WO2005084689A1 - Extrait de radix astragli, procede pour le produire et utilisation de celui-ci - Google Patents

Extrait de radix astragli, procede pour le produire et utilisation de celui-ci Download PDF

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WO2005084689A1
WO2005084689A1 PCT/CN2005/000216 CN2005000216W WO2005084689A1 WO 2005084689 A1 WO2005084689 A1 WO 2005084689A1 CN 2005000216 W CN2005000216 W CN 2005000216W WO 2005084689 A1 WO2005084689 A1 WO 2005084689A1
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astragalus
extract
solution
ethanol
resin
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PCT/CN2005/000216
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Chinese (zh)
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Zhengzhong Cao
Minzhu Chen
Bin Wang
Weiping Li
Yuan Cao
Yan Yang
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Hutchison Medipharma Enterprises Limited
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
    • C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
    • C07D311/30—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only not hydrogenated in the hetero ring, e.g. flavones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
    • C07H17/06—Benzopyran radicals
    • C07H17/065—Benzo[b]pyrans
    • C07H17/07—Benzo[b]pyran-4-ones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J17/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, having an oxygen-containing hetero ring not condensed with the cyclopenta(a)hydrophenanthrene skeleton

Definitions

  • the invention belongs to the field of traditional Chinese medicine pharmacy, in particular to an astragalus extract, a preparation method thereof and application in medicine. Background technique
  • Astragalus total saponins are the main extracts of Astragalus, which contains saponins and flavonoid glycosides.
  • Existing methods for preparing astragalus total saponins include water extraction method, alcohol extraction method, water extraction and alcohol precipitation method, resin adsorption and extraction, n-butanol extraction method, etc., but some of these methods are more complicated, and some extract yields Low, poor stability, and some remove many active ingredients, can not effectively play the medicinal value of astragalus. Summary of the invention
  • the object of the present invention is to avoid the deficiencies in the prior art mentioned above, and to provide an astragalus extract with reliable quality and good stability.
  • the astragalus extract contains the following weight percentages: astragalus total saponins 53-80%, astragalus flavonoids 10-25%.
  • the astragalus extract contains the following weight percentages of substances: astragalus total saponins 74-80%, astragalus flavone glycosides 10-20%.
  • the astragalus extract also contains 10-25% organic acids.
  • the astragalus extract can be prepared by the following steps:
  • the resin in the step c may be selected from D-101, AB-8 or SIP-1300 type resin, and preferably D-101 type resin.
  • the weight ratio of the amount of the extract to the resin in step c may be 1: 5-10.
  • the concentration of the ethanol aqueous solution in the step a is preferably 65-99%, and more preferably 80-99%.
  • the step a is preferably performed under heating and refluxing conditions.
  • the concentration of the ethanol aqueous solution in the step c is preferably 70-90%.
  • Another object of the present invention is to provide a preparation method that is relatively simple, can effectively remove impurities, and retains the astragalus extract as described above.
  • the preparation method includes the following steps:
  • the resin in the step c may be selected from D-101, AB-8 or SIP-1300 type resin, and preferably D-101 type resin.
  • the weight ratio of the amount of the extract to the resin in step c may be 1: 5-10.
  • the concentration of the ethanol aqueous solution in the step a is preferably 65-99%, and more preferably 80-99%.
  • the step a is preferably performed under heating and refluxing conditions.
  • the concentration of the ethanol aqueous solution in the step c is preferably 70-90%.
  • Another object of the present invention is to provide the application of Radix Astragali extract in pharmacy.
  • the medicine containing astragalus extract described in the application is made into an oral preparation.
  • Still another object of the present invention is to provide a method for treating or preventing immune-mediated chronic inflammatory diseases in individuals, and treating or preventing rheumatoid arthritis and / or erythema erythematosus in individuals.
  • the content of total saponins in astragalus extract provided by the invention is not less than 53%, up to 80%, of which astragaloside IV can reach 10%, reliable quality, good stability, low toxicity, high anti-inflammatory activity, provided preparation
  • the method is relatively simple, can effectively remove impurities, retain the astragalus extract to the maximum, and the average yield can reach 1.20-2.0%.
