Lespedeza serrata extract and its preparation method and application

Abstract

The present invention provides a Lespedeza serrata extract and a preparation method and application thereof, and belongs to the field of medical technology. The present invention crushes the Lespedeza serrata, adds a solvent to reflux and extract for 0.5 to 3 hours, filters, adds a solvent to the filter residue and repeatedly extracts 1 to 2 times, filters, combines the filtrate, and concentrates under reduced pressure to 5 to 15% of the volume of the filtrate to obtain a concentrated solution. The present invention performs purification treatments on the concentrated solution to obtain a Lespedeza serrata extract. The present invention can obtain a drug for treating otitis and / or nephritis by proportioning the Lespedeza serrata extract and medically acceptable excipients, and the obtained Lespedeza serrata extract has a protective effect on cell damage and can be used to prepare a drug for cell repair.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine, and in particular relates to a Lespedeza lepedunculata extract and a preparation method and application thereof. Background Art

[0002] Lespedeza serrata is the whole herb of Lespedeza serrata (Thunb.) DC. of the Leguminosae family. It is distributed in southern Liaoning, North China, Shaanxi and the Yangtze River Basin. It has the effects of clearing away heat and promoting diuresis, and preventing malaria. It is used to treat heatstroke, urinary problems, malaria, colds, hypertension and other diseases.

[0003] The whole plant of Lespedeza serrata can be used as medicine. The record of it was first found in "Compendium of Materia Medica for Famine Relief". The book states: "Lespedeza serrata is also known as Suijun Tea. It grows in swamps. There are two species, with leaves of different sizes. The large leaves are similar to black bean leaves, and the small leaves have stems like clover. The leaves are larger, the flowers are purple and white, the seeds are as big as millet grains, the smell is similar to Sophora japonica, and it is warm in nature." The records of the nature, flavor and meridians of Lespedeza serrata can be found in "Fujian Folk Herbs": sweet, flat, non-toxic; "Compendium of Materia Medica for Eastern Fujian": slightly bitter, flat in nature, enters the heart and liver meridians. Lespedeza has been used as medicine since ancient times, and its folk medicinal value is recorded in detail in the Materia Medica. It is used to treat lung heat cough and whooping cough: 1-2 liang of fresh Lespedeza, 5 qian of crystal sugar, stewed in boiling water for one hour, three times a day (Fujian Folk Herbs); "Mindong Materia Medica" records: it benefits the bones and muscles, strengthens the spleen and drives away dampness, treats dizziness, weakness, and snake bites; stewed with crystal sugar to treat epistaxis; "Jiangxi Folk Herbs" records that Lespedeza can treat women's leucorrhea: 1 liang of Lespedeza root, stewed with 4 liang of lean pork, and boiled in soup. The earliest use of Lespedeza to treat urinary system diseases was first seen in "Fujian Folk Herbs": moisturizing the lungs and relieving heat, promoting diuresis and relieving stranguria; to treat persistent urination, 1-2 liang of fresh Lespedeza, 5-8 qian of plantain, 1 liang of crystal sugar, boiled in water, and taken twice a day.

[0004] Since the late 1930s, domestic and foreign scholars have systematically studied the chemical components of Lespedeza serrata, and determined that it mainly contains alkaloids, flavonoids, sterols and other chemical components. Literature reports that Lespedeza serrata is effective for renal insufficiency, but there are no reports on the efficacy of Lespedeza serrata and its extracts on inflammatory diseases, especially nephritis, and the effects on cell repair function. In order to further develop and utilize traditional medicines, explain the role of traditional medicines in the treatment of inflammatory diseases, and provide theoretical basis and practical experience for finding more active anti-inflammatory drugs, we discovered and prepared the effective part of Lespedeza serrata inflammation inhibition activity through systematic pharmacological and chemical studies, and evaluated its anti-inflammatory and anti-nephritis activities in vitro and in vivo. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a method for preparing a Lespedeza gracilis extract, which can obtain a Lespedeza gracilis extract containing a large amount of pharmaceutically active ingredients.

[0006] The present invention also aims to provide the use of Lespedeza serrata extract in the preparation of drugs for treating otitis and nephritis and drugs for cell repair.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for preparing a Lespedeza serrata extract, comprising the following steps:

[0009] The fine-stalked Lespedeza is crushed, water or ethanol solvent is added and refluxed for extraction for 0.5 to 3 hours, filtered, the filter residue is added with solvent and the extraction is repeated 1 to 2 times, filtered, the filtrate is combined, and concentrated under reduced pressure to obtain a concentrate.

[0010] Preferably, the water is added in an amount of 2 to 5 L / kg, and the alcohol solution is a 20 to 60% alcohol solution, and its addition amount is 6 to 12 L / kg.

