CN111150751A - Method for preparing ormosia angustifolia extract, ormosia angustifolia extract and application thereof - Google Patents
Method for preparing ormosia angustifolia extract, ormosia angustifolia extract and application thereof Download PDFInfo
- Publication number
- CN111150751A CN111150751A CN202010101778.7A CN202010101778A CN111150751A CN 111150751 A CN111150751 A CN 111150751A CN 202010101778 A CN202010101778 A CN 202010101778A CN 111150751 A CN111150751 A CN 111150751A
- Authority
- CN
- China
- Prior art keywords
- ormosia
- treatment
- angustifolia extract
- solid
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/02—Breeding vertebrates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/20—Animals treated with compounds which are neither proteins nor nucleic acids
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/035—Animal model for multifactorial diseases
- A01K2267/0368—Animal model for inflammation
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
Abstract
The invention belongs to the field of medicines, and particularly relates to a method for preparing a ormosia angustifolia extract, which comprises the following steps: (1) extracting semen Adenantherae Pavoninae with water, and separating solid and liquid to obtain liquid phase; (2) salting out the liquid phase substance to obtain a mixture; (3) carrying out solid-liquid separation on the mixture to obtain a solid-phase substance; (4) dissolving the solid phase substance in water to obtain a solution; (5) dialyzing the solution, and collecting the permeate; wherein, a dialysis membrane with the aperture of 1000-20000 daltons is adopted for dialysis treatment; (6) carrying out chromatography treatment on the permeate, and collecting eluent, namely the ormosia angustifolia extract; wherein, DEAE sepharose gel column is adopted for chromatography treatment. The invention also relates to the prepared ormosia angustifolia extract and application thereof. The ormosia angustifolia extract can establish an inflammatory granuloma animal model by inducing inflammatory granuloma, has short induction period and good repeatability, and the animal model can be used for evaluating the curative effect of anti-inflammatory drugs.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to a method for preparing a ormosia angustifolia extract, the prepared ormosia angustifolia extract and a medical application thereof.
Background
The red beans (Adenantha pavonina Linn. var. micosperma), namely the five-color red beans, the red ormosia, the star-shaped large-eyed rosewood, the red beans, the red wood, the red globe of the red globe, the peacock beans, the amphibian trees, the jequirity, the acacia, the vermilion, the alopecurus deciduous tree, the high red beans, the tender branches, the two curly feathers, the leaf stalks and the leaf axes are slightly tender hair without glands, the plumes are 3-5 pairs, the small leaves are 4-7 pairs, the long round or long stems are 2.5-3.5 cm long and 1.5-2.5 cm wide, the blunt hair at both ends, the short stems, the general flowering heads are singly built in the oval leaves or arranged in the tops of the cones, the small flowers, the white or yellow flowers, the stems of the flowers, the long fruits, the stems of the flowers, the small-shaped stems of the roses, the red stems of the wild roses, the stems of the roses, the stems of the roses, the stems of the roses of the stems of the roses.
Inflammatory granuloma refers to a locally formed nodular chronic inflammatory lesion with clear boundaries mainly composed of macrophages and inflammatory cells, and is one of the important models for evaluating anti-inflammatory drugs. They are classified into heteroplastic granuloma and infectious granuloma according to inflammatory factors. At present, carrageenan, agar, plastic, croton oil, ganoderma lucidum spore powder cotton balls and the like are commonly used for inducing heterogenic granuloma; infectious granulomas were induced by subcutaneous injection of bcg. The foreign body induced granuloma has long period and irregular shape, and is difficult to detect and quantify; the induction of granuloma by BCG not only has long period, but also has cheese necrosis in the center of the granuloma tissue and poor repeatability.
At present, a method for constructing an inflammatory granuloma animal model with short induction period and high repeatability is needed to be used as a model for evaluating an anti-inflammatory drug.
Abrus (Abrus precatorius) of Abrus genus, which contains a highly toxic protein (isoabrins) with a molecular weight of 6.3-6.7 ten thousand daltons. The ormosia and the abrus belong to different species, and the academic names and chemical components of the ormosia and the abrus are completely different.