  • the provided astragalus extract has a variety of medicinal effects. Brief description of the drawings
  • Figure 1 shows that astragalus extract inhibits TNFa mRNA expression.
  • the present invention provides an astragalus extract, which contains the following weight percentages of substances: astragalus total saponins 53-80%, astragalus flavonoids 10-25%.
  • an astragalus extract containing the following weight percentage substances is provided: astragalus total saponins 74-80%, Astragalus flavonoids 10-20%.
  • the astragalus extract also contains 10-25% organic acid.
  • the astragalus extract can be prepared by the following steps:
  • the amount of the ethanol aqueous solution in the step a is preferably 6-10 times the weight of the astragalus raw material.
  • the weight ratio of the amount of the extract to the resin in step c may be 1: 5-10, and in some preferred embodiments of the present invention, the weight ratio is 1: 8-10.
  • the extract is at 50 ° C.
  • the relative specific gravity of C heat measurement is 1.10-1.45.
  • the invention also provides a method for preparing astragalus extract, comprising the following steps:
  • the amount of the ethanol aqueous solution used in the step a is 6-10 times the weight of the astragalus raw material.
  • the weight ratio of the amount of the extract to the resin in step c may be 1: 5-10, and in some preferred embodiments of the present invention, the weight ratio is 1: 8-10.
  • the extract is at 50 ° C.
  • the relative specific gravity of C heat measurement is 1.10-1.45.
  • the preparation steps and preparation methods of the astragalus extract may specifically include, but are not limited to, the following operations: a. Astragalus raw material is extracted by heating and refluxing for 1-3 times with 6-10 times the amount of 65-99% ethanol for 1-2 hours each time;
  • the extract is diluted with an equal volume of water and passed through a macroporous resin column.
  • the weight ratio of the amount of the extract to the resin is 1: 5-10.
  • the effluent should be negative for saponin, and then elute with water until the sugar is negative for the eluent. ; Finally elute with 70-90% ethanol, until the eluate is negative for sugar;
  • the astragalus extract is obtained with a yield of 1.20-2.0%.
  • the astragalus extract of the present invention can be used for preparing a medicament for treating or preventing an immune-mediated chronic inflammatory disease.
  • the medicament can be made into an oral preparation.
  • the astragalus extract of the present invention can be used for preparing a medicament for treating or preventing rheumatoid arthritis and / or lupus erythematosus.
  • the medicament can be made into an oral preparation.
  • the present invention provides a method for treating or preventing an immune-mediated chronic inflammatory disease, the method comprising administering to an individual an effective amount of the astragalus extract of the present invention.
  • the effective amount refers to a dose capable of achieving the desired effect of treating or preventing immune-mediated chronic inflammatory diseases.
  • the method comprises administering an effective amount of the yellow extract of the present invention.
  • the effective amount refers to a dose capable of achieving the desired effect of treating or preventing immune-mediated chronic inflammatory diseases.
  • the present invention is further described below through examples.
  • Example 1 Example 1:
  • the astragalus raw material was extracted by heating and refluxing 8 times the amount of 75% ethanol twice for 1 hour each time. The residue was filtered off, the extracts were combined, and the ethanol was recovered and concentrated to 50.
  • the specific gravity of C measured by Hunan is 1.15.
  • the extract is obtained. The extract is added with an equal volume of water. After dilution, it passes through a macroporous resin column. The weight ratio of the extract to the resin is 1: 5. The effluent saponin reaction is negative.
  • the astragalus raw materials were extracted by heating and refluxing twice with 10 times the amount of 90% ethanol for 2 hours each time. The residue was filtered off, the extracts were combined, and the ethanol was recovered and concentrated to 50%. The relative specific gravity of C was 1.35 to obtain an extract.
  • the extract was diluted with an equal volume of water and passed through a macroporous resin column. The weight ratio of the extract to the resin was 1: 10, and the saponin reaction in the effluent was negative. Then washed with water. After removing the eluent, the sugar reaction was negative, and finally the 90% ethanol eluent was used for the sugar reaction.
