear Inula Herb lactone are tested mice ear
Embodiment 4 the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the arthritic activity of Sheepear Inula Herb lactone antagonism Freund's complete adjuvant
(1) sample preparation
The southern regions of the Yunnan Province Sheepear Inula Herb total extract that embodiment 1 makes, petroleum ether part, methylene dichloride position, ethyl acetate extract and the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta, Sheepear Inula Herb lactone, become respectively the suspension of desired concn with the 0.5%Tween-80 solution preparation, for subsequent use.Before use the indomethacin sheet is mixed with the suspension of 210mg/kg as positive control.The administration volume of each group is 10mL/kg, oral administration.
(2) experimental technique
Male rat, 190 ~ 200g divides at random and does 22 groups, 10 every group.If 1 group model, 1 group of positive control and 20 groups of administration groups.After rat is bought the week that conforms, the beginning administration.Medication is: positive continuous oral administration 5 days, and once a day, dosage is 210mg/kg; Tested group of successive administration 5 days, once a day, abdominal injection, the southern regions of the Yunnan Province Sheepear Inula Herb total extract group dosage is 500mg/kg, 1000mg/kg, and sherwood oil, methylene dichloride, ethyl acetate extract and the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta, Sheepear Inula Herb lactone dosage are 100mg/kg, 200mg/kg.Model group successive administration 5 days, once a day, oral injection blank solvent.Cause inflammation after the last administration, survey Rat Right pawl solvent, every Rat Right group injection Freund's complete adjuvant 0.05mL surveys right sufficient volume again after measuring after 24 hours, tries to achieve the Rat Right paw swelling.
(3) judgement criteria and statistical method
Each is organized experimental result and carries out statistical study with SPSS software ONE WAY ANNOVA method.
(4) experimental result
Positive drug (indomethacin), the high dose group at the southern regions of the Yunnan Province Sheepear Inula Herb total extract, methylene dichloride position, and the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta, the Sheepear Inula Herb lactone is low, high dose group all can obviously suppress rat paw swelling, show that the southern regions of the Yunnan Province Sheepear Inula Herb total extract, methylene dichloride position and the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta, Sheepear Inula Herb lactone all have certain anti-inflammatory activity (P＜0.05, table 5).
Table 5. the southern regions of the Yunnan Province Sheepear Inula Herb total extract, each extract part and the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta, the arthritic activity of Sheepear Inula Herb lactone antagonism Freund's complete adjuvant
Claims (10)
1. the southern regions of the Yunnan Province Sheepear Inula Herb plant milk extract is characterized in that, described extract is the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the Sheepear Inula Herb lactone, and their structural formula and absolute configuration are as follows respectively:
The southern regions of the Yunnan Province Sheepear Inula Herb lactone first:
The southern regions of the Yunnan Province Sheepear Inula Herb lactone second:
The southern regions of the Yunnan Province Sheepear Inula Herb lactone third:
The southern regions of the Yunnan Province Sheepear Inula Herb lactone fourth:
The southern regions of the Yunnan Province Sheepear Inula Herb lactone penta:
The Sheepear Inula Herb lactone:
2. prepare the method that the southern regions of the Yunnan Province as claimed in claim 1 Sheepear Inula Herb is extracted, it is characterized in that,
The method comprises the following steps:
(1) the southern regions of the Yunnan Province Sheepear Inula Herb herb is dry, pulverize, extract 3～4 times with 1 ~ 10 times of amount 80～95% ethanol soaking at room temperature, each 12～24 hours, merge to get extracting solution;
(2) the concentrating under reduced pressure extracting solution gets water suspendible fluid extract to without the alcohol flavor, adds 1～3 times of water dilution, uses successively 2～6 times of sherwood oils, methylene dichloride, ethyl acetate extraction, and reclaim under reduced pressure respectively extracts part, gets without aqueous extract;
(3) silica gel column chromatography is used at above-mentioned methylene dichloride position, methylene chloride/methanol system gradient elution take volume ratio as 100:0～2:1, thin-layer chromatography detects, collect respectively and contain the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the flow point of Sheepear Inula Herb lactone, again through dextran gel column chromatography, methylene chloride/methanol wash-out take volume ratio as 1:1 finally by liquid chromatography preparation, gets the southern regions of the Yunnan Province Sheepear Inula Herb lactone first, second, third, fourth, penta and the Sheepear Inula Herb lactone.
3. the application of the southern regions of the Yunnan Province Sheepear Inula Herb extract as claimed in claim 1 in the preparation anti-inflammatory drug.
4. application as claimed in claim 3 is characterized in that, described medicine is the pharmaceutical composition that is comprised of as activeconstituents and pharmaceutical excipient the southern regions of the Yunnan Province Sheepear Inula Herb extract.
5. application as claimed in claim 3 is characterized in that, described medicine is the pharmaceutical composition that is comprised of as activeconstituents and pharmaceutical excipient the southern regions of the Yunnan Province Sheepear Inula Herb lactone.
6. application as claimed in claim 3 is characterized in that, described medicine is the pharmaceutical composition that is comprised of as activeconstituents and pharmaceutical excipient the southern regions of the Yunnan Province Sheepear Inula Herb lactone first.
7. application as claimed in claim 3 is characterized in that, described medicine is the pharmaceutical composition that is comprised of as activeconstituents and pharmaceutical excipient the southern regions of the Yunnan Province Sheepear Inula Herb lactone second.
8. application as claimed in claim 3 is characterized in that, described medicine is the pharmaceutical composition that is comprised of as activeconstituents and pharmaceutical excipient the southern regions of the Yunnan Province Sheepear Inula Herb lactone third.
9. application as claimed in claim 3 is characterized in that, described medicine is the pharmaceutical composition that is comprised of as activeconstituents and pharmaceutical excipient the southern regions of the Yunnan Province Sheepear Inula Herb lactone fourth.
10. application as claimed in claim 3 is characterized in that, described medicine is the pharmaceutical composition that is comprised of as activeconstituents and pharmaceutical excipient the southern regions of the Yunnan Province Sheepear Inula Herb lactone penta.
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| CN111714533A (en) * | 2020-07-21 | 2020-09-29 | 贵州光正制药有限责任公司 | Chrysanthemum indicum extract and preparation and application thereof |
| CN112402412A (en) * | 2020-11-18 | 2021-02-26 | 中国医学科学院药用植物研究所 | Application of inner ester compound of jingdao in preparing medicine for treating inflammation-caused diseases |
| CN112402412B (en) * | 2020-11-18 | 2022-03-18 | 中国医学科学院药用植物研究所 | Application of inner ester compound of jingdao in preparing medicine for treating inflammation-caused diseases |
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