CN116531424A - Lespedeza virgata extract and preparation method and application thereof - Google Patents
Lespedeza virgata extract and preparation method and application thereof Download PDFInfo
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- CN116531424A CN116531424A CN202210094727.5A CN202210094727A CN116531424A CN 116531424 A CN116531424 A CN 116531424A CN 202210094727 A CN202210094727 A CN 202210094727A CN 116531424 A CN116531424 A CN 116531424A
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- lespedeza
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- 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
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Abstract
The invention provides a lespedeza extract and a preparation method and application thereof, belonging to the technical field of medicines. The invention is characterized in that the lespedeza virgata is crushed, added with a solvent for reflux extraction for 0.5 to 3 hours, filtered, and the filter residue is added with the solvent for repeated extraction for 1 to 2 times, filtered, combined with the filtrate, and concentrated under reduced pressure until the volume of the filtrate is 5 to 15 percent, thus obtaining concentrated solution. The invention carries out separate purification treatment on the concentrated solution to obtain the lespedeza extract. The invention can obtain the medicine for treating otitis and/or nephritis by proportioning the lespedeza extract and the medically acceptable auxiliary materials, and the obtained lespedeza extract has the protection effect on cell injury and can be used for preparing the medicine for cell repair.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a lespedeza extract and a preparation method and application thereof.
Background
Lespedeza virgata is whole herb of Lespedezavirgata (thunder.) DC. Distributed in the south of Liaoning, north China, shaanxi to Yangtze river basin. Has effects of clearing summer-heat, promoting urination, and preventing malaria, and can be used for treating heatstroke, dysuria, malaria, common cold, hypertension, etc.
The whole herb of Lespedeza virginiana can be used as a medicine, and the description of the whole herb of Lespedeza virginiana is recorded in the 'salvage materia medica', and the book is: two kinds of the green and flat tea are called as the aliased green tea of the lespedeza, the leaf shape is big and small, the leaf is similar to black bean leaf, the leaf is similar to alfalfa leaf and grows up, the flower color is purple white, the knot is similar to maize grain, the smell is similar to that of the sophora japonica, and the nature is warm. The description of the characteristic taste and meridian tropism of the lespedeza is found in folk herbal medicine: sweet, flat, nontoxic; fujiandong Ben Cao (Mindong Ben Cao): slightly bitter in flavor, neutral in nature, entering heart and liver meridians. The lespedeza has been used as a medicine, and the folk medicinal value of the lespedeza is well documented in the herbal medicine and is used for treating cough due to lung heat and pertussis: one to two of the fresh lespedeza herb, five pieces of rock candy and boiled water are added for stewing for one hour, and the mixture is taken three times per day (Fujian folk herbal medicine); the "Mindong herbal" states that: has effects of strengthening tendons and bones, invigorating spleen, eliminating dampness, and treating dizziness, listlessness, and snake bite; stewing crystal sugar for treating epistaxis; the "Jiangxi folk herbal medicine" records that the lespedeza cure women's leucorrhea with reddish discharge: the lespedeza root is used for stewing the soup with lean pork for four times, and the lespedeza root is decocted with decoction for taking. Whereas the earliest use of lespedeza in the treatment of urinary system diseases was originally found in folk herbal medicine: moistening lung, relieving fever, promoting diuresis and treating stranguria; for treating dribbling urine, one or two of the fresh lespedeza herb, five or eight of the plantain herb and one or two of the rock candy are taken twice daily by decocting with water.
Since the last 30 th century, students at home and abroad systematically studied the chemical components of Lespedeza virginiana, and determined that the Lespedeza virginiana mainly contains various chemical components such as alkaloid, flavone, sterol and the like. The literature reports that the lespedeza has curative effect on renal insufficiency, but the curative effect on inflammatory diseases, especially nephritis and the influence on cell repair function are not reported on the lespedeza and the extract thereof. In order to deeply develop and utilize traditional medicines, explain the roles of the traditional medicines in treating inflammatory diseases, provide theoretical basis and practical experience for searching anti-inflammatory medicines with better activity, discover and prepare effective parts of the anti-inflammatory activity of Lespedeza virgata through pharmacological and chemical researches of a system, and evaluate the anti-inflammatory and anti-nephritis activities of the Lespedeza virgata in vitro and in vivo.
Disclosure of Invention
Accordingly, the present invention has been made in view of the above-mentioned problems, and an object of the present invention is to provide a method for producing an extract of Lespedeza virgata, which can give an extract of Lespedeza virgata containing a large amount of pharmaceutically active ingredients.