  • Astragalus extract contains 60% of astragalus saponins, 15% of organic acids, and 15% of astragalus flavonoids.
  • Astragalus raw materials are extracted by heating and refluxing 3 times with 10 times the amount of 85% ethanol for 2 hours each time.
  • the residue is filtered off, the extracts are combined, and the ethanol is recovered and concentrated to 50.
  • the relative specific gravity of C was 1.45, and the extract was obtained.
  • the extract was diluted with an equal volume of water and passed through a macroporous resin column.
  • the weight ratio of the amount of the extract to the resin was 1: 10, and the saponin reaction in the effluent was negative. Then washed with water. After removing the eluent, the sugar reaction was negative. Finally, the 80% ethanol eluent was used to recover the sugar reaction.
  • Astragalus extract contains 65% of astragalus total saponins, 13% of organic acids, and 20% of astragalus flavonoids.
  • the astragalus raw material was extracted by heating and refluxing three times with 70% ethanol for 6 times, each time for 1.5 hours. The residue was filtered, the extracts were combined, and the ethanol was recovered and concentrated to 50 ° C. The specific gravity of the heat was 1.25 to obtain a flow extract.
  • the extract was diluted with an equal volume of water and passed through the D101 column. The weight ratio of the flow extract to the resin was 1: 7, and the saponin reaction in the effluent was negative. Then the column was washed with water until the sugar reaction was negative. Finally, 70% ethanol was used to elute The sugar reaction was negative; the ethanol eluate was recovered, dried, and ground to obtain the astragalus extract with a yield of 2.0%. Astragalus extract contains 53.2% of total astragaloside saponins, 20% of organic acids, and 17% of astragaloside flavonoids.
  • the astragalus raw material was extracted by heating and refluxing 8 times with 75% ethanol 3 times. At 2 'each time, the drug residue was filtered and the extracts were combined. After recovering the ethanol, it was concentrated to 50 ° C and the specific gravity was 1.35.
  • the flow extract is diluted with an equal volume of water and passed through the D101 column. The weight ratio of the flow extract to the resin is 1: 5; the effluent has a negative saponin reaction; the column is washed with water until the sugar reaction is negative, and finally 90 ° / ethanol is used. The sugar reaction of the eluent was negative; the ethanol eluent was recovered, dried, and ground to obtain the yellow extract, and the yield was 1.9%.
  • Astragalus extract contains 63.3% of astragalus saponins, 15% of organic acids, and 15% of flavonoids.
  • Astragalus raw materials are extracted by heating and refluxing 3 times with 6 times the amount of 80% ethanol. At 2 'j, each time, the residue is filtered, the extracts are combined, and the ethanol is recovered and concentrated to 50 ° C. The specific gravity of the heat is 1.40.
  • the flow extract is diluted with an equal volume of water and passed through the D101 column. The weight ratio of the flow extract to the resin is 1: 10, and the effluent is soapy or the reaction is negative. Then the column is washed with water until the sugar reaction is negative, and finally 80 ethanol is used to The eluent had a negative sugar reaction; the ethanol eluent was recovered, dried, and ground to obtain the astragalus extract (1123), with a yield of 1.85%. Astragalus extract (1123) contains 74.9% of astragalus total saponins, 15% of organic acids, and 10% of astragalus flavonoids.
  • Astragalus raw material was extracted by heating and refluxing three times with 10 times the amount of 95% ethanol for 1.5 hours each time. The residue was filtered and the extracts were combined. The ethanol was recovered and concentrated to 50 ° C. The specific gravity was 1.30, and the flow extract was obtained. The flow extract is diluted with an equal volume of water and passed through the D101 column. The weight ratio of the flow extract to the resin is 1: 8; the effluent saponin reaction is negative; then the column is washed with water until the sugar reaction is negative, and finally 90% ethanol is used to wash. The dehydration sugar reaction was negative; the ethanol eluent was recovered, dried, and ground to obtain the astragalus extract with a yield of 1.75%.