Disclosure of Invention
The invention provides a method for preparing a ormosia angustifolia extract, wherein the prepared ormosia angustifolia extract can establish an inflammatory granuloma animal model by inducing inflammatory granuloma to be used as a model for evaluating an anti-inflammatory drug. On the basis, the invention also provides a ormosia angustifolia extract and a medical application thereof.
The invention relates to a method for preparing a ormosia angustifolia extract, which comprises the following steps:
(1) extracting semen Adenantherae Pavoninae with water, and separating solid and liquid to obtain liquid phase;
(2) salting out the liquid phase substance to obtain a mixture;
(3) carrying out solid-liquid separation on the mixture to obtain a solid-phase substance;
(4) dissolving the solid phase substance in water to obtain a solution;
(5) dialyzing the solution, and collecting the permeate; wherein, a dialysis membrane with the pore diameter of 1000-20000 daltons (such as 2000 daltons, 3000 daltons, 4000 daltons, 5000 daltons, 7000 daltons, 9000 daltons, 10000 daltons, 12000 daltons, 14000 daltons, 16000 daltons and 18000 daltons) is adopted for dialysis treatment;
(6) carrying out chromatography treatment on the permeate, and collecting eluent, namely the ormosia angustifolia extract; wherein, DEAE sepharose gel column is adopted for chromatography treatment.
In some embodiments of the first aspect of the present invention, in the step (4), the volume ratio of the solid phase to the water is 1 (1-6), for example, 1:2, 1:3, 1:4, 1: 5.
In certain embodiments of the first aspect of the present invention, in step (6), the elution is performed with Tris-HCl buffer followed by elution with sodium chloride solution.
In some embodiments of the first aspect of the present invention, in step (6), the weight ratio of the Tris-HCl buffer solution to the sodium chloride solution is 1 (0.7-2), such as 1:0.8, 1:1, 1: 1.5.
In certain embodiments of the first aspect of the present invention, the sum of the weight of the Tris-HCl buffer and the sodium chloride solution in step (6) is 1 to 8 times, e.g., 2, 3, 4, 5, 6, 7 times the weight of the water in step (4).
In some embodiments of the first aspect of the present invention, in step (6), the entire eluate is collected as the ormosia angustifolia extract.
In some embodiments of the first aspect of the present invention, the method has one or more of the following features a to S:
A. in the step (1), the weight ratio of water to ormosia is (1-10) to 1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1 and 9: 1;
B. in step (1), leaching is carried out at 1-17 deg.C, such as 2 deg.C, 3 deg.C, 4 deg.C, 5 deg.C, 6 deg.C, 7 deg.C, 8 deg.C, 9 deg.C, 10 deg.C, 12 deg.C, 14 deg.C, 16 deg.C, 15 deg.C;
C. in the step (1), leaching lasts for 0.5-5 hours, such as 1 hour, 2 hours, 3 hours and 4 hours;
D. in step (1), leaching is carried out one or more times, preferably three times, such as 1, 2, 3 times;
E. in the step (1), solid-liquid separation is performed by filtration, preferably by filtration through filter paper;
F. in the step (2), before salting out treatment, concentrating the liquid phase substance, and collecting the concentrate;
preferably, after concentration and before salting-out treatment, the concentrate is subjected to centrifugal separation, and the supernatant is collected for salting-out treatment;
preferably, concentration is performed at 1-17 deg.C, such as 2 deg.C, 3 deg.C, 4 deg.C, 5 deg.C, 6 deg.C, 7 deg.C, 8 deg.C, 9 deg.C, 10 deg.C, 12 deg.C, 14 deg.C, 16 deg.C, 15 deg.C;
preferably, the concentration is 0.1 to 0.9 times, e.g., 0.2 times, 0.3 times, 0.4 times, 0.5 times, 0.6 times, 0.7 times, 0.8 times, the original volume;
more preferably, the rotational speed of the centrifugal separation is 1000 to 10000rpm, such as 2000rpm, 3000rpm, 4000rpm, 5000rpm, 6000rpm, 7000rpm, 8000rpm, 9000 rpm;