The invention also aims at providing the application of the lespedeza extract in preparing medicines for treating otitis and nephritis and medicines for cell repair.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of a lespedeza extract, which comprises the following steps:
pulverizing Lespedeza virgata, adding water or ethanol solvent, reflux extracting for 0.5-3 h, filtering, adding solvent into filter residue, repeatedly extracting for 1-2 times, filtering, mixing filtrates, and concentrating under reduced pressure to obtain concentrated solution.
Preferably, the water is added in an amount of 2 to 5L/kg, the alcohol solution is a 20 to 60% alcohol solution, and the amount of the water is added in an amount of 6 to 12L/kg.
Preferably, the concentrate is further concentrated to dryness under reduced pressure.
Preferably, the concentrated solution is extracted by petroleum ether, ethyl acetate and n-butanol in sequence, and a water-soluble part is reserved and freeze-dried.
Preferably, the concentrated solution is extracted for 2-3 times by adopting n-butanol, a water-soluble part is reserved, and freeze drying is carried out.
Preferably, the water-soluble fraction is precipitated with 80% ethanol and the alcohol-soluble fraction is lyophilized.
Preferably, the water-soluble fraction is precipitated with 80% ethanol, the precipitated fraction is washed with absolute ethanol and/or acetone, and the precipitate remaining after washing is lyophilized.
The invention also provides application of the lespedeza extract obtained by the preparation method in preparing medicines for treating inflammation, wherein the inflammation comprises otitis and/or nephritis.
The invention also provides application of the lespedeza extract obtained by the preparation method in preparing medicines for cell repair.
Preferably, the medicament comprises an extract of Lespedeza virgata and pharmaceutically acceptable auxiliary materials.
The invention has the beneficial effects that:
the preparation method of the Lespedeza virgata extract is simple, the operability is strong, the yield of the obtained effective part is high, and the industrialization prospect is good through systematic chemical and pharmacological researches.
The lespedeza extract prepared by the invention has obvious anti-inflammatory effect, can obviously improve ear swelling of mice, has obvious curative effect on otitis of mice, and is expected to be developed into a new medicine for treating inflammatory diseases.
The Lespedeza virgata extract prepared by the invention can obviously reduce serum BUN and CRE levels and spleen indexes of nephritis mice after high-dose in vivo administration; has the trend of reducing the serum CHO level and the urine protein amount of nephritis mice for 24 hours; the Lespedeza virgata extract prepared by the invention can obviously relieve the damage of the doxorubicin induced rat renal tubular epithelial NRK-52E cells in vitro, and the cell recovery effect of the Lespedeza virgata extract is enhanced along with the increase of the concentration. In vivo and in vitro experimental results show that the lespedeza extract prepared by the invention has a certain cytoprotective effect. Has better curative effect on nephritis in mice.
Detailed Description
The invention provides a preparation method of a lespedeza extract, which comprises the following steps: pulverizing Lespedeza virgata, adding water or alcohol solvent, reflux extracting for 0.5-3 h, filtering, adding solvent into filter residue, repeatedly extracting for 1-2 times, filtering, mixing filtrates, concentrating under reduced pressure to 5-15% of filtrate volume to obtain concentrated solution.
The Lespedeza pedunculata used in the invention is whole herb of Lespedeza pedunculata (Chinese medicinal materials).
The invention firstly pulverizes the lespedeza, preferably passes through a 80-100 mesh sieve after pulverizes, and then adds solvent for reflux extraction. The present invention is not limited to a specific pulverizing method.
In the present invention, the solvent includes water, 20 to 60% alcohol solution, which may be aqueous ethanol solution or aqueous methanol solution. The invention can extract the lespedeza by adding water or 20-60% alcohol solution to obtain the lespedeza water extract or the lespedeza alcohol extract respectively. The concentration of the alcohol solution is preferably 30 to 50%, more preferably 40%; the alcohol solution is preferably an ethanol solution.
In the present invention, when the solvent is water, the amount of the solvent to be added is 2 to 5L/kg, preferably 3 to 4L/kg, more preferably 3.4L/kg, depending on the amount of the lespedeza.
In the present invention, when the solvent is a 40% ethanol solution, the amount of the solvent to be added is 6 to 12L/kg, preferably 8 to 10L/kg, more preferably 9L/kg, depending on the amount of the Lespedeza virgata.