  • Astragalus extract contains 79.1% of astragalus total saponins, 10% of organic acids, and 10% of astragalus flavonoids.
  • Example 8 Preparation of Astragalus Extract according to literature method (Han Lujia, Yan Qiaojuan, etc .: Journal of Agricultural Engineering, 16 (5), 118, 2000)
  • the astragalus raw material was extracted by heating and refluxing 6 times with 60% ethanol 3 times for 2 hours each time. The residue was filtered and the extracts were combined. After the ethanol was recovered, it was concentrated to 50 ° C and the specific gravity was 1.30. The extract was diluted with an equal volume of water and passed through the AB-8 column. The weight ratio of the flow extract to the resin was 1: 10, and the effluent solution had a negative saponin reaction. Then the column was washed with double the column volume of water and finally washed with 95% ethanol. The dehydration sugar reaction was negative; the ethanol eluent was recovered, dried, and ground to obtain the astragalus extract (15803), with a yield of 2.3%.
  • Astragalus extract (15803) contains 49.2% of total astragaloside, 8.3% of organic acid, and 7.1% of astragaloside.
  • Example 9 According to the inhibitory activity of astragalus extract on the expression of precursor inflammatory factor TNF cx mRNA, the anti-inflammatory activity of extracts from different processes was compared.
  • Astragalus membranaceus extract commercially available astragalus medicinal materials, respectively prepared according to the method of Example 6 (extract 1123) and the method of Example 8 (extract 15803); dissolved by adding 10% dimethylsulfite (DMSO), The storage solution concentration was 10 mg / ml.
  • DMSO dimethylsulfite
  • Normal human peripheral blood mononuclear cells take healthy human peripheral blood, heparin (62.5 U / ml) anticoagulated, Ficoll isolates blood cells, and resuspend the cells in IMDM medium containing 10% fetal bovine serum.
  • bDNA Nucleic Acid Quantification Kit GenoSpectra, USA, trade name is QuantiGene Kits. ProbeDesigner Software (Bayer, Germany) was used to design the nucleic acid probe against the TNF a mRNA sequence.
  • the probe sequence is as follows: Table A TNF a mRNA bDNA nucleic acid probe
  • TNFa32 906-925 BL aataaagggattggggcagg
  • TNFa33 926-945 BL gggtgtctgaaggagggggt
  • hTNFa34 1008-1031 BL cgaagtggtggtcttgttccttaa
  • Other reagents Cell culture medium and fetal bovine serum Gibco's products; Endotoxin (LPS), dexamethasone (DEX) and DMSO are products of Sigma, USA Real method:
  • TNF- ⁇ tumor necrosis factor
  • Freshly extracted human peripheral blood mononuclear cells were suspended in IMDM medium containing 10% fetal bovine serum, counted by placental blue staining exclusion method, and adjusted the cell suspension density to 3 X 10 3 cells / ml. In a round bottom 96-well culture plate, add 100 ⁇ l of cell suspension to each well, and the total number of cells in each cell is 2 ⁇ 10 4 .
  • the medicine of the present invention (hereinafter referred to as astragalus total soap ring) was prepared according to Example 6, and was used in vivo, and was formulated with 1% sodium carboxymethyl cellulose (CMC-Na). It was used in vitro, dissolved in dilute ethanol, and then used for nutrition. Dilute the solution to the required concentration (final ethanol concentration is less than 1%.).
  • Data are expressed as mean ⁇ S.
  • the t-test was used to compare the sample mean between the measurement data groups; the X 2 test was used to compare the count data.
  • Animals Wistar rats, both male and female, 2-3 months old, weighing 1.60 ⁇ 10s; BALB / C mice, both male and female, 6-8 weeks of age, weighing 18 ⁇ 2s: C 57 BL / 6J mice, Male, 6-8 weeks old, weighing 18 ⁇ 2s: Kunming mice, 7-8 weeks old, weighing 20 ⁇ 2s.
  • the real face is generally divided into three control groups (normal control, model control, positive control, and test drug) (some exceptions).