more preferably, centrifugation lasts 2 to 40 minutes, such as 5 minutes, 8 minutes, 10 minutes, 12 minutes, 14 minutes, 16 minutes, 18 minutes, 20 minutes, 22 minutes, 24 minutes, 26 minutes, 28 minutes, 30 minutes, 32 minutes, 34 minutes, 36 minutes, 38 minutes, 39 minutes;
G. in the step (2), salting-out treatment is carried out by adopting at least one salt selected from ammonium sulfate, sodium sulfate and sodium chloride, preferably ammonium sulfate;
H. in the step (2), salting-out treatment is carried out until the solubility of salt (in a liquid phase) reaches saturation;
I. in the step (3), before solid-liquid separation, the mixture is allowed to stand at 1 to 17 ℃ (e.g., 2 ℃, 3 ℃, 4 ℃, 5 ℃, 6 ℃, 7 ℃, 8 ℃, 9 ℃, 10 ℃, 12 ℃, 14 ℃, 15 ℃, 16 ℃);
preferably, the standing lasts for 0.5 to 9 hours, such as 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours;
J. in the step (3), solid-liquid separation is carried out through centrifugal treatment;
preferably, the rotation speed of the centrifugal treatment is 1000 to 10000rpm, such as 2000rpm, 3000rpm, 4000rpm, 5000rpm, 6000rpm, 7000rpm, 8000rpm, 9000 rpm;
preferably, the centrifugation treatment lasts 2 to 40 minutes, such as 5 minutes, 8 minutes, 10 minutes, 12 minutes, 14 minutes, 16 minutes, 18 minutes, 20 minutes, 22 minutes, 24 minutes, 26 minutes, 28 minutes, 30 minutes, 32 minutes, 34 minutes, 36 minutes, 38 minutes, 39 minutes;
K. in the step (5), the dialysis treatment lasts for 20 to 80 hours, such as 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours, 48 hours, 50 hours, 52 hours, 54 hours, 56 hours, 58 hours, 60 hours, 62 hours, 64 hours, 66 hours, 68 hours, 70 hours, 72 hours, 74 hours, 76 hours, 78 hours;
l. in the step (5), the solute concentration in the permeate is 1-10 mg/mL, such as 2, 3, 4, 5, 6, 7, 8, 9 mg/mL;
m, filtering the permeation solution before chromatography in the step (6);
preferably, a filter membrane with a pore size of 0.2-0.8 μm (e.g., 0.4 μm, 0.6 μm) is used for filtration;
n. in the step (6), the specification of the DEAE sepharose column is phi 35mm multiplied by L450 mm;
o. in the step (6), the DEAE-Sepharose column has a volume of 100 to 600mL, for example, 150mL, 200mL, 250mL, 300mL, 350mL, 400mL, 450mL, 500mL, 550 mL;
p. in step (6), the concentration of Tris-HCl buffer is 10-100 mM, such as 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90 mM;
in the step (6), the pH value of the Tris-HCl buffer solution is 7-8, such as 8;
in the step (6), the concentration of the sodium chloride solution is 0.1-1M, such as 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M;
s, in the step (6), a Tris-HCl buffer solution is adopted to balance a DEAE sepharose column;
preferably, the DEAE sepharose column is equilibrated with at least 2 column volumes (e.g., at least 3 or at least 4 column volumes) of Tris-HCl buffer;
preferably, the pH value of the Tris-HCl buffer is 7-8, such as 8;
preferably, the concentration of Tris-HCl buffer is 10-100 mM, such as 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90 mM.
In a second aspect, the present invention relates to a red bean extract, which is obtained by the method according to the first aspect of the present invention.
In some embodiments of the second aspect of the present invention, the ormosia extract comprises a protein and a polysaccharide.
In some embodiments of the second aspect of the present invention, the ormosia extract comprises a glycoprotein.
In some embodiments of the second aspect of the present invention, the extract of ormosia comprises a first glycoprotein having a weight average molecular weight of 20 to 32KD (e.g. 28KD) and/or a second glycoprotein having a weight average molecular weight of 37 to 43KD (40 KD).
The third aspect of the present invention relates to a pharmaceutical composition comprising the ormosia angustifolia extract according to the second aspect of the present invention.
