CN106974917B - Application of exodermis poria triterpene in preparation of medicine for treating nephropathy - Google Patents
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Abstract
Aiming at the problems that the effect of the current nephropathy treatment medicament on some cases is still not ideal and the toxicity of the traditional Chinese medicine extract is high, the invention provides the application of the poria peel triterpene with safety and obvious curative effect in preparing the nephropathy treatment medicament, wherein the poria peel triterpene is an ethanol extract of the poria peel. The exodermis triterpene can relieve the pathological change degree of the kidney, reduce the urine protein content caused by the kidney disease, reduce creatinine and urea nitrogen, and play a role in treating the kidney disease.
Description
Technical Field
The invention belongs to the field of medicinal preparations, and relates to application of poria peel triterpenes in preparation of a medicament for treating nephropathy.
Background
In recent years, the incidence of chronic nephritis has been on an increasing trend year by year with the increase of environmental pollution, the acceleration of the pace of people's work and life, the increase of professional competition, the change of dietary structure and the abuse of antibiotics. The survey results showed that the prevalence of chronic kidney disease in adults in our country was 10.8%, from which it was estimated that there were about 1.2 million patients with chronic kidney disease in people over 18 years old. The disease is a chronic disease, has long course of disease and great harm, seriously affects health and work, is used for economic loss of medical cost of chronic nephritis and the like every year in China, reaches hundreds of millions of yuan every year, and brings huge loss to social production and economy. Chronic nephritis is a disease mainly manifested by proteinuria, hematuria, edema and hypertension, which has occult onset and prolonged course of disease, and has significant difference in prognosis of different pathological types, most patients have slow disease progression, but the disease will eventually progress to renal failure, and no specific treatment method exists in modern medicine at present. At present, western medicine treatment is mainly used in clinical treatment, mainly hormone and cytotoxic drugs are used as therapeutic drugs, and the hormone has the property of yang rigidity, is easy to damage yin fluid and is easy to cause damp-heat; cytotoxic drugs can cause bone marrow suppression and liver function damage, and have poor treatment effect and great side effect. Researches show that the traditional Chinese medicine can remarkably relieve clinical symptoms of patients with chronic nephritis, improve the life quality of the patients, and improve the indexes of quantitative urine protein, hematuria, urine microalbumin and the like.
Nephrotic Syndrome (NS) is a group of clinical syndromes with kidney damage due to multiple etiologies, and is a common manifestation of glomerular disease. TCM has no name, and can be classified into edema, consumptive disease, lumbago and the like according to its clinical manifestations. The NS is mainly treated by glucocorticoid, immunosuppressant, symptomatic support and other methods in western medicine, and has long treatment course, easy recurrence, easy generation of hormone dependence and toxic and side effects, although the curative effect is good. Practice proves that the effect of the hormone alone or the immunosuppressant is still not ideal in some cases, and the recurrence rate is high. In addition, the use of hormones and immunosuppressants results in reduced immune function, which in turn increases infection and causes the disease to recur.
The traditional Chinese medicine has a long history of treating nephrotic syndrome, and the nephrotic syndrome can be classified into 4 types in traditional Chinese medicine, namely lung-spleen-kidney yang deficiency type, kidney-qi deficiency type, qi-yin deficiency type and liver-kidney yin deficiency type. The clinically used prescription for treating the disease comprises: the traditional Chinese medicine composition can be used for treating kidney-yang deficiency type by using Jisheng kidney-qi pills, lung-kidney-qi deficiency type by using Xiangsha Liujunzi decoction, liver-kidney-yin deficiency type by using Zhibai Dihuang decoction, qi-yin deficiency type by using Shenqi Dihuang decoction, damp-heat type by using Sanren decoction and wrinkled Gianthyssop Zuojin decoction, damp-damp type by using Wupi Yin and Wuling powder, damp-turbidity type by using Weiling decoction, and blood stasis type by using Yang tonifying and Wu Tang. The Chinese medicine for treating nephrotic syndrome is prepared with Tripterygium wilfordii as main component, and belongs to the field of toxic medicine. In recent years, the extracted tripterygium glycosides are adopted to treat the disease, and the tripterygium glycosides have good curative effect on eliminating proteinuria and have no drug resistance. The composition has effects of inhibiting immunity, relieving inflammation, inhibiting proliferation of mesangial cells, and improving permeability of glomerular filtration membrane. In clinical treatment, tripterygium glycosides are found to cause a small amount of leucopenia of patients, and the adverse reactions of nausea, stomach discomfort, pain, diarrhea and the like generally exist in the digestive system. In the case of an elderly patient suffering from renal insufficiency, the symptoms of renal insufficiency are aggravated and renal impairment is caused. Therefore, patients over 50 years old must be timely tested for liver and kidney function. Tripterygium glycosides have a great influence on reproductive systems, so that the application is limited, and development of new Chinese medicines which are safe, effective, stable in efficacy and convenient to take is urgently needed.