  • the overall experimental dose range is 5-100mg'kg, divided into low, medium and high dose groups according to 1: 2-1: 4 dose intervals, administered intragastrically (ig), normal and model control group ig 1% CMC -Na, equal capacity.
  • Positive control drugs were indomethacin (Indo); dexamethasone (Dex). Astragaloside (ASI); Tripterygium glycoside (TS); Gynostemma pentaphyllum (GPS); Ginsenoside (GS), etc. The choice depends on different models.
  • do is the day of creation, d-n ⁇ n or d-i! ⁇ + N Among -n is n days before modeling, and n or + n is 11 days after modeling.
  • FCA Freund's Complete Adjuvant
  • Each mouse was injected subcutaneously with 1% carrageenan (prepared by NS) in the left hindfoot plantar to induce inflammation. Paw pits were detected before and 3, 5 and 7 h after inflammation, and the difference between the volume before and after inflammation was taken as the swelling.
  • the plate was coated with 20 / zg'mr 1 type II collagen (C II) at 100 / d per well. 4 . C overnight, washed 3 times with 2% BSA 37. Incubate for 1 h at C, wash 3 times, add 1:40 dilution of the test solution, incubate at 37 ° C for 1 h, wash 3 times; add 1: 200 diluted # sheep anti-rat IgG-HRP 100 ⁇ , 37 ° Incubate for 1 h at C, wash 3 times; add substrate solution, protect from light at room temperature for 20 min, and stop the reaction with 2mol ⁇ L— 1 H 2 S0 4 . The measurement was performed at a wavelength of 492 nm in a microplate reader, and the results were expressed as the average A value of three complex wells.
  • the anticoagulated tail vein blood of each mouse was divided into 4 tubes of 0.1 ml each. Add 0.1ml OX2 1 (anti-human C3b, blank control), OX19 (CD3), W3 / 25 (CD4) and OX8 (CD8) monoclonal antibodies (blessed by Professor Puklavec, University of Oxford, UK) with 2% FCS-RPMI 1640 Make 1: 5 dilution, 4. C Warm for 40 min, add cell wash solution (2% FCS-RPMI 1640 medium). Each tube was 3ml, mixed well, centrifuged (1500rpm, 5min), and washed 3 times.
  • synovial membranes of the bilateral knee joints of the rats were aseptically removed, and the individual cells were broken down with type II collagenase and trypsin.
  • DMEM culture medium formulated synovial cell suspension (5xl0 5. Mr 1), added to 24-well culture plate, lml of each well, 37. C and 5% CO 2 were cultured for 48 h. Cell culture supernatants were collected and tested for IL-I activity and No 2 -content.
  • Group dosage Amount of granuloma (g / 100g body weight)
  • AA rats have severe synovitis (hyperemia of synovial tissue, edema and inflammatory cell proliferation, and hyperplasia of synovial tissue) on both knee and ankle joints, articular cartilage Damage (atrophy, thinning, or disappearance), and osteomyelitis (inflammation of inflammatory cells in the bone marrow cavity).
  • AST can significantly alleviate the above pathological changes.
  • Dex has a stronger effect on inhibiting secondary inflammatory reactions in AA rats than AST, the degree of lesion reduction is not as good as the latter.
  • the result of failure 5 showed that after the administration of AST, it could restore the thymus coefficient of AA rats, but had no significant effect on the spleen coefficient.
  • the results in Table 6 show that AST can reduce the proliferation of ConA in spleen cells of AA rats and restore normal levels of IL-1 secretion from excessive IL-1; Dex reduced both indicators.
  • the results in Table 7 show that AST can significantly reduce the excessive secretion of IL-I and NO by synovial cells of AA rats, while Dex reduces both to below normal levels.
  • FCA causes inflammation, dl2 ⁇ 19 ig solvent or drug.
  • x ⁇ S, n 10, * p ⁇ 0.05, ** p ⁇ 0.01, compared with AA control Table 5
  • ⁇ organ coefficient was: g weight AOOg organ weight, d 0 with FCA-induced inflammation, d 12 -i 9 ig solvent or medicament, d 24 taken out of each group The thymus and spleen of each rat were weighed.