The invention carries out reflux extraction on the lespedeza, the reflux extraction time is 0.5-3 h, the reflux extraction time is preferably 0.5-2 h when the solvent is water, more preferably 40-60 min, and the reflux extraction time is preferably 40-100 min, more preferably 50-60 min when the solvent is an alcohol solution. The invention can effectively extract the active pharmaceutical ingredients such as flavone, quercetin, kaempferia galangal acid, trifolium glycoside, isoquercitrin and the like from the lespedeza virginiana by reflux extraction of water or alcohol solution.
The invention filters after the reflux extraction is finished, the filtrate is reserved for standby, the filter residue is added with solvent to repeat the reflux extraction process again, and the repeated extraction times are preferably 1 time. The invention combines the filtrates obtained by each reflux extraction, and carries out reduced pressure concentration treatment on the filtrate.
The filtrate is concentrated to 5-15% of the original filtrate volume under reduced pressure, more preferably to 6-14% of the filtrate volume when the solvent is water, more preferably to 5-10% of the filtrate volume when the solvent is ethanol solution, and more preferably to 6-10% of the filtrate volume when the solvent is methanol solution.
The concentrated solution prepared by the method is continuously decompressed and concentrated to be dried to obtain the solid powdery lespedeza extract, and the extract contains more medicinal active ingredients and has pharmacological activity.
The concentrated solution prepared by the method is sequentially extracted by petroleum ether, ethyl acetate and n-butanol, a water-soluble part is reserved, and the solid powdery lespedeza extract is obtained by freeze drying. The invention removes impurity components which are soluble in petroleum ether, ethyl acetate and n-butanol through further extraction treatment of the concentrated solution, and further purifies to obtain the lespedeza extract with higher pharmacological activity. The invention is not limited to a specific extraction mode, extractant amount and freeze-drying mode.
The concentrated solution prepared by the method is repeatedly extracted for 2 to 3 times by adopting n-butanol, a water-soluble part is reserved, and the solid powdery lespedeza extract is obtained by freeze drying. The invention removes impurity components which are soluble in n-butanol through further extraction treatment of the concentrated solution, and further purifies to obtain the lespedeza extract with higher pharmacological activity. The invention is not limited to a specific extraction mode, extractant amount and freeze-drying mode.
The invention sequentially extracts petroleum ether, ethyl acetate and n-butanol or repeatedly extracts n-butanol to obtain a water-soluble part, precipitating the water-soluble part with 80% ethanol, and freeze-drying the alcohol-soluble part; the precipitate fraction is washed with absolute ethanol and/or acetone, and the precipitate remaining after washing is lyophilized. When washing is performed using both absolute ethanol and acetone, the present invention is not limited to a specific washing sequence. The invention can further purify the water-soluble part to obtain the Lespedeza virgata extract with higher pharmacological activity. The invention is not limited to a specific extraction mode, the using amount of the extractant and the using amount of 80% ethanol.
The invention also provides application of the obtained lespedeza extract in preparing medicines for treating otitis, and the lespedeza extract can obviously improve ear swelling of mice and has obvious anti-inflammatory effect.
The invention also provides application of the obtained lespedeza extract in preparing medicines for treating nephritis, the lespedeza extract can obviously reduce serum BUN and CRE levels and spleen indexes of nephritis mice, and has the trend of reducing serum CHO levels and reducing the urine protein amount of the nephritis mice for 24 hours. As an alternative embodiment, the nephritis of the invention is doxorubicin nephritis, and good anti-doxorubicin nephritis effect can be achieved.
The invention also provides application of the obtained lespedeza extract in preparing medicaments for cell repair, the lespedeza extract can obviously relieve the damage of the doxorubicin induced rat tubular epithelial NRK-52E cells, and the cell recovery effect of the lespedeza extract is enhanced along with the increase of the concentration, so that the lespedeza extract has good cell protection effect.
The invention also comprises a pharmaceutical composition prepared by using the lespedeza extract as a pharmaceutical active ingredient, wherein the pharmaceutical composition can comprise pharmaceutically acceptable auxiliary materials according to actual needs.
The pharmaceutical composition of the present invention may be any pharmaceutically acceptable dosage form including, but not limited to, drop pills, tablets, capsules, oral liquids, buccal agents, granules, electuaries, pills, powders, pastes, pellets, suspensions, powders, injections, suppositories, ointments, plasters, creams, sprays, drops or patches.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
In the experimental results table of the present invention: "" means that p < 0.05 compared to the model control group, "" means that p < 0.01 compared to the model control group, "" means that p < 0.001 compared to the model control group; "#" indicates that p < 0.05 compared to the blank, "#" indicates that p < 0.01 compared to the blank, and "#" indicates that p < 0.001 compared to the blank.
Example 1
The embodiment provides a preparation method of the water extract of the lespedeza virgata:
pulverizing 500g of Lespedeza virginiana, sieving with 80 mesh sieve, adding 1.7L of water, boiling, reflux extracting for 30min, filtering, and collecting filtrate; adding 1.7L water into the filter residue again, boiling, reflux extracting for 30min, and filtering; the two filtrates were combined and concentrated to 0.5L under reduced pressure.
Concentrating 0.25L of the concentrated solution to dryness to obtain water extract of Lespedeza virgata, and recording as LEVH-1.
Example 2
This example uses another 0.25L of the concentrate from example 1 to perform three successive extractions: extracting with 0.1L petroleum ether, respectively retaining the extractive solution and water soluble part; extracting the water-soluble part with 0.1L ethyl acetate again, and respectively retaining the extract and the water-soluble part; the water-soluble fraction was extracted with 0.1L of n-butanol, and the extract and the water-soluble fraction were retained, respectively. Concentrating the extractive solution obtained by three times of extraction under reduced pressure to dry, foaming into powder, and sequentially marking the products as LEVH-2, LEVH-3 and LEVH-4; the water-soluble part obtained after three times of extraction is directly frozen and dried into powder to obtain the water extract of the lespedeza, which is marked as LEVH-5.
The extraction records of the effective parts of the water extracts of the lespedeza virginiana obtained in the examples 1-2 are shown in the table 1. (extraction yield = amount of sample extracted +.The total mass of raw material × 100%)
TABLE 1 LEV extract record table
Example 3
10g of the water extract of Lespedeza virgata (LEVH-5) obtained in example 2 was taken, 50mL of 80% ethanol was added for precipitation, and the precipitate was separated by filtration, leaving an alcohol-soluble fraction and a precipitate fraction, respectively.
Concentrating and drying the alcohol-soluble part to obtain a solid product, which is marked as LEVH-6;
the sediment part is washed with 20mL of 80% ethanol, and the washing liquid is concentrated and dried to obtain a solid product which is marked as LEVH-8; the washed precipitate is directly freeze-dried to obtain the water extract of Lespedeza virgata, which is marked as LEVH-7.
The extraction records of the effective parts of the water extract of the lespedeza are shown in table 2. (extraction yield = amount of sample extracted +.The total mass of raw material × 100%)
TABLE 2 LEV extract record table
Example 4
Taking 3000g of lespedeza, crushing, adding 10.2L of water, boiling, extracting under reflux for 30min, and filtering to collect filtrate; adding 10.2L water into the residue again, boiling, reflux extracting for 30min, and filtering; the two filtrates were combined and concentrated to 3L under reduced pressure. Cooling the concentrated solution, extracting with 1L of n-butanol solution, retaining water-soluble part, and extracting the water-soluble part with n-butanol repeatedly for 2 times; combining the extracts, concentrating to dryness to obtain a solid product, and marking the solid product as LEVH-4b; concentrating the rest water solution to 1L, collecting 0.5L concentrate, concentrating to dry to obtain Lespedeza virgata water extract, and recording as LEVH-5b.
Example 5
An additional 0.5L of the concentrate obtained after the extraction of example 4 was taken, and precipitation was carried out by adding 80% ethanol 2L, and the precipitate was separated by filtration, leaving an alcohol-soluble fraction and a precipitate fraction, respectively.
Concentrating the alcohol-soluble part to dryness to obtain a solid product, which is marked as LEVH-6b;
the precipitated fraction was concentrated to dryness to give a solid product, designated LEVH-7b.
Example 6
5g (without water) of LEVH-7b sample prepared in example 5 was taken, washed successively with 20mL of absolute ethanol and 20mL of acetone, and the washings were concentrated to dryness to obtain the product LEVH-8b and the product LEVH-9b, respectively.
Example 7
The embodiment provides a preparation method of the ethanol extract of the lespedeza virgata:
taking 500g of lespedeza virginiana, crushing, putting into a 10L round-bottom flask, adding 40% ethanol water for 5L, extracting under reflux for 1h, and filtering to collect filtrate; adding 40% ethanol water 4L into the residue, refluxing for 1 hr, mixing the filtrates, and concentrating to 700ml.
Taking 350ml of concentrated solution, directly freeze-drying to obtain the Lespedeza virgata ethanol extract, and recording as LEVE-1.
Example 8
350mL of the residual concentrate from example 7 was taken and subjected to three successive extractions: extracting with 150mL petroleum ether, respectively retaining the extract and water-soluble part; extracting the water-soluble part with 100mL ethyl acetate again, and respectively retaining the extract and the water-soluble part; the water-soluble fraction was extracted with 100mL of n-butanol, and the extract and the water-soluble fraction were retained, respectively. Concentrating the extractive solution obtained by three times under reduced pressure to obtain solid products, respectively, and sequentially marking as LEVE-2, LEVE-3 and LEVE-4; and (5) continuously concentrating and drying the water-soluble part obtained after extraction to obtain an ethanol extract of the lespedeza, and recording as LEVE-5.
The records of the extraction of the effective parts of the ethanol extracts of Lespedeza virgata obtained in examples 7 to 8 are shown in Table 3. (extraction yield = amount of sample extracted +.The total mass of raw material × 100%)
TABLE 3 LEV extract record table
Experimental example 1
This example investigated the protective effect of Lespedeza virgata extract on otitis media in mice.
1. Mouse selection:
animal quality: BALB/c mice, male, SPF grade, 18-20g, beijing Vitolihua laboratory animal technologies Co., ltd., eligibility certificate: (Beijing) 2012-0001.
Animal feeding environment: air-conditioning, wherein the room temperature is 21-25 ℃; the humidity is 45 plus or minus 20 percent, and the fresh air is ventilated for 10 times per hour. Artificial light was applied for 12 hours. Water is fed freely.
Pre-experimental adaptation period: after the animals are introduced and inspected according to the requirements, the animals are bred in separate cages, 5 animals are adapted to observe for 3 days, the general condition of the animals is observed, and the weight is weighed after the adaptation period is finished.
The croton oil is adopted to induce a mouse otitis model, a carrageenan otitis model and a Dinitrofluorobenzene (DNFB) is adopted to induce a mouse delayed hypersensitivity (DTH) model.
2. Experimental instrument:
analytical balance: BS 224S type, sactorius; enzyme-labeled instrument: power Wave XS2, bioTek; blood biochemical analyzer: acute 40R, TOSHIBA.
3. The experimental method comprises the following steps:
group 3.1 and dose setting:
blank control group: injecting an equal dose of vehicle (physiological saline);
model control group: injecting an equal dose of vehicle (physiological saline);
high dose group: 200mg/kg body weight, subcutaneously;
medium dose group: 100mg/kg body weight, subcutaneously;
low dose group: 20mg/kg body weight, subcutaneously;
the high, medium and low dose groups adopt 1g of the lespedeza fine extract powder prepared in the examples 1-4, and are respectively dissolved in 5, 10 and 50mL of physiological saline to obtain the injection.
3.2 modeling and drug administration:
3.2.1 model of soybean oil-induced otitis in mice
BALB/c mice were randomly grouped, 10 animals per group, subcutaneously injected with Lespedeza virgata extract (200 mg/kg) 1 time, and the control group was given an equal volume of drug vehicle. After the last dose for 1h, mice were coated with 0.02mL of baba oil on both sides of the right ear and the left ear served as a normal control. Mice were sacrificed after 1h of inflammation and cervical dislocation, the same position of the left and right ears were removed with a 6mm diameter punch, the left and right ear pieces were weighed, the mass difference of the left and right ear pieces was indicative of the inflammatory swelling degree, and the inhibition rate (IR%) of each group against croton oil otitis in mice was calculated using the swelling degree = (model group average ear swelling degree-administration group ear swelling degree)/model group average ear swelling group×100% ], and the results are shown in table 4.
Table 4 inhibition of croton oil otitis in mice by LEV extract
As can be seen from Table 4, compared with the model group, LEVE-1 (p < 0.01), LEVE-5 (p < 0.05), LEVH-1 (p < 0.001), LEVH-2 (p < 0.01), LEVH-4 (p < 0.001), LEVH-5 (p < 0.001), LEVH-6 (p < 0.001), and LEVH-7 (p < 0.001).
The results show that administration of LEVE-1, LEVE-5, LEVH-1, LEVH-2, LEVH-4, LEVH-5, LEVH-6, and LEVH-7 samples significantly improved croton oil otitis in mice, and no significant effect was seen on croton oil otitis in mice after administration of the other groups.
3.2.2DNFB-induced mouse DTH model
BALB/c mice were randomly grouped, 10 animals per group, subcutaneously injected with Lespedeza virgata extract (100 mg/kg) 1 time, and the control group was given an equal volume of drug vehicle. After 1h of administration, the control group was treated with DNFB-free solvent and the DTH group was modeled by 5g/L DNFB abdominal sensitization and auricle challenge. The same position of the left and right ears were removed with a 6mm diameter punch 48h after excitation, and the ears were weighed, the mass difference between the left and right ears indicated the inflammatory swelling degree, the inhibition rate (IR%) was calculated for each group using the swelling degree, and the re-screening results for the mice DTH for each group were shown in table 5.
TABLE 5 inhibition of the DTH model by LEV extract
As can be seen from Table 5, compared with the model group, LEVE-1 (p < 0.05), LEVH-2 (p < 0.05), and LEVH-7 (p < 0.01).
The results in Table 5 show that LEVE-1, LEVH-2, and LEVH-7 samples administered significantly improved the ear swelling in mice, and that no significant effect was seen on the ear swelling in mice after administration of the other groups.
3.2.3 carrageenan-induced model of otitis in mice
BALB/c mice were randomly grouped, 10 animals per group, subcutaneously injected with lespedeza extract (20 mg/kg of administered dose of lespedeza extract 1 time per day for 6 consecutive days), and control group was given an equal volume of drug vehicle. After the last administration for 1h, carrageenan was applied to both sides of the right ear of the mice for 0.02mL of inflammation, and the left ear was used as a normal control. Mice were sacrificed after cervical dislocation after 1h of inflammation, the same position of the left and right ears were removed with a 6mm diameter punch, the ears were weighed, the difference in mass of the left and right ears indicated the inflammatory swelling degree, the inhibition (IR%) of each group was calculated using the swelling degree, and the carrageenan inflammation rescreening results of each group for mice were shown in table 6.
TABLE 6 effect of LEV extract on model of carrageenan inflammation in mice
As can be seen from table 6, administration of the LEVH-7 samples significantly improved ear swelling (p < 0.05) in mice compared to the model group, and no significant effect was seen on ear swelling in mice after administration of the other groups.
Taken together, the results show that LEVE-1, LEVE-5, LEVH-1, LEVH-2, LEVH-4, LEVH-5, LEVH-6, LEVH-7 can significantly improve croton oil otitis in mice; in a Dinitrofluorobenzene (DNFB) induced delayed hypersensitivity (DTH) model of mice, LEVE-1, LEVH-2 and LEVH-7 sample administration can significantly improve ear swelling of mice after dose group administration of Lespedeza extract; in the mouse carrageenan otitis model, LEVH-7 sample administration can significantly improve mouse ear swelling after administration of the Lespedeza extract low dose group. Therefore, the lespedeza extract has a certain curative effect on the otitis media of mice.
Experimental example 2
This example investigated the protective effect of Lespedeza virgata extract on doxorubicin nephritis in mice.
1. Mouse selection:
animal quality: BALB/c mice, male, SPF grade, 18-20g, beijing Vitolihua laboratory animal technologies Co., ltd., eligibility certificate: (Beijing) 2012-0001.
Animal feeding environment: air-conditioning, wherein the room temperature is 21-25 ℃; the humidity is 45 plus or minus 20 percent, and the fresh air is ventilated for 10 times per hour. Artificial light was applied for 12 hours. Water is fed freely.
Pre-experimental adaptation period: after the animals are introduced and inspected according to the requirements, the animals are bred in separate cages, 5 animals are adapted to observe for 3 days, the general condition of the animals is observed, and the weight is weighed after the adaptation period is finished.
2. Doxorubicin: purity 98%, cat No. T1020, CAS No. 25316-40-9, TARGET MOL company.
3. The experimental method comprises the following steps:
group 3.1 and dose setting:
blank control group: an equivalent dose of vehicle (normal saline);
model time alignment group: an equivalent dose of vehicle (normal saline);
high dose group: 5.0g/Kg,0.5g/ml,0.1ml/10g;
low dose group: 2.5g/Kg,0.25g/ml,0.1ml/10g;
( In the high dose group, 5.0g/Kg is the administration mass per kilogram of mice; 0.5g/mL means that 0.5g of extract is dissolved per mL of vehicle; 0.1mL/10g means 0.1mL of vehicle per 10g of mice; the low dose group was the same. The LEVH-7 prepared in example 3 was selected for the drug in the high and low dose groups )
3.2 modeling and drug administration:
BALB/c mice were randomly divided into 4 groups of 10, each, and weighed. The animals of each group were once injected with doxorubicin (10 mg/Kg) at tail vein except for the blank control group (administration of an equal volume of vehicle) to establish a mouse doxorubicin nephritis model; starting gastric lavage administration on 8 th day after molding, 1 time a day, and continuously 21 days; after the last administration, urine is collected, mice are sacrificed after 24 hours, and materials are obtained for index detection.
4. Experimental results:
4.1 general growth of animals:
animals were weighed 1 time every 7 days, weight 0 was the initial weight before molding, weight 1 was the weight after molding, weight 2 was the weight after the first week, weight 3 was the weight after the second week, weight 4 was the weight after the third week, and the effect of the drug on the weight was observed, and the results are shown in Table 7.
Table 7. Influence of lev extract on body weight of doxorubicin nephritis mice (mean±std, n=10
As can be seen from table 7, the weight of the model control group was significantly reduced (p < 0.05 or 0.01) after modeling, compared to the blank control group; compared with the model group, the Lespedeza virgata extract has no significant influence (p is more than 0.05) on the reduction of the weight of the model after the administration.
4.2 24h urine protein amount:
the results of 24-hour urine protein amount detection of the Lespedeza virginiana extract on doxorubicin nephritis mice are shown in Table 8.
Table 8. Influence of lev extract on the amount of 24h urine protein of nephritis mice (mean±std, n=3
As can be seen from Table 8, the urine protein level of mice was significantly increased (p < 0.01) after doxorubicin manufacturing as compared with the blank control group; whereas the Lespedeza virginiana extract reduced the urine protein content of nephritis mice for 24 hours, but no significant difference (p > 0.05) was seen.
4.3 biochemical blood detection:
the results of the biochemical index measurements of the serum of mice are shown in Table 9.
Table 9. Influence of lev extract on biochemical detection results of serum of nephritis mice (mean±std, n=10)
As can be seen from Table 9, the serum BUN, CRE and CHO levels of the doxorubicin nephritis mice were significantly elevated (p < 0.05 or 0.01) compared to the blank control group; serum BUN, CRE and CHO levels were reduced in nephritis mice following administration of Lespedeza virgata extract, wherein the high dose groups BUN and CRE were significantly different from the model groups (p < 0.05 or 0.01).
4.4 organ index:
the indices of kidney, thymus and spleen organs of the mice were examined, and the results are shown in Table 10.
Table 10. Influence of lev extract on organ index of nephritis mice (mean±std, n=10)
As can be seen from table 10, compared with the blank control group, the spleen index of the mice with doxorubicin nephritis was significantly increased, while the spleen index of the mice with nephritis was significantly decreased (p < 0.05 or 0.01) after administration of the lespedeza extract; the kidney index and thymus index of nephritis mice are not obviously affected after the lespedeza extract is administrated.
In conclusion, the results show that the high-dose group administration of the lespedeza extract can obviously reduce serum BUN and CRE levels and spleen indexes of nephritis mice; has the functions of reducing the serum CHO level and reducing the urine protein quantity of nephritis mice for 24 hours. The Lespedeza pedunculata extract has a certain curative effect on the doxorubicin induced nephritis in mice.
Experimental example 3
This example investigated the protective effect of Lespedeza virgata extract on rat renal tubular epithelial NRK-52E cell injury.
1. Laboratory instrument and materials
DMSO: sorabio D8371, dimethyl sulfoxide, cell culture;
doxorubicin: purity 98%, cat No. T1020, CAS No. 25316-40-9, targetmol company;
dexamethasone: dexamethasone, cas No. 50-02-2, cat No. B76795, innochem,1G;
one ten thousandth electronic balance: ME-104, mettler; ultrasonic cleaner: deep and ultra-clean.
2. Construction of experimental model
Cell line: NRK-52E cell
The rat renal tubular epithelial NRK-52E cell injury model was induced with doxorubicin.
3. The experimental method comprises the following steps:
3.1 experimental procedure:
NRK-52E cells in the logarithmic growth phase were adjusted to a density of 0.5x105/mL, 1 x 105/mL, inoculated into 96-well plates at a density of 100 μl per well, and cultured in an incubator at 37 ℃ with 5% co2 for 4h adherence; adding an equal dose of solvent or a Lespedeza extract group into each hole respectively for pre-incubation for 1h; adding the vehicle in the blank control group with the same dosage, and adding 0.5ug/ml doxorubicin in the other groups for 24 hours; CCK8 detects cellular activity.
Group 3.2 and dose setting:
blank control group: an equivalent dose of vehicle (normal saline);
model control group: an equivalent dose of vehicle (normal saline);
positive control group: 10um dexamethasone;
extract low dose group: 10ug/mL;
low dose group in extract: 50ug/mL;
high dose group in extract: 100ug/mL;
extract high dose group: 200ug/mL;
(extraction Process section for all extracts LEVH-7 prepared in example 3)
4. Experimental results
The effect of Lespedeza virginiana extract on doxorubicin-induced rat tubular epithelial NRK-52E cell injury model is shown in Table 11.