In some embodiments of the third aspect of the present invention, the pharmaceutical composition further comprises a pharmaceutical excipient. Such as pharmaceutical excipients, which are conventional in the pharmaceutical field.
The fourth aspect of the present invention relates to the use of an ormosia angustifolia extract according to the second aspect of the present invention or a pharmaceutical composition according to the third aspect of the present invention for the preparation of a medicament for inducing inflammatory granuloma.
In some embodiments of the fourth aspect of the present invention, the inflammatory granuloma is an infectious granuloma.
The fifth aspect of the present invention relates to a method for establishing an inflammatory granuloma animal model, comprising the steps of:
obtaining an extract of ormosia according to the second aspect of the invention or a pharmaceutical composition according to the third aspect of the invention;
injecting the ormosia angustifolia extract of the second aspect of the present invention or the pharmaceutical composition of the third aspect of the present invention into a test animal.
In some embodiments of the fifth aspect of the present invention, the experimental animal is a rat.
In some embodiments of the fifth aspect of the invention, the injection is subcutaneous.
In some embodiments of the fifth aspect of the invention, the injected dose per animal is 20-150. mu.g/kg, such as 30. mu.g/kg, 40. mu.g/kg, 50. mu.g/kg, 60. mu.g/kg, 70. mu.g/kg, 80. mu.g/kg, 90. mu.g/kg, 100. mu.g/kg, 110. mu.g/kg, 120. mu.g/kg, 130. mu.g/kg, 140. mu.g/kg.
In some embodiments of the fifth aspect of the invention, the inflammatory granuloma is formed in the subject on days 3-11 of injection (e.g., days 4, 5, 6, 7, 8, 9, 10). Wherein the day of injection is day 1 of injection.
In some embodiments of the fifth aspect of the invention, the inflammatory granuloma is an infectious granuloma.
The sixth aspect of the present invention relates to a kit comprising the ormosia angustifolia extract of the second aspect of the present invention or the pharmaceutical composition of the third aspect of the present invention, and a syringe.
In some embodiments of the sixth aspect of the present invention, the syringe is a syringe for subcutaneous injection.
The seventh aspect of the present invention relates to the use of the ormosia angustifolia extract according to the second aspect of the present invention, the pharmaceutical composition according to the third aspect of the present invention, or the kit according to the sixth aspect of the present invention for establishing an animal model of inflammatory granuloma or for evaluating the efficacy of a drug for treating inflammatory granuloma.
In some embodiments of the seventh aspect of the present invention, the inflammatory granuloma is an infectious granuloma.
In some embodiments of the seventh aspect of the present invention, the animal is a rat.
In some embodiments of the seventh aspect of the invention, the agent for treating inflammatory granuloma includes, but is not limited to, dithetasone and lisinopyrin.
In the invention, the Tris-HCl buffer solution is a Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer solution.
In the present invention, the sodium chloride solution is a sodium chloride aqueous solution.
In the present invention, unless otherwise specified, wherein:
the term "salting out" refers to a process in which a large amount of electrolyte (e.g., inorganic salts) is added to a solution, and strong hydration is generated due to a large amount of ions, with the result that an originally hydrated compound (e.g., a polymer compound) is dehydrated to cause coagulation.
The term "dialysis" refers to a process of separating molecules of different sizes using a semi-permeable membrane. A semi-permeable membrane is a type of membrane having a pore size. Molecules smaller than their pore size can pass freely. Molecules larger than their pore size cannot pass through.
The term "chromatography" refers to a technique that uses different proportions of substances in a stationary phase to a mobile phase to achieve separation.
The term "DEAE sepharose" is a weak anion exchange medium formed by bonding diethylaminoethyl groups on a fast flow rate sepharose.
The invention has the following beneficial effects:
the ormosia angustifolia extract prepared by the method can establish an inflammatory granuloma animal model by inducing inflammatory granuloma, the induction period is short, the repeatability is good, and the established inflammatory granuloma animal model can be used for evaluating the curative effect of anti-inflammatory drugs.