  • X ⁇ S, n 10. * p ⁇ 0.05, ** p ⁇ 0.01, compared with the normal control group; "" p ⁇ 0.01 compared with the AA control.
  • mice were sensitized with a 1% dinitrofluorobenzene (DNFB) acetone solution in 25 ⁇ 1 and intensified once the next day.
  • DNFB dinitrofluorobenzene
  • the right ear was coated with 1% DNFB 10 ⁇ l and challenged. After 24 hours, the mice were sacrificed. Each of the left and right ear pieces with a diameter of 8 mm was weighed. The difference between the weights of the two ear pieces was taken as the swelling degree.
  • cyclophosphamide Cy, 180mg ⁇ kg- ⁇ 3 days (d 3) prior to or sensitized ip Cy (250mg ⁇ kg)
  • the mice can induce DTH responses were lower and higher.
  • mice BALB / ip C mice 10% SRBC suspension 0.2ml (4xl0 8 cells / mouse) sensitization, each mouse simultaneously sc Cy 80 mg ⁇ kg in mice induced immune function.
  • a spleen cell suspension (K mr 1 cells) was prepared routinely. On a 96-well culture plate, add 100 ⁇ l of cell suspension (final concentration 5 ⁇ 10 6 ⁇ mr 1 ), 50 ⁇ ⁇ ConA (final concentration ⁇ ml) or LPS (final concentration 6 / xg-ml -1 ) to each well, and different Concentration AST or nutrient solution, at 37. C, 5% C0 2 and saturated humidity for 48h, 6h before termination, add [ 3 H] TdR (1.4xl0 4 Bq) to each well. After the culture was completed, cells were harvested, and the radioactivity was measured with a FJ-2107 liquid scintillation counter (produced by Xi'an 262 Factory), and the results were expressed as the average value of cpm of 3 multiple holes.
  • Rat peritoneal cell suspension (2 ⁇ 10 ⁇ ⁇ ⁇ 1 ) was routinely prepared with RPMI 1640 medium »added 24 In a well culture plate, set 1 ml / well at 37. C and 5% CO 2 were cultured for 2 h to obtain monolayer macrophages (M ⁇ ), and 0.8 ml of RPMI 1640 culture solution, 100 ⁇ L of LPS (final concentration. Mr 1 ), and AST or nutrient solution of 100 ⁇ l in different concentrations were added, and set at 37. C, 5% CO 2 for 6 h, discard the nutrient solution containing the drug and LPS, and wash the cells twice with the pre-warmed nutrient solution.
  • mice thymocyte proliferation method was used to express the activity of IL-1 in terms of [ 3 H] TdR incorporation.
  • Routine preparation of rat spleen cell suspension ( ⁇ 7 ⁇ ⁇ 1 ) »On a 24-well culture plate, add 0.5 ml of cell suspension, 0.25 ml of ConA (final concentration ⁇ ml — 1 ), and different concentrations of drugs or nutrition Solution (final volume 1 ml). ⁇ 37 ⁇ Set 37. C, 5% CO 2 for 6 h. Wash the cells twice with Hank's solution containing 5% calf serum to remove the drug and ConA, reconstitute the cell suspension, 1ml per well, and continue to culture for 48h. After the incubation, collect the supernatant at -20. CSave the test. The activated mouse spleen cell method was used to express the activity of IL-2 in terms of [ 3 H] TdR incorporation.
  • Group 12 and ig were administered according to Table 12 for 7 consecutive days, and the proliferation and response of ConA and LPS of spleen lymphocytes of each group were measured in vitro at d 8 .
  • the results showed that the three doses of AST had no significant effect on the proliferation response of ConA and LPS in splenocytes of normal mice.
  • Table 12 Effects of AST on mitogen response of splenic lymphocytes in normal mice [ 3 H] TdR incorporation (cpmx 10- "
  • AS1 Astragalus saponin tincture, dO ip Cy 180mg ⁇ kg -1 ; d0 ⁇ 4ig solvent or medicine.