Influence of LEV extract on doxorubicin-induced rat tubular epithelial NRK-52E cell injury model (mean+ -SD, n=6)
As can be seen from Table 11, the NRK-52E cell activity was significantly reduced (p < 0.001) after doxorubicin induction compared to the normal control group; compared with a model control group, the positive drug dexamethasone can significantly increase NRK-52E cell activity (p < 0.001) after doxorubicin induction; compared with a model control group, the low, medium, low, medium and high concentrations of the lespedeza extract can obviously reduce the damage (p < 0.001) of the doxorubicin induced rat renal tubular epithelial NRK-52E cells, and the better the action effect of the lespedeza extract along with the increase of the concentration; and the Lespedeza virgata extract does not show obvious cytotoxicity under the conditions of low concentration, medium concentration and high concentration. The result shows that the lespedeza extract can obviously relieve the damage of the doxorubicin induced rat renal tubular epithelium NRK-52E cells, and the cell recovery effect of the lespedeza extract is enhanced along with the increase of the concentration.
Experimental example 4
The embodiment provides a capsule containing the lespedeza extract: taking 20g of lespedeza extract, adding 29.5g of dextrin and 0.5g of magnesium stearate, fully and uniformly stirring, preparing 1000 granules according to a capsule preparation method, and filling into 50mg of capsules, wherein each granule contains 20mg of lespedeza extract.
Experimental example 5
The present example provides a tablet comprising an extract of Lespedeza virgata: mixing the extract of Lespedeza virginiana 20g with starch 20g, dextrin 16g and sucrose 3g, stirring, granulating, drying at below 60deg.C, adding appropriate amount of magnesium stearate and pulvis Talci, mixing, pressing into 1000 tablets, and sugar coating or film coating.
Experimental example 6
The embodiment provides a granule containing the lespedeza extract: taking 20g of lespedeza extract, adding a proper amount of sucrose and dextrin, granulating according to a granule preparation method, and drying to obtain 1000g.
Experimental example 7
The embodiment provides an oral liquid containing the lespedeza extract: dissolving 20g of Lespedeza virgata extract in water, regulating the pH value to 7.0 with 5% sodium hydroxide, stirring, refrigerating at 4 ℃ for 48 hours, filtering, adding 60g of sucrose into the filtrate, stirring to dissolve, adding a proper amount of essence, regulating the pH value to 7.0, adding water to prepare 1000ml, stirring, standing for 12 hours, filtering, filling and sterilizing to obtain the Lespedeza virgata extract.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. The preparation method of the lespedeza extract is characterized by comprising the following steps of:
pulverizing Lespedeza pedunculata, adding water or alcohol solvent, reflux extracting for 0.5-3 hr, filtering, adding solvent into the filter residue, repeatedly extracting for 1-2 times, filtering, mixing filtrates, and concentrating under reduced pressure to obtain concentrated solution.
2. The method according to claim 1, wherein the water is added in an amount of 2 to 5L/kg, the alcohol solution is 20 to 60% alcohol solution, and the amount of the alcohol solution added is 6 to 12L/kg.
3. The method of claim 1, wherein the concentrate is further concentrated to dryness under reduced pressure.
4. The method according to claim 1, wherein the concentrated solution is extracted with petroleum ether, ethyl acetate and n-butanol in this order, and the water-soluble fraction is retained and freeze-dried.
5. The method according to claim 1, wherein the concentrated solution is extracted 2 to 3 times with n-butanol, and the water-soluble fraction is retained and freeze-dried.
6. The method of any one of claims 4 to 5, wherein the water soluble fraction is precipitated with 80% ethanol and the alcohol soluble fraction is lyophilized.
7. The method according to any one of claims 4 to 5, wherein the water-soluble fraction is precipitated with 80% ethanol, the precipitated fraction is washed with absolute ethanol and/or acetone, and the precipitate remaining after washing is lyophilized.
8. Use of the extract of Lespedeza virgata obtained by the preparation method according to any one of claims 1 to 7 for the preparation of a medicament for the treatment of inflammation, preferably, the inflammation comprises otitis and/or nephritis.
9. Use of the extract of Lespedeza virgata obtained by the preparation method of any one of claims 1 to 7 in the preparation of a medicament for cell repair.
10. A pharmaceutical composition comprising an effective amount of the extract of lespedeza virgata obtained by the preparation method of any one of claims 1-7 and pharmaceutically acceptable excipients.
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