The poria peel is dry sclerotium peel of poria cocos (poricos (Schw.) Wolf) of polyporaceae, the traditional Chinese medicine considers that poria cocos is sweet, light and neutral in taste and has the effects of promoting diuresis and excreting dampness, benefiting spleen and harmonizing stomach, and calming heart and tranquilizing, the prior research shows that the poria peel has the effects of immunoregulation, anti-tumor, anti-convulsion, bacteriostasis and anti-inflammation, etc. the research on the pharmacodynamics and pharmacokinetics of the poria cocos ethanol extract on nephrotic syndrome in the research on the pharmacodynamics and pharmacokinetics of nephrotic syndrome in the research on the poria cocos ethanol extract of China lao-hoku (university of Chinese medicine 2012) of Shen has carried out on the pharmacodynamics and pharmacokinetics of the poria cocos ethanol extract on the nephrotic syndrome, and shows that dehydro terra acid, dehydro pachyma acid, dehydro cocos acid and pachyma acid are the basis of exerting the pharmacodynamics ethanol extract of the efficacy in poria cocos triterpene, however, compared with the poria cocos peel, the chemical components are complex, the polysaccharides, the triterpene, the research shows that the poria peel is different from the application of a method for extracting effective component CN-21-84 of poria cocos peel, namely, the effective component of poria cocos peel is disclosed in a patent application, namely, a method for extracting effective component of poria cocos peel-3, and a method for extracting effective component for a method for extracting a.
Disclosure of Invention
Aiming at the problems that the effect of the current nephropathy treatment medicament on some cases is still not ideal and the toxicity of the traditional Chinese medicine extract is high, the invention provides the application of the poria peel triterpene with safety and obvious curative effect in preparing the medicine for treating nephropathy.
In order to achieve the purpose, the invention adopts the following technical scheme.
An application of the triterpene of Poria peel in preparing the medicines for treating nephrosis is disclosed, wherein the triterpene of Poria peel contains 3 β -hydroxy-lanosta-7, 9(11), 24-triene-21-acid and dehydroeiric acid as main active components.
The nephropathy is nephrotic syndrome and nephritis.
The total content of the 3 β -hydroxy-lanosta 7,9(11), 24-triene-21-acid and dehydroeiric acid in the poria peel triterpene is more than 50% w.t.
The poria peel triterpene is an ethanol extract of poria peel, and is extracted by the following method:
the poria peel triterpene is ethanol extract of poria peel
(1) Washing exodermis Poria with 1.0-2.0 wt% hydrochloric acid, washing with water to neutral, and air drying;
(2) reflux-extracting the poria peel treated in the step (1) for 2 times by using 70% ethanol solution in volume fraction, wherein the extraction time is 0.5h each time, the dosage of the poria peel and the ethanol solution is 1:10(g/ml), combining the extracting solutions, and recovering ethanol from the extracting solution until the ethanol is dried;
(3) dissolving the dry matter obtained in the step (2) into a solution by using water, adding a mixture of n-butyl alcohol and ethyl acetate in a volume ratio of 7:1.5 for extraction, wherein the volume of the mixture of n-butyl alcohol and ethyl acetate is 1.5-2 times that of the aqueous solution, and recovering the solvent from the organic phase until the organic phase is dried to obtain a crude product;
(4) dissolving the crude product with 100-120 times of ethanol by mass to prepare a solution, loading the obtained solution on a neutral alumina chromatographic column, wherein the mass of the neutral alumina is 15-20 times of that of the crude product, then eluting with a mixture of ethanol and ethyl acetate in a volume ratio of 2:5, and collecting the eluent, wherein the use amount of the mixture of ethanol and ethyl acetate is 2 times of that of the alumina;
(5) adding active carbon into the eluate, wherein the amount of active carbon is 2-3% of the crude product, heating to boil, maintaining for 30min, filtering while hot, washing with ethanol, mixing the filtrate and washing solution, concentrating to 1/3, standing for precipitation, filtering, and drying to obtain Poria peel triterpene.