  • GS ginsenoside, d-3 ip Cy 250 mg ⁇ kg " 1 ; d-3 ⁇ d + 4 ig solvent or drug
  • d 5 was used to detect the amount of IgM produced by spleen cells in each group of mice.
  • the results showed that AST had a dose-dependent antagonistic effect on Cy inhibiting IgM production in mice, and the effect in the high-dose group was significantly better than total gypenosides (GPS :). Effect of AST on Cytosolic IgM Production in Mouse Spleen Cells
  • AST can significantly promote the suboptimal dose of ConA (3 / g ⁇ ml) and LPS (6 / g ⁇ ⁇ 1 ) were induced proliferative response of mouse spleen lymphocyte and a suboptimal dose of LPS-induced promotion of large Murine peritoneal macrophages produce IL-1, and suboptimal ConA induces rat splenocytes to produce IL-2.
  • Their dose-response curves are bell-shaped. It shows that AST has a concentration-dependent two-way regulation effect on the proliferation or secretion function of three immune cells.
  • Experiment 3 Analgesic effect of astragalus saponins on mice
  • the qualified female mice were placed at 55 ⁇ 0.5.
  • C The metal plate of the water bath takes the mouse's hind leg as a signal of pain response, and the latency of the pain response is used as an indicator of pain severity.
  • Hot plate method is divided into groups and administered according to Table 16. The latency of pain response was measured before and 2h, 4h and 6h after administration. All three doses of AST significantly prolonged the pain latency, with effects occurring 2 h after administration, peaking at 4 h, and then decreasing. Table 16 Analgesic effect of AST (mouse hot plate method) Latent period (s)
  • AST also has a significant effect on the pain model of mice caused by heat (hot plate method) and acetic acid (twisted body method). Pain effect.
  • AST has both anti-inflammatory and analgesic effects and function-dependent two-way immunomodulatory effects.
  • the in vitro experiments on the function of major immune cells show that AST can promote the proliferation response of mouse spleen lymphocytes induced by suboptimal ConA and LPS, and the production of IL-1 and IL-2 in the abdominal cavity of rats. It is bell-shaped. It shows that AST has a concentration-dependent two-way regulation effect on the proliferation and secretion function of the above immune cells. Studies have shown that AST is an anti-inflammatory immunomodulator with both immunomodulatory and anti-inflammatory and analgesic effects.
  • AST not only indicate that it can be used for the treatment of immune-mediated chronic inflammatory diseases such as rheumatoid arthritis and / or lupus erythematosus, but also can be widely used to correct various immune dysfunctions.
  • AST with a content of 54.40%, was prepared with a 1% sodium carboxymethylcellulose sodium solution and ground to prepare a suspension with a maximum concentration of 125 mg ⁇ ml "for ig administration.
  • AST was prepared with dilute ethanol and the maximum dissolved concentration was 10 mg ⁇ Ml " 1 , ethanol concentration below 10%, for ip administration.
  • mice Forty Kunming mice, weighing 20 ⁇ lg, male and female, were divided into two groups of 20 rats each.
  • ip AST 250mg 'kg (equivalent to 16.65g ⁇ kg of crude drug — ⁇ observe the appearance, behavior, mental state, breathing, body temperature of mice immediately and continuously for 14 days after administration , Manure and daily feed consumption.
  • ig AST 5g ⁇ kg 1 or ip AST 250mg ⁇ kg 1 had no significant effect on the weight gain of mice at 7 and 14 days, and had normal posture, shiny hair, behavioral activity, mental state, breathing, body temperature, and feces were normal. None of the animals died.
  • mice 60 Wistar rats, 7-8 weeks old, female rats weighing 150 ⁇ s 15g, male rats weighing 170 ⁇ s 15go, rats were randomly divided into 3 groups of 20 rats each, male and female, which is low in the present invention High-dose group (ig astragalus total saponin 90mg ⁇ kg ' 1 , d " 1 and 900mg ⁇ kg" 1. (T 1 ) and a control group (i g 1% carboxymethyl cellulose sodium solution). Ig drug or vehicle was started at 8:00 am every day for 90 consecutive days. During the experiment, the behavior, activity, hair, skin color, stool, and breathing of the rats were observed daily. The food intake was weighed once a day and the weight was weighed once a week.