Drawings
In order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the embodiments of the present disclosure taken in conjunction with the accompanying drawings, in which
FIG. 1 is a diagram showing a gel electrophoresis pattern of a sample (LY) in Experimental example 1 of the present invention;
FIG. 2 is a photograph showing granuloma in Experimental example 2 of the present invention;
FIG. 3 is a pathological section of granuloma in Experimental example 2 of the present invention.
Detailed Description
EXAMPLES preparation of test article (LY)
(1) Adding distilled water in an amount which is 3 times the weight of the red ormosia into 1kg of the red ormosia, extracting for 1 hour at 4 ℃, and filtering to respectively obtain a liquid phase substance and a solid phase substance; repeating the extraction operation twice on the solid phase substance, and combining the liquid phase substances obtained three times;
(2) concentrating the combined liquid phase at 4 deg.C to 1/2 to obtain concentrate;
(3) centrifuging the concentrate at 5000rpm for 20 min, and collecting supernatant;
(4) adding ammonium sulfate into the supernatant at room temperature, and mixing until the ammonium sulfate in the liquid is saturated to obtain a mixture;
(5) standing the mixture obtained in the step (4) at 4 ℃ for 1 hour, then carrying out centrifugal separation at the rotating speed of 5000rpm for 20 minutes, and collecting precipitates;
(6) dissolving the precipitate in 200mL of water, wherein the volume ratio of the precipitate to the water is 1:3, so as to obtain a solution;
(7) dialyzing the solution at room temperature for 48 hours, wherein the dialysis aperture is 5000-10000 daltons, and collecting the permeation solution; the solute concentration in the permeate was about 3mg/mL as determined by an ultraviolet spectrophotometer (Shimadzu ultraviolet-visible spectrophotometer UV-1900);
(8) filtering the permeate with a filter membrane with the pore diameter of 0.4 mu m, and collecting the filtrate;
(9) loading 300mL of DEAE Sepharose into a column of 35 mM. times.L 450mM, and equilibrating the column with at least 4 column volumes of Tris-HCl (50 mM, pH 8.0); then, the filtrate is chromatographically separated by adopting the chromatographic column, 300g of Tris-HCl solution (with the concentration of 50mM and the pH value of 8.0) is firstly eluted, then 300g of NaCl solution with the concentration of 0.2-0.5M is eluted, and 100mg of eluent is collected to be used as a test sample (LY).
Experimental example 1 qualitative detection of sample (LY)
(1) The test sample (LY) obtained in example 1 was qualitatively tested by the Folin phenol method (protein characterization) and the sulphuric acid phenol method (polysaccharide characterization).
The results showed strong positive, indicating that the sample (LY) contained both protein and polysaccharide.
(2) The sample (LY) obtained in example 1 was subjected to polyacrylamide gel electrophoresis.
The results show that: as shown in FIG. 1, it was confirmed by electrophoresis (molecular weight comparison with MARKER) that the sample (LY) consisted mainly of a glycoprotein having a molecular weight of 28KD (nomenclature: aderin-I) and a glycoprotein having a molecular weight of 40KD (nomenclature: aderin-II).
EXAMPLE 2 inflammatory Effect of test sample (LY)
Rats were injected subcutaneously with LY at doses of 25. mu.g/kg, 50. mu.g/kg and 100. mu.g/kg, respectively, 6 rats per dose, after which the rats were observed. The inflammatory response was localized on the day, granulomas were localized on the fourth day, mice were sacrificed on the tenth day, free granulomas were isolated and weighed, the weight of granulomas is shown in table 1, a photograph of granulomas is shown in fig. 2, and a pathological section of granulomas is shown in fig. 3.
TABLE 1
As can be seen from Table 1, the 25. mu.g/kg, 50. mu.g/kg and 100. mu.g/kg dose groups induced granulomas with average weights of 0.60. + -. 0.11g, 1.01. + -. 0.167g and 2.05. + -. 0.34g, respectively. FIG. 2 shows that granuloma tissue has formed. The pathological section of fig. 3 shows that the granuloma has new blood vessels, spindle-shaped fibroblasts, plasma cells and infiltration of acute and chronic inflammatory cells. This demonstrates the successful establishment of an animal model of inflammatory granuloma. Furthermore, the 50. mu.g/kg and 100. mu.g/kg dose groups induced granuloma with better effect.