[0011] Preferably, the concentrated solution is further concentrated under reduced pressure to dryness.

[0012] Preferably, the concentrate is extracted with petroleum ether, ethyl acetate and n-butanol in sequence, and the water-soluble portion is retained and freeze-dried.

[0013] Preferably, the concentrate is extracted 2 to 3 times with n-butanol, the water-soluble portion is retained, and freeze-dried.

[0014] Preferably, the water-soluble portion is precipitated with 80% ethanol, and the alcohol-soluble portion is freeze-dried.

[0015] Preferably, the water-soluble part is precipitated with 80% ethanol, the precipitated part is washed with anhydrous ethanol and / or acetone, and the remaining precipitate after washing is freeze-dried.

[0016] The present invention also provides the use of the Lespedeza serrata extract obtained by the above preparation method in preparing a medicine for treating inflammation, wherein the inflammation includes otitis and / or nephritis.

[0017] The present invention also provides the use of the Lespedeza serrata extract obtained by the above preparation method in the preparation of a medicine for cell repair.

[0018] Preferably, the medicament comprises a Lespedeza serrata extract and a pharmaceutically acceptable excipient.

[0019] Beneficial effects of the present invention:

[0020] Through systematic chemical and pharmacological research, the invention has a simple preparation method for the Lespedeza serrata extract, strong operability, a high yield of the obtained effective parts, and good industrialization prospects.

[0021] The Lespedunculate lespeduncle extract prepared by the invention has a significant anti-inflammatory effect, can significantly improve ear swelling in mice, has a significant therapeutic effect on mouse otitis, and is expected to be developed into a new drug for treating inflammatory diseases.

[0022] The pedunculate lespedeza extract prepared by the present invention can significantly reduce the serum BUN, CRE levels and spleen index of nephritis mice after high-dose administration in vivo; it has a tendency to reduce serum CHO levels and reduce the 24-hour urine protein amount of nephritis mice; the pedunculate lespedeza extract prepared by the present invention can significantly alleviate doxorubicin-induced rat renal tubular epithelial NRK-52E cell damage in vitro, and with the increase of concentration, the cell recovery effect of the pedunculate lespedeza extract is enhanced. The in vivo and in vitro experimental results show that the pedunculate lespedeza extract prepared by the present invention has a certain cell protection effect. It has a good therapeutic effect on mouse nephritis. DETAILED DESCRIPTION

[0023] The invention provides a method for preparing a Lespedeza serrata extract, comprising the following steps: crushing the Lespedeza serrata, adding water or an alcohol solvent to perform reflux extraction for 0.5 to 3 hours, filtering, adding a solvent to the filter residue to perform repeated extraction for 1 to 2 times, filtering, combining the filtrate, and concentrating under reduced pressure to 5 to 15% of the volume of the filtrate to obtain a concentrated solution.

[0024] The Lespedeza serrata used in the present invention is the whole herb of Lespedeza serrata (Chinese medicinal material).

[0025] The present invention firstly grinds the fine-stalked Lespedeza, preferably passes through a 80-100 mesh sieve after grinding, and then adds a solvent for reflux extraction. The present invention does not limit the specific grinding method.

[0026] In the present invention, the solvent includes water and a 20-60% alcohol solution, and the alcohol solution can be an aqueous ethanol solution or an aqueous methanol solution. The present invention can extract Lespedeza serrata by adding water or a 20-60% alcohol solution to obtain a Lespedeza serrata water extract or a Lespedeza serrata alcohol extract. The concentration of the alcohol solution is preferably 30-50%, more preferably 40%; the alcohol solution is preferably an ethanol solution.

[0027] In the present invention, when the solvent is water, the amount of the solvent added is 2 to 5 L / kg, preferably 3 to 4 L / kg, and more preferably 3.4 L / kg, according to the amount of Lespedeza gracilis used.

[0028] In the present invention, when the solvent is a 40% ethanol solution, the amount of solvent added is 6 to 12 L / kg, preferably 8 to 10 L / kg, and more preferably 9 L / kg, according to the amount of Lespedeza gracilis used.

[0029] The present invention performs reflux extraction on Lespedeza serrata, and the reflux extraction time is 0.5 to 3 hours. When the solvent is water, the reflux extraction time is preferably 0.5 to 2 hours, more preferably 40 to 60 minutes. When the solvent is an alcohol solution, the reflux extraction time is preferably 40 to 100 minutes, more preferably 50 to 60 minutes. The present invention can effectively extract the medicinal active ingredients such as flavonoids, quercetin, kaempferic acid, trifolioside and isoquercitrin from Lespedeza serrata through reflux extraction with water or alcohol solution.