  • High-dose group ig astragalus total saponin 90mg ⁇ kg ' 1 , d " 1 and 900mg ⁇ kg" 1.
  • T 1 a control group
  • Ig drug or vehicle was started at 8:00 am every day for
  • the dose was adjusted according to the change in body weight. After 90 days of administration, 1/2 of the rats in each group was collected for blood testing, and the femoral artery was sacrificed to sacrifice the rats. The blood was collected to determine the blood biochemical indicators.
  • Stomach in the pylorus, cardia, the small curve and the large curve are taken from each of the four tissues, and other organs are taken from the same tissue. 10% neutral formalin solution, conventional embedding, sectioning, and staining.
  • n 10, A; ⁇ S. Compared with the control group, p> 0.05.
  • ATS aspartate aminotransferase
  • ALT alanine aminotransferase
  • ALP alkaline phosphatase
  • BUN urea nitrogen
  • TP total protein
  • ALB albumin
  • GLU blood glucose T-BIL: total bilirubin

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Abstract

La présente invention concerne un extrait de radix astragli, un procédé pour le produire, ainsi que l'utilisation de celui-ci. Un tel extrait de radix astragli comprend principalement de la saponine d'astragalus, un acide organique et un glycoside flavonoïde d'astragalus. Le procédé pour le produire consiste à effectuer une extraction à reflux à chaud des matières premières d'astragalus dans de l'alcool, à effectuer une concentration afin d'obtenir l'extrait, à diluer l'extrait avec de l'eau, à le faire passer à travers une colonne en résine macroporeuse, jusqu'à ce que l'effluent soit négatif à la réaction de saponine, puis à effectuer l'élution avec de l'eau, jusqu'à ce que l'éluent soit négatif à la réaction de saccharide, à effectuer l'élution avec de l'alcool, jusqu'à ce que l'éluent soit négatif à la réaction de saccharide, à récupérer l'alcool, puis à effectuer un séchage et un écrasement, de manière à obtenir l'extrait de radix astragli. La présente invention concerne également l'utilisation d'un tel extrait de radix astragli pour produire divers médicaments.
PCT/CN2005/000216 2004-02-23 2005-02-23 Extrait de radix astragli, procede pour le produire et utilisation de celui-ci WO2005084689A1 (fr)

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CN107812120A (zh) * 2017-11-28 2018-03-20 孙志强 一种用于治疗前列腺炎疾病的复方中药组合物
CN112076244A (zh) * 2020-09-27 2020-12-15 内蒙古大唐药业股份有限公司 蒙古黄芪有效成分的提取方法
CN114748490B (zh) * 2022-04-24 2023-12-12 南方科技大学 一种用于治疗卵巢早衰的药物组合物、应用及其制备方法
CN116637134B (zh) * 2023-06-12 2024-10-08 云南中医药大学 一种不丹黄芪提取物及其在制备治疗慢性咽炎药物中的应用

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CN1430966A (zh) * 2003-01-10 2003-07-23 安徽天洋药业有限公司 一种含黄芪有效部位的药物组合物
CN1444949A (zh) * 2003-04-29 2003-10-01 中国药科大学 一种防治心血管病的中药黄芪有效部位
CN1470241A (zh) * 2002-07-22 2004-01-28 江苏正大天晴药业股份有限公司 黄芪甲甙药物组合物及其制备方法

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CN1425674A (zh) * 2002-12-19 2003-06-25 上海博泰医药科技有限公司 黄芪总皂苷的制备方法
CN1430966A (zh) * 2003-01-10 2003-07-23 安徽天洋药业有限公司 一种含黄芪有效部位的药物组合物
CN1444949A (zh) * 2003-04-29 2003-10-01 中国药科大学 一种防治心血管病的中药黄芪有效部位

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103630500A (zh) * 2013-11-29 2014-03-12 广西医科大学 树脂脱色用于比色法测定α-淀粉酶抑制剂活性的方法

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