The invention has the following advantages:
the poria peel triterpene has the activities of resisting tumor, resisting inflammation, regulating immunity, enhancing insulin and the like, can relieve the pathological change degree of the kidney, reduce the urine protein content caused by the nephropathy, reduce creatinine and urea nitrogen, and has a treatment effect on the nephropathy.
Drawings
FIG. 1 is a liquid chromatogram of 3 β -hydroxy-lanosta 7,9(11), 24-triene-21-oic acid and dehydroeiric acid on a pachyman triterpene standard;
FIG. 2 is a liquid chromatogram of the triterpene extract from Poria peel;
FIG. 3 is a graph showing the effect of pachyman peel triterpene on the change in body weight of doxorubicin hydrochloride-induced nephrotic syndrome in rats.
Detailed Description
The present invention will be further described with reference to the following examples and drawings, but the present invention is not limited to the following examples.
Example 1 extraction preparation of a triterpene from hoelen skin.
(1) Washing exodermis Poria with 1.5% hydrochloric acid, washing with water to neutral, and air drying;
(2) reflux-extracting exodermis Poria with 70% ethanol solution for 2 times (each time for 0.5 hr) at a ratio of 1:10(g/ml), mixing extractive solutions, and recovering ethanol from the extractive solution to dry;
(3) dissolving the dry matter obtained in the step (1) into a solution by using water, adding a mixture of n-butyl alcohol and ethyl acetate in a volume ratio of 7:1.5 for extraction, wherein the volume of the mixture of n-butyl alcohol and ethyl acetate is 2 times that of the aqueous solution, and recovering the solvent from the organic phase until the solvent is dried to obtain a crude product;
(4) dissolving the crude product with 100-120 times of ethanol by mass to prepare a solution, loading the obtained solution on a neutral alumina chromatographic column, wherein the mass of the neutral alumina is 15 times of that of the crude product, then eluting with a mixture of ethanol and ethyl acetate in a volume ratio of 2:5, and collecting the eluent, wherein the dosage of the mixture of ethanol and ethyl acetate is 2 times of that of the alumina;
(5) adding active carbon into the eluate, heating to boil and keeping for 30min, filtering while hot, washing with ethanol, mixing the filtrate and washing solution, concentrating to 1/3, standing for precipitation, filtering, and drying to obtain Poria peel triterpene.
Example 2 determination of effective ingredients in a triterpene from hoelen peel.
The contents of hydroxy-lanostane 7,9(11), 24-triene-21-acid and dehydroeiric acid in the extract are determined by the following method:
instruments and reagents: waters high performance liquid chromatography system, Water600 pump, 996 diode array detector, M32 chromatographic work station, Lichrospher-C18 column, 200X 4.6mm, 5 mu (product of Jiangsu Hanbang science Co., Ltd.).
The reagents are acetonitrile (chromatographic pure grade), 3 β -hydroxy-lanostane 7,9(11), 24-triene-21-acid and dehydroeibriic acid (prepared by a novel traditional Chinese medicine preparation center in Shandong province, and the content of the preparation is more than 98.0 percent).
Detection conditions are as follows: mobile phase: acetonitrile: 0.5% acetic acid solution (80:20), detection wavelength 242 nm.
Preparation of control solution by precisely weighing 3 β -hydroxy-7, 9(11), 24-triene-21-acid control, placing in 10mL volumetric flask, diluting with methanol to scale, and making into 228 μ g/mL-1The control solution of (4); accurately weighing dehydroeiric acid reference substance, placing into 10mL volumetric flask, diluting with methanol to scale, and making into 130 μ g/mL-1The control solution of (4).
Preparation of a test solution: precisely weighing about 40mg of the poria peel triterpene in example 1, placing the poria peel triterpene in a 100mL volumetric flask, dissolving the poria peel triterpene in methanol, diluting the poria peel triterpene to a scale mark, shaking the poria peel triterpene uniformly, filtering the solution by using a 0.45-micron microporous membrane, and taking filtrate to obtain the poria peel triterpene.
The determination method comprises precisely sucking 10 μ L of each of the reference solution and the sample solution, respectively injecting into a liquid chromatograph to obtain chromatogram 1 of the reference and chromatogram 2 of the extract, wherein according to peak area calculation, the content of 3 β -hydroxy-lanostane 7,9(11), 24-triene-21-acid is 30.15%, the content of dehydroeiubic acid is 28.19%, and the total content of the two substances is 58.34%.
Example 3 Effect of Poria peel triterpenes on rat Doxorubicin nephrotic syndrome.