Experimental example 3 Observation of Effect of drugs on LY-induced inflammationObserve
Dexamethasone and aspirin lysine have significant effect on LY inflammation, 5 groups of 6 rats (male) were tested, respectively, control group, dexamethasone 0.1mg/kg and 0.4mg/kg, aspirin lysine 100mg/kg and 400mg/kg, and LY 100 μ g/kg was injected subcutaneously to make a model, which was administered on day 7, control group was injected intravenously with physiological saline, and the remaining groups were injected intravenously with different doses of dexamethasone and aspirin lysine, 2 times a day, 3 consecutive days, day 10, dissected granulation tissue was sacrificed and weighed, and the results are shown in Table 2.
TABLE 2
As can be seen from Table 2, different doses of dexamethasone and different doses of lysine opipramine basically have the effect of treating LY-induced subcutaneous granuloma in rats, and the medicament dose and the treatment effect are in positive correlation, which shows that the inflammatory granuloma animal model established by the invention can be successfully used for evaluating the medicament curative effect and evaluating the relationship between the medicament dose and the curative effect.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (10)
1. A method for preparing a ormosia angustifolia extract comprises the following steps:
(1) extracting semen Adenantherae Pavoninae with water, and separating solid and liquid to obtain liquid phase;
(2) salting out the liquid phase substance to obtain a mixture;
(3) carrying out solid-liquid separation on the mixture to obtain a solid-phase substance;
(4) dissolving the solid phase substance in water to obtain a solution;
(5) dialyzing the solution, and collecting the permeate; wherein, a dialysis membrane with the aperture of 1000-20000 daltons is adopted for dialysis treatment;
(6) carrying out chromatography treatment on the permeate, and collecting eluent, namely the ormosia angustifolia extract; wherein, DEAE sepharose gel column is adopted for chromatography treatment.
2. The method according to claim 1, wherein in the step (4), the volume ratio of the solid phase to the water is 1 (1-6).
3. The method according to claim 1, wherein in step (6), elution is performed with a Tris-HCl buffer solution, followed by elution with a sodium chloride solution;
preferably, the weight ratio of the Tris-HCl buffer solution to the sodium chloride solution is 1 (0.7-2).
4. The method according to any one of claims 1 to 3, characterized by one or more of the following A to S:
A. in the step (1), the weight ratio of water to ormosia is (1-10) to 1;
B. in the step (1), leaching is carried out at the temperature of 1-17 ℃;
C. in the step (1), leaching lasts for 0.5-5 hours;
D. leaching for one or more times, preferably three times in the step (1);
E. in the step (1), solid-liquid separation is carried out by filtration;
F. in the step (2), before salting out treatment, concentrating the liquid phase substance, and collecting the concentrate;
preferably, after concentration and before salting-out treatment, the concentrate is subjected to centrifugal separation, and the supernatant is collected for salting-out treatment;
preferably, the concentration is carried out at a temperature of 1 ℃ to 17 ℃;
preferably, the concentration is 0.1 to 0.9 times of the original volume;
more preferably, the rotation speed of centrifugal separation is 1000-10000 rpm;
more preferably, the centrifugation lasts for 2-40 minutes;
G. in the step (2), salting-out treatment is carried out by adopting at least one salt selected from ammonium sulfate, sodium sulfate and sodium chloride, preferably ammonium sulfate;
H. in the step (2), salting out is carried out until the solubility of salt reaches saturation;
I. in the step (3), before solid-liquid separation, the mixture is kept stand at the temperature of 1-17 ℃;
preferably, standing lasts for 0.5-9 hours;
J. in the step (3), solid-liquid separation is carried out through centrifugal treatment;
preferably, the rotation speed of centrifugal treatment is 1000-10000 rpm;
preferably, the centrifugation treatment lasts for 2-40 minutes;
K. in the step (5), the dialysis treatment lasts for 20-80 hours;
l, in the step (5), the concentration of solute in the permeation solution is 1-10 mg/mL;
m, filtering the permeation solution before chromatography in the step (6);
preferably, a filter membrane with the aperture of 0.2-0.8 mu m is adopted for filtration treatment;
n. in the step (6), the specification of the DEAE sepharose column is phi 35mm multiplied by L450 mm;
o, in the step (6), the volume of the DEAE sepharose column is 100-600 mL;
p, in the step (6), the concentration of the Tris-HCl buffer solution is 10-100 mM;
in the step (6), the pH value of a Tris-HCl buffer solution is 7-8;
r, in the step (6), the concentration of the sodium chloride solution is 0.1-1M;
s, in the step (6), a Tris-HCl buffer solution is adopted to balance a DEAE sepharose column;
preferably, the DEAE sepharose column is equilibrated with at least 2 column volumes of Tris-HCl buffer.