[0030] The present invention performs filtration after the reflux extraction, retains the filtrate for standby use, adds solvent to the filter residue and repeats the aforementioned reflux extraction process again, and the number of repeated extractions is preferably 1. The present invention combines the filtrates obtained from each reflux extraction and performs a reduced pressure concentration treatment on the filtrate.

[0031] The present invention concentrates the filtrate under reduced pressure to 5-15% of the original filtrate volume, 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 an ethanol solution, and more preferably to 6-10% of the filtrate volume when the solvent is a methanol solution.

[0032] The present invention further reduces the pressure and concentrates the prepared concentrated solution to dryness to obtain a solid powdery Lespedeza pedunculata extract, which contains more active pharmaceutical ingredients and has pharmacological activity.

[0033] The present invention sequentially extracts the prepared concentrated solution with petroleum ether, ethyl acetate and n-butanol, retains the water-soluble part, and freeze-dries to obtain a solid powdered Lespedeza pedunculata extract. The present invention further extracts the concentrated solution to remove impurities soluble in petroleum ether, ethyl acetate and n-butanol, and further purifies to obtain a Lespedeza pedunculata extract with higher pharmacological activity. The present invention does not limit the specific extraction method, the amount of the extractant and the freeze-drying method.

[0034] The present invention uses n-butanol to repeatedly extract the prepared concentrated solution 2 to 3 times, retains the water-soluble part, and freeze-dries to obtain a solid powdered Lespedeza pedunculata extract. The present invention further extracts the concentrated solution to remove impurities soluble in n-butanol, and further purifies to obtain a Lespedeza pedunculata extract with higher pharmacological activity. The present invention does not limit the specific extraction method, the amount of the extractant, and the freeze-drying method.

[0035] The present invention sequentially extracts the petroleum ether, ethyl acetate and n-butanol or repeatedly extracts the n-butanol to obtain a water-soluble part, precipitates it with 80% ethanol, and freezes the alcohol-soluble part; washes the precipitate with anhydrous ethanol and / or acetone, and freezes the remaining precipitate after washing. When anhydrous ethanol and acetone are used for washing at the same time, the present invention does not limit the specific washing sequence. The present invention can further purify the Lespedunculate pedunculate extract with higher pharmacological activity by further processing the water-soluble part. The present invention does not limit the specific extraction method, the amount of the extractant and the amount of 80% ethanol.

[0036] The present invention also provides the use of the prepared Lespedeza lepedunculata extract in the preparation of a drug for treating otitis. The Lespedeza lepedunculata extract can significantly improve ear swelling in mice and has a significant anti-inflammatory effect.

[0037] The present invention also provides the use of the prepared Lespedeza pedunculata extract in the preparation of a drug for treating nephritis. The Lespedeza pedunculata extract can significantly reduce the serum BUN, CRE levels and spleen index of nephritis mice, and has a tendency to reduce serum CHO levels and reduce the 24h urine protein amount of nephritis mice. As an optional embodiment, the nephritis described in the present invention is doxorubicin nephritis, and a good anti-doxorubicin nephritis effect can be achieved.

[0038] The present invention also provides the use of the Lespedeza pedunculata extract prepared above in the preparation of a cell repair drug. The Lespedeza pedunculata extract can significantly alleviate doxorubicin-induced rat renal tubular epithelial NRK-52E cell damage, and as the concentration increases, the cell recovery effect of the Lespedeza pedunculata extract is enhanced, showing a good cell protection effect.

[0039] The present invention also includes a pharmaceutical composition prepared using the Lespedeza serrata extract of the present invention as a pharmaceutical active ingredient, and the pharmaceutical composition may include pharmaceutically acceptable excipients according to actual needs.

[0040] The pharmaceutical composition of the present invention can be in any pharmaceutically acceptable dosage form, including but not limited to pills, tablets, capsules, oral liquids, lozenges, granules, granules, pills, powders, pastes, pills, suspensions, powders, injections, suppositories, ointments, plasters, creams, sprays, drops or patches.

[0041] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0042] In the experimental result table of the present invention: "*" indicates p < 0.05 compared with the model control group, "**" indicates p < 0.01 compared with the model control group, and "***" indicates p < 0.001 compared with the model control group; "#" indicates p < 0.05 compared with the blank control group, "##" indicates p < 0.01 compared with the blank control group, and "###" indicates p < 0.001 compared with the blank control group.

[0043] Example 1

[0044] This example provides a method for preparing the water extract of Lespedeza serrata:

[0045] Take 500g of fine-stalked Lespedeza, crush it through an 80-mesh sieve, add 1.7L of water, boil it, reflux extract it for 30 minutes, and filter and collect the filtrate; add 1.7L of water to the filter residue again, boil it, reflux extract it for 30 minutes, and filter it; combine the two filtrates and concentrate them under reduced pressure to 0.5L.