1 materials and methods
1.1 instruments and drugs
Ultraviolet spectrophotometer model WFZ UV-PC (yuniko (shanghai) instruments ltd); DXL-DL metabolic cage: von experimental animal facilities ltd, Jiangsu province; calf serum albumin: west ampere walson biotechnology limited; coomassie Brilliant blue G-250: shanghai chemical reagents, China pharmaceutical group; urine protein test paper: golbao biotechnology, Guangdong; doxorubicin hydrochloride: zhejiang Haizheng pharmaceutical industry Co., Ltd, 10 mg/count, lot No. 130201, when in use, injection of normal saline as solvent, prepared into 2mg/ml solution; the sample in example 1 is adopted for the poria peel triterpene, and olive oil is used as a solvent for preparation of a solution of 10mg/ml for use.
1.2 animals
Healthy male Wistar rats with the weight of 158 +/-13 g are selected and provided by the experimental animal center of Qingdao city (the qualification number: SCXK (lu) 2014-.
1.3 preparation of the model
25 Wistar rats are male, are fed for three days in compliance, have no adverse reaction, randomly select 20 rats to be injected with 6.5mg/kg of doxorubicin hydrochloride in the tail vein, are additionally injected with 4mg/kg of doxorubicin hydrochloride after one week, detect the molding condition by using urine protein test paper during the molding period, two weeks after molding, collecting urine for 24 hours, measuring the urine protein content by a Coomassie brilliant blue method, rejecting a molding group and a normal group without difference, and randomly dividing the successfully molded rats into a normal control group (normal saline 10 ml/kg. d), a model control group (normal saline 10 ml/kg. d), a small-dose group (poria skin triterpene 42 mg/kg. d), a medium-dose group (the poria skin triterpene is 84 mg/kg. d) and a high-dose group (the poria skin triterpene is 168 mg/kg. d). The administration was continued for 30 days.
1.4 index detection
The white rats are respectively placed in clean metabolism cages on the first 1 day before molding, the 7 th day after molding and the 14 th day after molding, and the 7 th day after administration, the 14 th day, the 21 st day and the 30 th day after administration, 24h urine samples are left under the conditions of fasting and free drinking, the content of urine protein is measured by protein test paper, and the weight change and the general index changes such as food intake, body hair, stool, mental state, edema and the like of the rats are measured at the same time.
1.5 statistical analysis
SPSS19.0 software is adopted for statistical treatment, the measured data is represented by mean plus or minus standard deviation (X plus or minus s), the data statistics is analyzed by single-factor variance, and pairwise comparison is carried out among groups. The difference is marked by p < 0.05.
2 results
2.1 Effect on rat body weight
The effect of exodermis poria triterpene on the change of body weight of rat nephrotic syndrome caused by doxorubicin hydrochloride is shown in table 1 and figure 3. Compared with the normal group, the model group rats showed a significant decrease in body weight after doxorubicin hydrochloride injection (p < 0.01). On the 7 th day after intragastric administration, the weight of the perilladerm triterpenoid intragastric administration group is obviously increased compared with that of the model group. Compared with the model group, the difference of the medication group is not obvious.
TABLE 1 Effect of Poria peel triterpenes on Doxorubicin hydrochloride-induced changes in body weight in nephrotic syndrome in rats
| Group of | Normal (g) | Model (g) | Low dose group (g) | Medium dose group (g) | High dose group (g) |
| 1 day before molding | 163.6±4.7 | 172.7±8.1 | 158.0±10.5 | 162.0±12.1 | 162.3±10.5 |
| 7 days after molding | 208.0±5.96 | 180.0±2.8** | 174.7±23.8* | 180.0±17.4* | 166.0±12.1* |
| 14 days after molding | 239.4±5.0 | 187.0±12.2** | 170.0±28.1* | 194.5±23.1* | 184.3±7.5* |
| 7 days after the administration | 252.8±3.6 | 187.2±16.9** | 172.6±73.6* | 224.6±23.1*△ | 218.0±3.4*△ |
| 14 days after the administration | 252.8±6.5 | 180.7±27.7** | 173.6±36.4* | 228.6±33.5* | 218.0±3.4*△ |
| 21 days after the administration | 261.6±9.3 | 173.0±28.8** | 173.6±36.4*△ | 223.3±64.0* | 252.0±10.1*△ |
| 30 days after the administration | 268.6±5.0 | 169.2±29.8** | 181.3±25.5* | 226.6±57.7*△ | 255.3±5.6*△△ |
P compared to normal group<0.05,**p<0.01, compared to the model set,△P<0.05,△△P<0.01。
2.2 Effect on general indicators of nephrotic syndrome in rats
Different degrees of emotional agitation appear on the 7 th day of 20 rats injected with doxorubicin hydrochloride by tail vein, the food consumption is reduced, the body hair is messy and dull, the tail part is red and swollen, and the tail rot phenomenon appears on the 10 th day. The symptoms of each treatment group are light in the middle period of administration, and the health condition of the treatment group is not obviously different from that of the normal group in the later period of administration.
2.3 Effect on rat urinary protein
The effect of exodermis poria triterpene on rat urokinase protein of doxorubicin hydrochloride-induced rat nephrotic syndrome is shown in tables 2-8. As can be seen from the data in the table, after doxorubicin hydrochloride injection, the urinary protein of rats was significantly increased compared to the normal group, and the typical nephrotic syndrome manifestation appeared 14 days after administration. After the tuckahoe peel triterpene is perfused into the stomach, the urine protein amount of the tuckahoe peel triterpene perfused into the stomach is reduced compared with that of a model group, wherein the high dose group is obviously improved. In tables 2 to 8, "+" indicates that urine protein is present in urine at 0.3 g/L; "+ +" indicates that urine protein is 1.0g/L in urine; "+ + + +" indicates that there is 3.0g/L urine protein in urine; "+ ++" indicates that 20g/L of urine protein is present in the urine.
TABLE 2 results of 24h urine protein detection 1 day before molding
| Is normal | Model (model) | Is low in | In | Height of | |
| 1 | - | - | - | - | - |
| 2 | - | - | - | - | - |
| 3 | - | - | - | - | - |
| 4 | - | - | - | - | - |
| 5 | - | - | - | - | - |
TABLE 3 results of 24h urine protein assay 7 days after molding
| Is normal | Model (model) | Is low in | In | Height of | |
| 1 | - | + | + | + | + |
| 2 | + | + | + | ++ | + |
| 3 | - | + | + | + | + |
| 4 | - | ++ | + | + | + |
| 5 | - | + | ++ | + | + |
TABLE 4 results of 24h urine protein assay 14 days after molding
| Is normal | Model (model) | Is low in | In | Height of | |
| 1 | - | ++ | ++ | ++ | +++ |
| 2 | - | +++ | +++ | +++ | +++ |
| 3 | - | +++ | ++ | ++ | ++ |
| 4 | - | +++ | +++ | +++ | ++ |
| 5 | - | +++ | +++ | +++ | ++ |
TABLE 5 results of 24h urine protein assay 7 days after drug administration
| Is normal | Model (model) | Is low in | In | Height of | |
| 1 | - | +++ | +++ | +++ | ++ |
| 2 | - | +++ | ++ | +++ | ++ |
| 3 | - | +++ | + | +++ | +++ |
| 4 | - | +++ | ++ | +++ | ++ |
| 5 | - | +++ | +++ | +++ | ++ |
TABLE 6 results of 24h urine protein assay 14 days after drug administration
| Is normal | Model (model) | Is low in | In | Height of | |
| 1 | - | +++ | +++ | ++ | ++ |
| 2 | - | ++++ | ++ | ++ | ++ |
| 3 | - | ++++ | + | ++ | +++ |
| 4 | - | Death was caused by death | +++ | ++ | ++ |
| 5 | - | ++++ | +++ | +++ | ++ |
TABLE 7 results of 24h urine protein assay 21 days after drug administration
| Is normal | Model (model) | Is low in | In | Height of | |
| 1 | + | ++++ | +++ | ++ | ++ |
| 2 | - | ++++ | +++ | ++ | ++ |
| 3 | - | ++++ | + | ++ | ++ |
| 4 | - | Death was caused by death | +++ | +++ | ++ |
| 5 | - | ++++ | ++ | ++ | Death was caused by death |
Table 8 results of 24h urine protein assay 30 days after drug administration.
| Is normal | Model (model) | Is low in | In | Height of | |
| 1 | - | ++++ | +++ | +++ | ++ |
| 2 | - | ++++ | +++ | +++ | ++ |
| 3 | - | ++++ | Death was caused by death | ++ | ++ |
| 4 | -- | Death was caused by death | +++ | ++ | ++ |
| 5 | - | ++++ | +++ | +++ | Death was caused by death |
The doxorubicin hydrochloride induces the nephrotic syndrome of rats, which belongs to tiny pathological nephropathy, and gradually develops into focal glomerulosclerosis later. After 2 times of tail vein injection of doxorubicin hydrochloride, the rat can be caused to have tail rot, diet reduction, emotional restlessness, weight reduction and other general indexes changed. Wherein, the body weight is an important non-specific observation index in animal experiments and can comprehensively reflect the toxic reaction of animal organisms. After doxorubicin hydrochloride is injected in an experiment, the weight growth speed of a rat is obviously slowed down, the phenomenon of urine protein appears, the weight growth speed is recovered after gastric lavage, the urine protein level can be obviously reduced in three dose groups of low (42 mg/kg-d), medium (84 mg/kg-d) and high (168 mg/kg-d), and particularly, the effect is most obvious in the high dose group. The results show that the poria peel triterpenes can play a role in treating nephrotic syndrome by relieving the pathological change degree of the kidney and reducing the urine protein content.
Example 4 drug effects of a triterpene from hoelen skin on rat nephritis.
1 test Material
1.1 instruments and drugs
Metabolic cages, von experimental animal facilities ltd, suzhou; PL303 electronic balance manufactured by Mettler-Totado Co; UV-2100 type ultraviolet-visible spectrophotometer, shanghai syneli instruments ltd; DDL-5 low-speed freezing centrifuge, produced by Shanghai' an pavilion scientific instrument factory; S-DK-600 electric heating constant temperature three-purpose water temperature box, produced by Shanghai Hede experiment equipment Limited company; thermo adjustable pipettor, shanghai leber analytical instruments ltd.
The triterpene is provided by a preparation room of a institute of traditional Chinese medicine in Shandong province; dexamethasone acetate tablets, Zhejiang Xianju pharmaceutical products GmbH, batch number: 120615; freund's complete adjuvant, Sigma product, usa, lot number: CAS9007-81-2, Specification: 10 mL/count, supplied by Jinan Kai Qi Biotechnology Co., Ltd; sodium pentobarbital: union import split charging, Shanghai chemical reagent purchase supply station split charging, batch number: 86-01-22; 0.9% sodium chloride injection, manufactured by Shandong Lukangxin pharmacy Co., Ltd, lot number 20111011; visual urine protein test paper, manufactured by Golbao Biotechnology Ltd, Huadu Golbao, Guangzhou, Australia, lot number 20120702; urine protein quantification kit, lot No. 20121214; urea nitrogen kit (urease method), lot No. 20130110; creatinine kit, batch No. 20130111, was provided by Nanjing institute of bioengineering.
1.2 animals
Wistar rats 85, SPF grade, male and female halves, weight 180 + -20 g, provided by the Experimental animal center of Shandong university, license number: SCXK (lu) 20090001. The urine protein tests were all negative.
2 test method
2.1 preparation of Kidney cortex plus Freund's complete adjuvant suspension
Taking Wistar rats, half of male and female, and 30mg/kg of 1% sodium pentobarbital for intraperitoneal injection anesthesia, dissecting the abdominal cavity under aseptic conditions, ligating renal arteries, intubating the kidneys from the ligated parts, cutting off renal veins, repeatedly washing the kidneys with physiological saline through the intubations to make the kidneys grey-white, taking off the kidneys, taking out the renal cortex, grinding the renal cortex into homogenate by using a homogenizer, taking out 10g of the cortex homogenate each time, adding 20ml of Freund's complete adjuvant, slowly adding 40ml of the physiological saline, placing the mixture in a mortar for even grinding, and completely emulsifying uniformly.
2.2 active Heymann nephritis model establishment
Taking 180-220 g Wistar rats, half each of male and female, selecting 50 patients with urine protein all negative through urine examination, reserving 10 rats as a normal control group, and carrying out intraperitoneal injection on the rats of the other groups with 2 mL/rat, and carrying out injection for 1 time in 2 weeks for 5 times. After 5 times of injection of the suspension of complete adjuvant of renal cortical Freund's kidney, each group of rats is placed in a metabolism cage to collect urine for 24 hours, the quantitative determination of 24 hours of urine protein of each group of rats is determined by a spectrophotometry, and the occurrence of proteinuria in animals indicates that the molding is successful.
2.3 grouping and administration
The rats successfully molded are randomly divided into a model control group, a dexamethasone positive control group and a poria peel triterpene low and high dose group, and each group contains 8 rats. The low-dosage and high-dosage groups of the poria peel triterpene are respectively 0.162g/kg and 0.486g/kg, the dexamethasone group is 5mg/kg, each drug group is administrated by gastric gavage, the administration volume is 10mL/kg, the administration time is 1 time per day, the administration time is 15 days, and the normal control group and the model control group are administrated with distilled water with equal volume.
The normal control group and the model control group are infused with distilled water with the same volume, and the administration is carried out for 1 time per day and for 15 days continuously.
2.3 Observation index
2.3.124 h urine protein quantification
Respectively placing the rats in clean metabolism cages 5d, 10d and 15d after the successful administration treatment of model building, collecting urine for 24h under the conditions of fasting and free drinking, recording urine volume, measuring the concentration of the urine protein of the rats by adopting a spectrophotometry, and calculating the content of the urine protein for 24 h.
2.3.2 detection of renal function index
After administration for 15 days, fasting for 12 hours without water prohibition, taking 6mL of blood from abdominal aorta, placing 3mL of blood into a heparin sodium anticoagulation tube, carrying out 3000r/min, centrifuging for 15min, and separating plasma; and after 3mL of blood is subjected to self-coagulation, centrifuging for 15min at 3000r/min, separating serum, storing in a freezer at the temperature of-20 ℃, and preparing for measuring the creatinine content and the urea nitrogen content in the blood serum.
2.4 statistical treatment
All experimental data are expressed as mean ± standard deviation (X ± s), statistical analysis is performed on the data using SPSS19.0 statistical software package, two-by-two comparison is performed using t-test, and P <0.05 is taken as a difference significance threshold.
3 results of the experiment
3.1 Effect of Fuling Peel triterpenes on the quantification of 24h urine protein in nephritis model rats
As can be seen from the results of table 9: compared with the normal control group, the content of the urine protein of the model control group rats at each time point for 24 hours is obviously higher than that of the normal control group, which indicates that the nephritis model is successfully replicated. Compared with the model control group rats, the urinary protein content of the rats in the poria peel triterpene high-dose group after administration for 15d is obviously reduced after 24h, which shows that the poria peel triterpene has a reducing effect on the hemamn nephritis urinary protein.
TABLE 9 influence of Poria peel triterpenes on the quantification of 24h urine protein in nephritis model rats
Comparison with model control group: p <0.05, p < 0.001.
3.2 Effect of Fuling skin triterpenes on Urea Nitrogen and creatinine in nephritis model rats
From the results in table 10 it can be seen: compared with a normal control group, the serum creatinine and the plasma urea nitrogen content of the rat in the model control group are obviously increased (P is less than 0.001), which indicates that the model building is successful. Compared with a model control group, the serum creatinine and the plasma urea nitrogen of the rat in the poria peel triterpene high-dose group are obviously reduced (P is less than 0.05), and the poria peel triterpene can reduce the creatinine and the urea nitrogen of the nephritis rat.
TABLE 10 influence of Fuling skin triterpenes on Urea Nitrogen and creatinine in nephritis model rats
Compared with the model control group, p is <0.05, and p is < 0.001.
Example 5 preparation of a dripping pill of triterpene from hoelen peel.
Preparing the poria peel triterpene dripping pill by a melting method: adding the triterpene into molten matrix, stirring, and dripping into condensing tube filled with condensing agent. The preparation method comprises the following steps:
(1) 60g of PEG6000 and 7g of Tween-80 were weighed and melted in a water bath at 90 ℃ to prepare a matrix, and 30g of the poria peel triterpene extracted in example 1 was added.
(2) Dripping at a speed of 25 granules/min to obtain dripping pill with a dripping distance of 5cm, dimethyl silicon oil at 20 deg.C as condensing agent, and cooling column height of 65 cm.
The prepared dripping pill is round, relatively round, has no tailing and adhesion, and has good hardness. The pill weight difference and the disintegration time limit accord with the regulations of Chinese pharmacopoeia on the pill.
Example 6 preparation of dispersible tablets of poria peel triterpene.
Weighing the raw materials, sieving with a 150-mesh sieve, weighing the raw materials according to the following formula, mixing uniformly, adding into a hopper of a tablet press, adjusting the pressure of the tablet press, and tabletting to obtain the poria peel triterpene dispersible tablet.
30.0g of poria peel triterpene;
2.3g of cross-linked sodium carboxymethyl starch;
4.7g of crospovidone-XL;
3.0g of micro silica gel powder;
30.0g of microcrystalline cellulose;
cellulose lactose 30.0 g.
The weight of the prepared dispersible tablet is 0.5g +/-5%, and the hardness is 100N; the tablet weight difference, the shedding rate, the disintegration time limit and the dissolution rate all accord with the regulations of Chinese pharmacopoeia on dispersible tablets.
Example 7 preparation of an emulsion for injection of poria peel triterpene.
(1) Weighing 2g of poria peel triterpene, 12g of soybean lecithin and 4g of oleic acid, adding 100g of soybean oil for injection, heating to 60 ℃, and uniformly mixing to obtain an oil phase;
(2) weighing 20g of Pluronic F68 and 22g of glycerol, adding 840ml of water, and uniformly mixing and dissolving into a water phase;
(3) slowly adding the water phase obtained in the step (2) into the oil phase obtained in the step (1) under high-speed shearing, homogenizing under high pressure for 15 times, filtering with 0.45 μm micro-membrane, introducing nitrogen, and sterilizing to obtain the poria peel triterpene injection emulsion.
The prepared emulsion has no demulsification, and is stable after being stored for 90 days at normal temperature.
Claims (2)
1. The application of the poria peel triterpene in preparing the medicine for treating the nephropathy is characterized in that the main effective components of the poria peel triterpene are 3 β -hydroxy-lanosta-7, 9(11), 24-triene-21-acid and dehydroeiric acid, wherein the total content of the 3 β -hydroxy-lanosta-7, 9(11), 24-triene-21-acid and the dehydroeiric acid in the poria peel triterpene is more than 50% w.t;
the nephropathy is nephrotic syndrome and nephritis;
the poria peel triterpene is an ethanol extract of poria peel, and is extracted by the following method:
(1) washing exodermis Poria with 1.0-2.0 wt% hydrochloric acid, washing with water to neutral, and air drying;
(2) reflux-extracting the poria peel treated in the step (1) for 2 times by using an ethanol solution with the volume fraction of 70%, wherein the extraction time is 0.5h each time, the dosage of the poria peel and the ethanol solution is 1:10(g/ml), combining the extracting solutions, and recovering ethanol from the extracting solution until the extracting solution is dry;
(3) dissolving the dry matter obtained in the step (2) into a solution by using water, adding a mixture of n-butyl alcohol and ethyl acetate in a volume ratio of 7:1.5 for extraction, wherein the volume of the mixture of n-butyl alcohol and ethyl acetate is 1.5-2 times that of the aqueous solution, and recovering the solvent from the organic phase until the organic phase is dried to obtain a crude product;
(4) dissolving the crude product with 100-120 times of ethanol by mass to prepare a solution, loading the obtained solution on a neutral alumina chromatographic column, wherein the mass of the neutral alumina is 15-20 times of that of the crude product, then eluting with a mixture of ethanol and ethyl acetate in a volume ratio of 2:5, and collecting the eluent, wherein the dosage of the mixture of ethanol and ethyl acetate is 2 times of that of the alumina;
(5) adding active carbon into the eluate, wherein the amount of active carbon is 2-3% of the crude product, heating to boil, maintaining for 30min, filtering while hot, washing with ethanol, mixing the filtrate and washing solution, concentrating to 1/3, standing for precipitation, filtering, and drying to obtain Poria peel triterpene.
2. The use of claim 1, wherein the pharmaceutical dosage form for treating renal disease comprises oral dosage form and injection dosage form.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1084395A (en) * | 1992-09-25 | 1994-03-30 | 关岩华 | A kind of production method for the treatment of medicine for curing nephritis |
| CN101020040A (en) * | 2007-03-19 | 2007-08-22 | 殷彬 | Chinese medicine for treating hepatorenal syndrome |
| CN101361954A (en) * | 2008-09-28 | 2009-02-11 | 韩俊杰 | Traditional Chinese medicine formulation for treating elderly nephritis |
| CN105617051A (en) * | 2014-10-29 | 2016-06-01 | 蒋才维 | Traditional Chinese medicine for treating acute nephritis |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1084395A (en) * | 1992-09-25 | 1994-03-30 | 关岩华 | A kind of production method for the treatment of medicine for curing nephritis |
| CN101020040A (en) * | 2007-03-19 | 2007-08-22 | 殷彬 | Chinese medicine for treating hepatorenal syndrome |
| CN101361954A (en) * | 2008-09-28 | 2009-02-11 | 韩俊杰 | Traditional Chinese medicine formulation for treating elderly nephritis |
| CN105617051A (en) * | 2014-10-29 | 2016-06-01 | 蒋才维 | Traditional Chinese medicine for treating acute nephritis |
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