5. A ormosia angustifolia extract produced by the method of any one of claims 1 to 4.
6. A pharmaceutical composition comprising the ormosia angustifolia extract of claim 5.
7. Use of the ormosia angustifolia extract of claim 5 or the pharmaceutical composition of claim 6 for the manufacture of a medicament for inducing inflammatory granuloma;
preferably, the inflammatory granuloma is an infectious granuloma.
8. A method for establishing an animal model of inflammatory granuloma, comprising the steps of:
obtaining an ormosia angustifolia extract of claim 5 or a pharmaceutical composition of claim 6;
injecting the ormosia angustifolia extract of claim 5 or the pharmaceutical composition of claim 6 into a laboratory animal;
preferably, the experimental animal is a rat;
preferably, the injection is subcutaneous;
preferably, the injection dose of each animal is 20-150 mu g/kg;
preferably, inflammatory granulomas develop in the experimental animals on days 3-11 of injection.
9. A kit comprising the ormosia angustifolia extract of claim 5 or the pharmaceutical composition of claim 6, and a syringe.
10. Use of the ormosia angustifolia extract of claim 5, the pharmaceutical composition of claim 6 or the kit of claim 9 for establishing an animal model of inflammatory granuloma or for evaluating the efficacy of a medicament for the treatment of inflammatory granuloma.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010101778.7A CN111150751B (en) | 2020-02-19 | 2020-02-19 | Method for preparing ormosia angustifolia extract, ormosia angustifolia extract and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010101778.7A CN111150751B (en) | 2020-02-19 | 2020-02-19 | Method for preparing ormosia angustifolia extract, ormosia angustifolia extract and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111150751A true CN111150751A (en) | 2020-05-15 |
| CN111150751B CN111150751B (en) | 2021-08-17 |
Family
ID=70566134
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010101778.7A Active CN111150751B (en) | 2020-02-19 | 2020-02-19 | Method for preparing ormosia angustifolia extract, ormosia angustifolia extract and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111150751B (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080160118A1 (en) * | 2003-05-16 | 2008-07-03 | Philippe Moser | Treatment of skin with cosmetic and dermatological preparations containing extracts from seeds of plants of the genus adenanthera |
| CN102432681A (en) * | 2011-12-04 | 2012-05-02 | 汕头市中乾网络科技有限公司 | Method for separating and purifying trypsin inhibitor from mung bean |
| CN104255531A (en) * | 2014-10-17 | 2015-01-07 | 南京帝道农业科技有限公司 | Rapid propagation methods for adenantherapavonlna suspension cell and regeneration plant |
| CN105924498A (en) * | 2016-07-05 | 2016-09-07 | 中国农业科学院作物科学研究所 | Preparation method for mung bean protein with effect of reducing blood fat |
| CN106800587A (en) * | 2017-01-23 | 2017-06-06 | 嵊州市派特普科技开发有限公司 | A kind of method for extracting red bean activated protein |
| CN110256547A (en) * | 2019-07-04 | 2019-09-20 | 天津科技大学 | A method of extracting β-conglutin from Australian sweet tea feather fan bean powder |
-
2020
- 2020-02-19 CN CN202010101778.7A patent/CN111150751B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080160118A1 (en) * | 2003-05-16 | 2008-07-03 | Philippe Moser | Treatment of skin with cosmetic and dermatological preparations containing extracts from seeds of plants of the genus adenanthera |
| CN102432681A (en) * | 2011-12-04 | 2012-05-02 | 汕头市中乾网络科技有限公司 | Method for separating and purifying trypsin inhibitor from mung bean |