[0046] Take 0.25L of the concentrate and continue concentrating to dryness to obtain the water extract of Lespedeza serrata, which is recorded as LEVH-1.

[0047] Example 2

[0048] In this example, another 0.25L of concentrated solution prepared in Example 1 was extracted three times in succession: extracted with 0.1L petroleum ether, and the extract and the water-soluble part were retained respectively; the water-soluble part was extracted again with 0.1L ethyl acetate, and the extract and the water-soluble part were retained respectively; the water-soluble part was extracted with 0.1L n-butanol, and the extract and the water-soluble part were retained respectively. The extracts obtained by the three extractions were respectively concentrated to dryness under reduced pressure and foamed into powders, and the products were successively recorded as LEVH-2, LEVH-3, and LEVH-4; the water-soluble part obtained after the three extractions was directly freeze-dried into powder to obtain the water extract of Lespedeza serrata, recorded as LEVH-5.

[0049] The extraction record of the effective parts of the water extract of Lespedeza serrata prepared in Examples 1 and 2 is shown in Table 1. (Extraction rate = the amount of sample extracted / the total mass of raw materials × 100%)

[0050] Table 1 LEV extract record

[0051]

[0052] Example 3

[0053] Take 10 g of the water extract of Lespedeza serrata (LEVH-5) obtained in Example 2, add 50 mL of 80% ethanol for precipitation, filter and separate the precipitate, and retain the alcohol-soluble part and the precipitate part respectively.

[0054] The alcohol-soluble part was concentrated and dried to obtain a solid product, which was recorded as LEVH-6;

[0055] The precipitate was further washed with 20 mL of 80% ethanol, and the washing solution was concentrated and dried to obtain a solid product, which was recorded as LEVH-8. The precipitate after washing was directly freeze-dried to obtain a water extract of Lespedeza serrata, which was recorded as LEVH-7.

[0056] The extraction records of each effective part of the water extract of Lespedeza serrata are shown in Table 2. (Extraction rate = the amount of sample extracted ÷ the total mass of raw materials × 100%)

[0057] Table 2 LEV extract record

[0058]

[0059] Example 4

[0060] 3000g of Lespedeza serrata was taken, crushed, added with 10.2L water, boiled, refluxed for 30min, filtered and collected the filtrate; 10.2L water was added to the filter residue again, boiled, refluxed for 30min, filtered; the two filtrates were combined and concentrated to 3L under reduced pressure. After the concentrated solution was cooled, it was extracted with 1L of n-butanol solution, the water-soluble part was retained, and the water-soluble part was repeatedly extracted with n-butanol twice; the extracts were combined and concentrated to dryness to obtain a solid product, which was recorded as LEVH-4b; the remaining aqueous solution was further concentrated to 1L, and 0.5L of the concentrated solution was further concentrated to dryness to obtain a water extract of Lespedeza serrata, which was recorded as LEVH-5b.

[0061] Example 5

[0062] Take another 0.5 L of the concentrated solution obtained after extraction in Example 4, add 2 L of 80% ethanol to precipitate, filter and separate the precipitate, and retain the alcohol-soluble part and the precipitate part respectively.

[0063] The alcohol-soluble part was concentrated to dryness to obtain a solid product, which was recorded as LEVH-6b;

[0064] The precipitate was concentrated to dryness to obtain a solid product, which was named LEVH-7b.

[0065] Example 6

[0066] Take 5 g (without water) of the LEVH-7b sample prepared in Example 5, wash it with 20 mL of anhydrous ethanol and 20 mL of acetone in sequence, and concentrate the washing liquid to dryness to obtain the product LEVH-8b and the product LEVH-9b respectively.

[0067] Example 7

[0068] This example provides a method for preparing the ethanol extract of Lespedeza serrata:

[0069] Take 500g of Lespedeza serrata, crush it and put it into a 10L round-bottom flask, add 5L of 40% ethanol water, reflux extraction for 1h, and filter and collect the filtrate; add 4L of 40% ethanol water to the residue, reflux for 1h, combine the two filtrates, and concentrate to a volume of 700ml.

[0070] 350 ml of the concentrated solution was directly freeze-dried to obtain the ethanol extract of Lespedeza serrata, which was recorded as LEVE-1.

[0071] Example 8

[0072] Take 350 mL of the remaining concentrated solution of Example 7 and perform three extraction treatments in succession: extract with 150 mL of petroleum ether, retain the extract and the water-soluble part respectively; extract the water-soluble part again with 100 mL of ethyl acetate, retain the extract and the water-soluble part respectively; extract the water-soluble part with 100 mL of n-butanol, retain the extract and the water-soluble part respectively. The extracts obtained by the three extractions were concentrated to dryness under reduced pressure to obtain solid products, which were recorded as LEVE-2, LEVE-3, and LEVE-4 respectively; the water-soluble part obtained after extraction was further concentrated and dried to obtain an ethanol extract of Lespedeza serrata, which was recorded as LEVE-5.

[0073] The extraction records of the effective parts of the ethanol extract of Lespedeza serrata prepared in Examples 7 to 8 are shown in Table 3. (Extraction rate = the amount of sample extracted / the total mass of raw materials × 100%)

[0074] Table 3 LEV extract record

[0075]

[0076] Experimental Example 1

[0077] This example studies the protective effect of Lespedeza lepedunculata extract on otitis media in mice.

[0078] 1. Mouse selection:

[0079] Animal quality: BALB / c mice, male, SPF grade, 18-20 g, Beijing Weitonglihua Experimental Animal Technology Co., Ltd., certificate number: (Beijing) 2012-0001.

[0080] Animal breeding environment: air-conditioning, room temperature 21-25℃; humidity 45±20%, ventilation 10 times / hour, fresh air. Artificial lighting for 12 hours. Free access to food and water.

[0081] Pre-experimental adaptation period: After the animals were introduced and inspected as required, they were housed in cages, 5 animals per cage, and observed for 3 days to observe their general condition. Their body weight was measured at the end of the adaptation period.

[0082] The mouse otitis model, carrageenan otitis model and dinitrofluorobenzene (DNFB) delayed-type hypersensitivity (DTH) model were induced by croton oil.

[0083] 2. Experimental instruments:

[0084] Analytical balance: BS 224S, Satorius; ELISA reader: Power Wave XS2, BioTek; Blood biochemistry analyzer: Accute 40R, TOSHIBA.

[0085] 3. Experimental methods:

[0086] 3.1 Group and dosage setting:

[0087] Blank control group: injected with an equal dose of vehicle (normal saline);

[0088] Model control group: injected with an equal dose of vehicle (normal saline);

[0089] High-dose group: 200 mg / kg body weight, subcutaneous injection;

[0090] Medium dose group: 100 mg / kg body weight, subcutaneous injection;

[0091] Low-dose group: 20 mg / kg body weight, subcutaneous injection;

[0092] The high, medium and low dose groups all used 1 g of the Lespedeza pedunculata extract powder prepared in Examples 1 to 4, which was dissolved in 5, 10 and 50 mL of normal saline, respectively, to obtain injections.

[0093] 3.2 Model establishment and drug administration:

[0094] 3.2.1 Croton oil-induced mouse otitis model

[0095] BALB / c mice were randomly divided into groups, 10 in each group, and subcutaneously injected with Lespedeza pedunculata extract (200 mg / kg) once, and the control group was given an equal volume of drug solvent. One hour after the last administration, 0.02 mL of croton oil was applied to both sides of the right ear of the mouse to cause inflammation, and the left ear was used as a normal control. One hour after the inflammation, the mice were killed by cervical dislocation, and the ear pieces at the same position of the left and right ears were removed with a 6 mm diameter puncher, and weighed. The difference in the mass of the left and right ear pieces represented the degree of inflammatory swelling. The swelling was used to calculate the inhibition rate (IR%) of each group on mouse croton oil otitis [mean ear swelling degree of the model group-ear swelling degree of the drug administration group) / mean ear swelling degree of the model group × 100%, and the results are shown in Table 4.

[0096] Table 4 Inhibitory effect of LEV extract on croton oil otitis in mice

[0097]

[0098] 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).

[0099] The results showed that the administration of LEVE-1, LEVE-5, LEVH-1, LEVH-2, LEVH-4, LEVH-5, LEVH-6, and LEVH-7 samples could significantly improve croton oil otitis in mice, while the administration of other groups had no significant effect on croton oil otitis in mice.

[0100] 3.2.2 DNFB-induced DTH model in mice

[0101] BALB / c mice were randomly divided into groups, 10 mice in each group, and subcutaneously injected with Lespedeza pedunculata extract (100 mg / kg) once, and the control group was given an equal volume of drug solvent. One hour after administration, the control group was treated with a solvent without DNFB, and the DTH group was sensitized with 5g / L DNFB in the abdomen and stimulated by the auricle to establish a model. 48 hours after stimulation, the ear pieces at the same position of the left and right ears were removed with a 6mm diameter puncher and weighed. The difference in the mass of the left and right ear pieces indicated the degree of inflammatory swelling. The inhibition rate (IR%) of each group was calculated using the swelling degree. The results of the re-screening of DTH in mice in each group are shown in Table 5.

[0102] Table 5 Inhibitory effect of LEV extract on mouse DTH model

[0103]

[0104] As shown in Table 5, compared with the model group, LEVE-1 (p < 0.05), LEVH-1 (p < 0.05), LEVH-2 (p < 0.05), and LEVH-7 (p < 0.01).

[0105] The results in Table 5 show that the administration of LEVE-1, LEVH-1, LEVH-2, and LEVH-7 samples can significantly improve ear swelling in mice, while administration of other groups had no significant effect on ear swelling in mice.

[0106] 3.2.3 Carrageenan-induced mouse otitis model

[0107] BALB / c mice were randomly divided into groups, 10 in each group, and subcutaneously injected with Lespedeza pedunculata extract (20 mg / kg of Lespedeza extract was subcutaneously injected once a day for 6 consecutive days), and the control group was given an equal volume of drug solvent. 1 hour after the last administration, 0.02 mL of carrageenan was applied to both sides of the right ear of the mouse to induce inflammation, and the left ear was used as a normal control. After 1 hour of inflammation, the mice were killed by cervical dislocation, and the ear pieces at the same position of the left and right ears were removed with a 6 mm diameter puncher, and weighed. The difference in the mass of the left and right ear pieces indicated the degree of inflammatory swelling. The swelling degree was used to calculate the inhibition rate (IR%) of each group. The results of the re-screening of carrageenan inflammation in mice in each group are shown in Table 6.

[0108] Table 6 Effects of LEV extract on carrageenan inflammation model in mice

[0109]

[0110] As shown in Table 6, compared with the model group, administration of LEVH-7 sample can significantly improve ear swelling in mice (p < 0.05), while administration of other groups had no significant effect on ear swelling in mice.

[0111] The above results show that LEVE-1, LEVE-5, LEVH-1, LEVH-2, LEVH-4, LEVH-5, LEVH-6, and LEVH-7 can significantly improve croton oil otitis in mice; in the delayed-type hypersensitivity (DTH) model of mice induced by dinitrofluorobenzene (DNFB), after the administration of the medium-dose group of Lespedeza extract, the administration of LEVE-1, LEVH-1, LEVH-2, and LEVH-7 samples can significantly improve the ear swelling of mice; in the carrageenan otitis model of mice, after the administration of the low-dose group of Lespedeza extract, the administration of LEVH-7 samples can significantly improve the ear swelling of mice. It can be seen that Lespedeza extract has a certain therapeutic effect on mouse otitis.

[0112] Experimental Example 2

[0113] This example studies the protective effect of Lespedeza pedunculata extract on doxorubicin nephritis in mice.

[0114] 1. Mouse selection:

[0115] Animal quality: BALB / c mice, male, SPF grade, 18-20 g, Beijing Weitonglihua Experimental Animal Technology Co., Ltd., certificate number: (Beijing) 2012-0001.

[0116] Animal breeding environment: air-conditioning, room temperature 21-25℃; humidity 45±20%, ventilation 10 times / hour, fresh air. Artificial lighting for 12 hours. Free access to food and water.

[0117] Pre-experimental adaptation period: After the animals were introduced and inspected as required, they were housed in cages, 5 animals per cage, and observed for 3 days to observe their general condition. Their body weight was measured at the end of the adaptation period.

[0118] 2. Doxorubicin: purity 98%, product number T1020, CAS number 25316-40-9, TARGET MOL.

[0119] 3. Experimental methods:

[0120] 3.1 Group and dosage setting:

[0121] Blank control group: equal dose of vehicle (normal saline);

[0122] Model time group: equal dose of vehicle (normal saline);

[0123] High dose group: 5.0g / Kg, 0.5g / ml, 0.1ml / 10g;

[0124] Low dose group: 2.5g / Kg, 0.25g / ml, 0.1ml / 10g;

[0125] (In the high-dose group, 5.0 g / Kg is the dosage per kilogram of mice; 0.5 g / ml means 0.5 g of extract is dissolved per mL of solvent; 0.1 ml / 10 g means 0.1 mL of solvent is used per 10 grams of mice; the same is true for the low-dose group. The drugs in both the high and low-dose groups are LEVH-7 prepared in Example 3)

[0126] 3.2 Model establishment and drug administration:

[0127] 40 BALB / c mice were randomly divided into 4 groups, 10 mice in each group, and weighed. Except for the blank control group (administered with equal volume of solvent), each group of animals was injected with doxorubicin (10 mg / Kg) through the tail vein once to establish the mouse doxorubicin nephritis model; oral administration began on the 8th day after modeling, once a day, for 21 consecutive days; after the last administration, urine was collected, and the mice were killed 24 hours later, and samples were taken for index detection.

[0128] 4. Experimental results:

[0129] 4.1 General growth of animals:

[0130] The animals were weighed once every 7 days, with weight 0 being the initial weight before modeling, weight 1 being the weight after modeling, weight 2 being the weight after the first week, weight 3 being the weight after the second week, and weight 4 being the weight after the third week. The effects of the drugs on body weight were observed, and the results are shown in Table 7.

[0131] Table 7. Effect of LEV extract on body weight of mice with doxorubicin nephritis (Mean±Std, n=10)

[0132]

[0133] As shown in Table 7, compared with the blank control group, the body weight of the model control group was significantly reduced after modeling (p < 0.05 or 0.01); compared with the model group, the administration of Lespedeza serrata extract had no significant effect on the reduction of modeling body weight (p > 0.05).

[0134] 4.2 24h urine protein:

[0135] The results of the effect of Lespedeza gracilis extract on 24-hour urine protein in doxorubicin nephritis mice are shown in Table 8.

[0136] Table 8. Effect of LEV extract on 24h urine protein in nephritis mice (Mean±Std, n=3)

[0137]

[0138] As shown in Table 8, compared with the blank control group, the amount of urine protein in mice after doxorubicin modeling was significantly increased (p < 0.01); and compared with the model control group, the extract of Lespedeza pedunculatea could reduce the amount of 24-hour urine protein in nephritis mice, but no significant difference was observed (p > 0.05).

[0139] 4.3 Blood biochemical test:

[0140] The biochemical indexes of mouse serum were tested, and the results are shown in Table 9.

[0141] Table 9. Effect of LEV extract on serum biochemical test results of nephritis mice (Mean±Std, n=10)

[0142]

[0143] As shown in Table 9, compared with the blank control group, the serum BUN, CRE and CHO levels of doxorubicin nephritis mice were significantly increased (p < 0.05 or 0.01); administration of Lespedeza pedunculata extract can reduce the serum BUN, CRE and CHO levels of nephritis mice, among which, the BUN and CRE in the high-dose group were significantly different from those in the model group (p < 0.05 or 0.01).

[0144] 4.4 Organ index:

[0145] The organ indexes of kidney, thymus and spleen of mice were tested. The results are shown in Table 10.

[0146] Table 10. Effects of LEV extract on organ indexes of nephritis mice (Mean±Std, n=10)

[0147]

[0148] As shown in Table 10, compared with the blank control group, the spleen index of doxorubicin nephritis mice was significantly increased, while the administration of Lespedeza pedunculata extract could significantly reduce the spleen index of nephritis mice (p < 0.05 or 0.01); the administration of Lespedeza pedunculata extract had no significant effect on the renal index and thymus index of nephritis mice.

[0149] The above results show that the high-dose group of Lespedeza pedunculata extract can significantly reduce the serum BUN, CRE levels and spleen index of nephritis mice, and has a tendency to reduce serum CHO levels and 24h urine protein in nephritis mice. This shows that the Lespedeza pedunculata extract has a certain therapeutic effect on doxorubicin-induced nephritis in mice.

[0150] Experimental Example 3

[0151] This example studies the protective effect of Lespedeza pedunculata extract on the injury of rat renal tubular epithelial NRK-52E cells.

[0152] 1. Experimental instruments and materials

[0153] DMSO: solarbio D8371, dimethyl sulfoxide, cell culture;

[0154] Doxorubicin: purity 98%, product number T1020, CAS number 25316-40-9, TARGET MOL;

[0155] Dexamethasone: Dexamethasone, cas number 50-02-2, product number B76795, innochem, 1G;

[0156] 1 / 10,000 electronic balance: ME-104, Mettler; ultrasonic cleaning machine: Deep Ultra Clean.

[0157] 2. Experimental model construction

[0158] Cell line: NRK-52E cell

[0159] Doxorubicin was used to induce rat renal tubular epithelial NRK-52E cell injury model.

[0160] 3. Experimental methods:

[0161] 3.1 Experimental steps:

[0162] The density of NRK-52E cells in the logarithmic growth phase was adjusted to 0.5×105 / mL and 1×105 / mL, and 100 μL was inoculated into 96-well plates per well and cultured in an incubator at 37°C and 5% CO2 for 4 h to adhere to the wall; an equal dose of solvent or Lespedeza extract was added to each well for pre-incubation for 1 h; an equal dose of solvent was added to the blank control group, and 0.5 ug / ml doxorubicin was added to the other groups for treatment for 24 h; CCK8 was used to detect cell activity.

[0163] 3.2 Groups and dosage settings:

[0164] Blank control group: equal dose of vehicle (normal saline);

[0165] Model control group: equal dose of vehicle (normal saline);

[0166] Positive control group: 10 μm dexamethasone;

[0167] Low-dose extract group: 10ug / mL;

[0168] Low-dose group of extract: 50ug / mL;

[0169] Medium and high dose group of extract: 100ug / mL;

[0170] High-dose extract group: 200ug / mL;

[0171] (The extracts are all LEVH-7 prepared in Example 3 of the extraction process)

[0172] 4. Experimental Results

[0173] The effects of Lespedeza serrata extract on doxorubicin-induced rat renal tubular epithelial NRK-52E cell injury model are shown in Table 11.

[0174] Table 11. Effects of LEV extract on doxorubicin-induced rat renal tubular epithelial NRK-52E cell injury model (mean ± SD, n = 6)

[0175]

[0176] As shown in Table 11, compared with the normal control group, the activity of NRK-52E cells induced by doxorubicin was significantly reduced (p < 0.001); compared with the model control group, the positive drug dexamethasone can significantly increase the activity of NRK-52E cells induced by doxorubicin (p < 0.001); compared with the model control group, the low, medium-low, medium-high and high concentrations of the extract of Lespedeza serrata can significantly reduce the damage of rat renal tubular epithelial NRK-52E cells induced by doxorubicin (p < 0.001), and as the concentration increases, the effect of the extract of Lespedeza serrata is better; and at low, medium-low, medium-high and high concentrations, the extract of Lespedeza serrata did not show obvious cytotoxicity. It shows that the extract of Lespedeza serrata can significantly alleviate the damage of rat renal tubular epithelial NRK-52E cells induced by doxorubicin, and as the concentration increases, the cell recovery effect of the extract of Lespedeza serrata is enhanced.

[0177] Experimental Example 4

[0178] This embodiment provides a capsule containing Lespedeza serrata extract: take 20 g of Lespedeza serrata extract, add 29.5 g of dextrin and 0.5 g of magnesium stearate, mix well, make 1000 capsules according to the capsule preparation method, fill them into 50 mg capsules, each capsule contains 20 mg of Lespedeza serrata extract.

[0179] Experimental Example 5

[0180] This embodiment provides a tablet containing Lespedeza pedunculata extract: take 20g of Lespedeza pedunculata extract, add 20g of starch, 16g of dextrin, and 3g of sucrose, stir and mix thoroughly to make granules, dry below 60°C, add appropriate amount of magnesium stearate and talcum powder, mix well, press into 1000 tablets, and coat with sugar or film to obtain.

[0181] Experimental Example 6

[0182] This embodiment provides a granule containing Lespedeza serrata extract: 20 g of Lespedeza serrata extract is added with appropriate amounts of sucrose and dextrin, granulated according to a granule preparation method, and dried to obtain 1000 g.

[0183] Experimental Example 7

[0184] This embodiment provides an oral liquid containing Lespedeza serrata extract: take 20g of Lespedeza serrata extract, add water to dissolve, adjust the pH value to 7.0 with 5% sodium hydroxide, stir well, refrigerate at 4°C for 48 hours, filter, add 60g of sucrose to the filtrate, stir well to dissolve, then add appropriate amount of flavor and adjust the pH value to 7.0, add water to make 1000ml, stir well, let stand for 12 hours, filter, fill, sterilize, and obtain.

[0185] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a Lespedeza serrata extract, characterized in that: The following steps are involved: The fine-stalked Lespedeza was crushed, water was added and refluxed for extraction for 0.5 to 3 hours, filtered, water was added to the filter residue and the extraction was repeated 1 to 2 times, filtered, the filtrate was combined, and concentrated under reduced pressure to obtain a concentrate, the concentrate was extracted with petroleum ether, ethyl acetate and n-butanol in sequence, the water-soluble part was retained, the water-soluble part was precipitated with 80% ethanol, the precipitated part was washed with anhydrous ethanol, and the remaining precipitate after washing was freeze-dried to obtain the fine-stalked Lespedeza extract.

2. The preparation method according to claim 1, characterized in that: The amount of water added is 2 to 5 L / kg.

3. Use of the Lespedeza serrata extract obtained by the preparation method according to any one of claims 1 to 2 in the preparation of drugs for treating inflammation.

4. The use according to claim 3, characterized in that: The inflammation is nephritis.

5. A pharmaceutical composition for treating nephritis, characterized in that: The pharmaceutical composition contains an effective dose of the Lespedeza serrata extract obtained by the preparation method according to any one of claims 1 to 2 and pharmaceutically acceptable excipients.

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