| CN104255531A (en) * | 2014-10-17 | 2015-01-07 | 南京帝道农业科技有限公司 | Rapid propagation methods for adenantherapavonlna suspension cell and regeneration plant |
| CN105924498A (en) * | 2016-07-05 | 2016-09-07 | 中国农业科学院作物科学研究所 | Preparation method for mung bean protein with effect of reducing blood fat |
| CN106800587A (en) * | 2017-01-23 | 2017-06-06 | 嵊州市派特普科技开发有限公司 | A kind of method for extracting red bean activated protein |
| CN110256547A (en) * | 2019-07-04 | 2019-09-20 | 天津科技大学 | A method of extracting β-conglutin from Australian sweet tea feather fan bean powder |
Non-Patent Citations (4)
| Title |
|---|
| DYSON S.W.等: "大豆诱发的间质性肉芽肿性皮炎", 《世界核心医学期刊文摘（皮肤病学分册）》 * |
| 华隼: "红豆-海红豆-相思子", 《云南林业》 * |
| 钟玉绪，等: "相思豆毒素的内化作用", 《中国药理学与毒理学杂志》 * |
| 钟玉绪，等: "相思豆毒素诱导肿瘤细胞凋亡及其机制", 《中国药理学与毒理学杂志》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111150751B (en) | 2021-08-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102847134B (en) | Preparation method for chlorella polypeptide microcapsule | |
| CN103204904B (en) | Dasyatis akajei chondroprotein polypeptide capable of resisting prostate cancer, and preparation method and application thereof | |
| CN102920736B (en) | Preparation method of traditional Chinese medicine leech extract | |
| NO146818B (en) | PROCEDURE FOR AA DANNING CHANNELS WITH HIGH FLUID CONDUCTIVITY IN A FORMATION AROUND A DRILL | |
| CN107778376A (en) | A kind of preparation method and applications of radix tetrastigme polysaccharide | |
| CN101343651A (en) | Mushroom ferment pure protein with antineoplastic activity, extracting method and formulation | |
| JP2002538214A (en) | Pharmaceutical composition for treating cardiovascular disease and method for producing the same | |
| CN111150751A (en) | Method for preparing ormosia angustifolia extract, ormosia angustifolia extract and application thereof | |
| CN106883306A (en) | A kind of extraction separation method of the sunflower stem core polysaccharide with antitumor activity | |
| CN112521443B (en) | Preparation method and application of pseudo-ginseng flower protein | |
| CN104327158B (en) | Antitumor active polypeptides extracted from garlic | |
| CN106317203A (en) | Method of extracting and storing high-purity tachyplesin | |
| JP3072321B2 (en) | Anti-HIV active substance and method for producing the same | |
| JP4480204B2 (en) | Anti-tumor fraction of Kawariharatake | |
| CN100371347C (en) | Active material of polypeptide proteins in bloody clam, preparation method and usage | |
| CN116731213B (en) | Polygonum multiflorum polysaccharide, extraction method thereof and application of polygonum multiflorum polysaccharide in preparation of liver protecting medicine | |
| CN105294879B (en) | A kind of ramulus mori antitumor activity polysaccharide RMPW 2 preparation method | |
| CN103638057A (en) | Preparation method and medical application of nephila spider extract anticancer part | |
| CN105861474B (en) | Sipunculus nudus active protease with antithrombotic effect and preparation method and application thereof | |
| CN108610397B (en) | Application of angelica sinensis protein in preparation of liver injury prevention medicine | |
| CN105646658B (en) | Ray porosa chondroprotein antioxidant peptide and application thereof | |
| JPS5927829A (en) | Antitumor agent and preparation thereof | |
| JPH0314841B2 (en) | ||
| CN105648006B (en) | Preparation method of dasyatis akajei chondroprotein antioxidant peptide | |
| Silva et al. | Anti-inflammatory effect of Mastruz (Chenopodium ambrosioides) extract in respiratory distress syndrome |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |