CN116115679B - Application of radix Tripterygii Wilfordii extract in preparing medicine for treating sepsis related encephalopathy - Google Patents
Application of radix Tripterygii Wilfordii extract in preparing medicine for treating sepsis related encephalopathy Download PDFInfo
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Abstract
The invention discloses an application of a trigeminal picrorhiza extract, in particular to an application of total flavonoids in preparing medicines for treating sepsis-related encephalopathy. The invention provides an application of the trigeminal picrorhiza in preparing medicaments for treating sepsis-related encephalopathy, and provides a new idea for developing medicaments for treating the sepsis-related encephalopathy. The trigeminal bitter is easy to obtain, and can drive the medicinal material resources to be fully developed and utilized, thereby bringing beneficial effects.
Description
Technical Field
The invention relates to a medicament for treating brain injury caused by sepsis, in particular to application of trigeminal bitter general flavone (Melicope pteleifolia totalflavone, MPTF) in preparing medicaments for treating sepsis-related encephalopathy.
Background
Sepsis-related encephalopathy is central nervous system dysfunction caused by sepsis, and is one of the common encephalopathy in ICU wards. Sepsis-associated brain diseases can occur at any stage of the sepsis course, clinically manifested as brain dysfunction from delirium to deep coma. Sepsis-associated encephalopathy is characterized by changes in behavior, cognition, arousal, and consciousness, and long-term cognitive dysfunction in a substantial proportion of patients. In ICU ward, the incidence rate is 9% -71%, no effective treatment means exists at present, and the clinical treatment mainly aims at the overall treatment, symptomatic treatment and support treatment of basic disease sepsis.
The name Melicope teifolia, the family Rutaceae, is widely distributed in the southern provinces in China such as Guangdong, fujian, hainan, guangxi, yunnan, guizhou and other provinces. It is bitter and cold in nature and has the actions of clearing heat and removing toxicity, dispelling wind and removing dampness. Pharmacological studies show that the extract of the trigeminal picrorhiza herb or the compound separated from the extract has the effects of anti-inflammatory, antioxidant, antibacterial and the like. Research shows that factors such as inflammation, oxidative stress and the like are involved in the course of sepsis-related encephalopathy. However, no research on treating sepsis-related encephalopathy by trigeminal bitter has been made so far.
Disclosure of Invention
The invention aims to provide an application of total flavonoids (Melicopepteleifolia total flavonoids, MPTF) of trigeminal bitter in preparing medicaments for treating sepsis-related encephalopathy, provides a new thought for developing medicaments for treating sepsis-related encephalopathy, is easy to obtain, and can drive full development and utilization of medicinal material resources.
The technical scheme of the invention is as follows: application of radix Tripterygii Wilfordii extract in preparing medicine for treating sepsis related encephalopathy is provided.
The application of the trigeminal bitter extract in preparing the medicine for treating sepsis related encephalopathy is provided, wherein the trigeminal bitter extract is total flavone of the trigeminal bitter.
The application of the trigeminal bitter extract in preparing the medicine for treating sepsis related encephalopathy is disclosed, and the preparation method of the total flavonoids of the trigeminal bitter is carried out according to the following steps:
(1) Pulverizing folium Folium Alternantherae, sieving, soaking in 95% ethanol overnight, concentrating under reduced pressure with rotary evaporator to obtain product A;
(2) Loading the product A on an HPD-100 type macroporous resin column pretreated by 95% ethanol, loading 4300mL of sample volume, eluting with 3500mL of distilled water, flushing the column with 4500mL of 70% ethanol, and collecting 70% ethanol column-passing fraction to obtain product B;
(3) And (3) repeating the step (2) for one time, collecting 70% ethanol, passing through a column, and evaporating to dryness to obtain the total flavonoids extract powder of the trigeminal picrorhiza.
In the step (1), the weight ratio of the trigeminal bitter to the 95% ethanol is about 1:5-10, and the filtrate is concentrated to 1/5-1/4 of the original volume under reduced pressure.
In the step (1), the pressure is reduced to 400-600 mmHg by a rotary evaporator.
A medicine for treating sepsis-related encephalopathy comprises trigeminal bitter total flavonoids.
A medicine for treating sepsis-related encephalopathy is mainly prepared from total flavonoids of trigeminal picrorhiza.
The preparation method of the medicine for treating sepsis related encephalopathy comprises the steps of combining the trigeminal bitter general flavone with acceptable auxiliary materials in the medicine, and processing the combined medicine according to a conventional method to prepare the corresponding medicine.
The medicine is tablet, capsule, granule, pill or oral liquid.
Compared with the prior art, the invention has the following beneficial effects:
1. the pathogenesis of sepsis-associated encephalopathy is not known so far, and thus no drug effective in treating sepsis-associated encephalopathy has been developed yet. The invention finds that the trigeminal bitter general flavone can be used for treating sepsis related encephalopathy, as shown in the subsequent experimental evidence of the invention: in vitro, the total flavonoids of Tripichia (14.5. Mu.g/ml) significantly protected LPS (50 ng/ml) in combination with INF-gamma (200U/ml) stimulated rat cortical astrocytes in vitro.
2. Compared with the CLP model group, the total flavonoids (30, 60 mg/kg) of the trigeminal picrorhiza obviously increase the survival number of the sepsis mice, and the difference has statistical significance. Compared with CLP group, the total flavonoids (60 mg/kg) obviously reduces brain tissue injury of sepsis mice, reduces inflammatory infiltration, maintains normal cell structure, and the like.
3. In surviving sepsis mice, no one of the mice was found to develop sepsis-related encephalopathy symptoms relative to the dexamethasone group.
Currently, the application of trigeminal bitter to the treatment of sepsis-related encephalopathy is not known in the prior art, and therefore, the clinical value of the trigeminal bitter as a medicament for research cannot be realized. The invention proves that the trigeminal bitter can be used for treating sepsis related encephalopathy through the following experiments.
Experiments prove that:
extraction and purification of 1MPTF
1.1 pretreatment of macroporous resin
HPD-100 type macroporous resin is soaked in 95% ethanol overnight, washed with 95% ethanol until no turbidity is caused, washed with distilled water until no alcohol smell is caused, and wet-packed into a column with column height of 52cm and diameter of 9.3cm (BV) and column volume of 3530.52cm 3 。
1.2 extraction
Weighing 1.1kg of the three-fork bitter crude drug, preparing a total flavone extract by adopting the method, concentrating the total flavone extract by using a rotary evaporator until no alcohol smell exists, loading 4300ml (1.2 BV) of total volume, eluting the total flavone extract by 3500ml of distilled water, flushing a column by 70% ethanol (4500 ml,1.3 BV) at 2BV/h, collecting 70% ethanol column-passing fractions, obtaining a first column-passing sample, and purifying the first obtained sample for the second time according to the method.
2 determination of total flavone content of purified sample
Concentrating and evaporating to dryness to obtain total flavone extract powder, dissolving 0.1222g sample in 200ml 70% ethanol, collecting 0.5ml sample, placing into a test tube with plug, and referring to Na 2 NO 2 -Al(NO 3 ) 3 -NaOH chromogenic assay for total flavone content.
According to the standard curve and the formula Y (%) = [ (cV 10) -3 )D/m]*100, the total flavone content of the sample is calculated to be 81.3%.
Y is the total flavone content of the trigeminal picrorhiza, mg/g; c is the measured mass concentration of the sample, mg/mL; v is the volume of filtrate, mL; d is dilution multiple; m is the mass of the raw material, 1.1546g and 1.1593g of the total flavonoids of the trigeminal picrorhiza are weighed in parallel and respectively named as ZHT-1 and ZHT-2, and the contents of the total flavonoids of the trigeminal picrorhiza and the ZHT-2 are calculated according to the relation between a standard curve and absorbance (table 2).
TABLE 1 concentration and absorbance of standard curves
TABLE 2 sample concentration and absorbance
3HPLC analysis of MPTF
A suitable amount of MPTF sample was taken for HPLC analysis. Experimental conditions: agilent C18 column (250 mm. Times.4.6 mm,5 μm), column temperature 30 ℃, detection wavelength 240nm, flow rate 1ml/min, sample injection amount 2. Mu.l, mobile phase: methanol/water.
The results showed a distinct and single peak on the HPLC profile at 18min, analyzed as trigeminal total flavonoids (fig. 1).
4 cell experiment
4.1 toxic Effect of MPTF on rat cortical astrocytes
The toxicity of the trigeminal bitter total flavonoids with different concentrations to rat cortex astrocytes was determined by CCK-8 method. The administration concentrations of the total flavonoids of the trigeminal picrorhiza in the cytotoxin effect experiment are 232, 116, 58, 29, 14.5 and 7.25 mug/ml respectively.
Rat cortical astrocytes, purchased from green flag (Shanghai) biotechnology development limited.
Collecting logarithmic phase cells, and adjusting cell suspension concentration to 1×10 6 Per ml, inoculated into 96-well cell culture plates, 10ml of cell suspension, 5% CO were added to each well 2 Incubate at 37 ℃. When the cell confluence in the hole reaches about 90%, the trigeminal total flavonoids (dissolved in DMSO) with different concentrations are added at the same time, so that the final concentrations respectively reach 232, 116, 58, 29, 14.5 and 7.25 mug/ml, and 3 compound holes are arranged in each gradient. At the same time, a blank group (cells only) was established. After 24h incubation, 10. Mu.l of CCK-8 reagent was added separately and the incubation was continued in an incubator. Taking out the 96-well plate after 1.5h, measuring the OD value of each sample to be tested at 450nm by using an enzyme-labeled instrument, and calculating the inhibition rate, wherein the inhibition rate= (OD) Blank space -OD Treatment of )/OD Blank space X 100% and the effect of total flavonoids of trigeminal bitter on astrocyte proliferation of rat cortex was analyzed.
Results
CCK-8 results showed that compared with the blank, the results showed that the total flavonoids had no significant cytotoxicity to rat cortical astrocytes at a concentration of less than or equal to 29. Mu.g/ml, and had significant inhibitory effect (P < 0.01) to rat cortical astrocytes at a concentration of greater than or equal to 58. Mu.g/ml (Table 3).
TABLE 3 Effect of different concentrations of Tripicrin on rat cortical astrocyte proliferation
* P<0.05， ** P<0.01vs. blank.
4.2 protective effect of Tripicrin on LPS-combined INF-gamma-stimulated rat cortical astrocytes
The protection effect of different concentrations of trigeminal bitter total flavonoids on LPS combined with INF-gamma stimulated rat cortical astrocytes was determined by CCK-8 method.
The administration concentrations of the total flavonoids of the trigeminal picrorhiza in the cytoprotective effect experiment are 7.25, 14.5 and 29 mug/ml respectively.
Cell incubation was as above. When the cell confluency in the culture hole reaches about 90%, LPS and INF-gamma are added simultaneously, so that the final concentration of LPS in the hole reaches 50ng/ml, and the INF-gamma reaches 200U/ml. Simultaneously, the total flavonoids of the trigeminal bitter with different concentrations are immediately added to ensure that the concentrations respectively reach 7.25, 14.5 and 29 mug/ml. 3 complex wells were set for each gradient. At the same time, a blank group (cells only) was established. After 24h incubation, 10. Mu.l of CCK-8 reagent was added, OD450 was read as above, and the cell proliferation rate was calculated.
Results
Compared with the blank, the LPS combined with INF-gamma obviously inhibits the proliferation of rat cortical astrocytes (P < 0.05); compared with the LPS combined with INF-gamma, 14.5 mug/ml of the total flavonoids of the trigeminal picrorhiza significantly promotes cell proliferation (P < 0.05), and the difference has statistical significance (Table 4), which shows that the total flavonoids of the trigeminal picrorhiza significantly protects cell damage caused by the LPS combined with INF-gamma.
TABLE 4 protection of LPS-associated INF-gamma stimulated rat cortical astrocytes by Trimeresurus flavone
* P<0.05vs. blank; # P<0.05vs.LPS+INF-γ。
5 animal experiment
5.1 Tripicropodophyllin protection of CLP-induced sepsis mice
The difference in survival rate of mice between the comparative groups was determined using the Kaplan-Meier method.
The administration dosage of the total flavonoids of the trigeminal bitter is 15mg/kg, 30mg/kg and 60mg/kg respectively.
SPF-grade ICR male mice were randomly assigned to vehicle groups, CLP groups, low-dose groups of total trigeminal flavone (15 mg/kg), medium-dose groups of total trigeminal flavone (30 mg/kg), high-dose groups of total trigeminal flavone (60 mg/kg), dexamethasone groups (5 mg/kg), and 12 animals per group. Reference a Cecal Ligation Punch (CLP) method was used to replicate a sepsis mouse model while a sham group was established. After molding, each dosing group was dosed continuously for three days, once daily, with an equal volume of purified water given to the CLP group and the sham group. Animals were observed for general status, and differences in death within 168h were recorded and compared for each group of mice.
Results
The remaining groups of mice, except the sham operated group, were dead. Wherein the death rate of animals in the CLP group within 72 hours is 100 percent, and death concentration occurs in 24-48 hours, which indicates that the modeling is successful. Compared with CLP group, trigeminal total flavonoids (30, 60 mg/kg) significantly increased survival number of sepsis mice, and the differences were statistically significant (P < 0.05) (fig. 2, table 5).
TABLE 5 number of animal survivors (only) for each group at different time points
5.2 Total Flavonoids reduce the incidence of sepsis-related encephalopathy
In addition, the probability of obvious central nervous system symptoms (animals continuously rotate in a cage for more than 10 minutes) of few sepsis mice which survive treatment by dexamethasone is obviously increased (about 20-30%), and the sepsis-related encephalopathy is primarily judged. However, after the treatment of the trigeminal bitter total flavone, no living animals are observed for the appearance of the nervous system symptoms. This result suggests that the total flavonoids of trigeminal picrorhiza reduce the incidence of sepsis-related encephalopathy in sepsis mice (table 6).
TABLE 6 sepsis mice sepsis-associated brain disease occurrence
5.3MPTF reduces sepsis mice brain tissue pathological lesions
H & E section pathology examination suggests that the neurons of the animals of the model group are arranged in disorder, the cytoplasm is deeply stained, and the nucleus is contracted. The trigeminal total flavonoids significantly reduced pathological damage to brain tissue of sepsis mice, including reduced inflammatory infiltration, maintenance of normal cell structure, etc. (figure 3).
To sum up: the experimental result of the invention shows that the proliferation rate of rat cortical astrocytes stimulated by LPS combined with INF-gamma is obviously increased in vitro by the total flavonoids of trigeminal picrorhiza. The survival rate of the CLP-induced sepsis mice is improved in vivo, and the pathological damage of brain tissues of the sepsis mice is obviously reduced.
Therefore, the invention provides the application of the trigeminal picrorhiza in the medicines for treating sepsis related encephalopathy, and provides a new idea for the medicine treatment of sepsis. The preparation method has the advantages that the trigeminal bitter is easy to obtain, can drive the full development and utilization of medicinal material resources, and has beneficial effects.
Drawings
Fig. 1: HLPC analysis of total flavonoids of trigeminal pichia;
fig. 2: number of animals living in each group. * P<0.05, ** P<0.01vs.CLP(Kaplan-Meier)；
Fig. 3: brain histopathological observation (H & E) of CLP-induced sepsis mice.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to be limiting.
Example 1:
(1) Extraction of the extract powder of the total flavonoids of the trigeminal sowthistle: pulverizing 1.12kg of Folium Alternantherae, sieving, soaking in 95% ethanol overnight, concentrating to 1/4 of the original volume with rotary evaporator at a weight ratio of Folium Alternantherae to ethanol of 1:8; loading on HPD-100 type macroporous resin column pretreated by 95% ethanol; loading 4300ml of total volume, eluting with 3500ml of distilled water, washing the column with 70% ethanol, collecting 70% ethanol column-passing fraction to obtain first column-passing sample, and purifying the first obtained sample for the second time according to the above method; evaporating to dryness to obtain extract powder of total flavonoids of herba Trifolii Pratentis.
(2) The preparation process of the tablet comprises the following steps: adding appropriate amount of conventional adjuvants into the total flavonoids, mixing, granulating, drying, tabletting, and making into tablet. Each tablet contains 75mg of total flavonoids of trigeminal bitter (taking 30mg/kg of mice as an example, the dosage for human use is 30/12.33 (conversion coefficient) =2.43 mg/kg. calculated as 60kg of adults, and the daily dosage for human use is 2.43×60=145.8 mg).
The usage amount is as follows: the preparation is administered 2 times daily, 2 tablets each time, and can be used for treating sepsis-related encephalopathy.
Example 2:
(1) Extraction of the extract powder of the total flavonoids of the trigeminal sowthistle: pulverizing 1.12kg of Folium Alternantherae, sieving, soaking in 95% ethanol overnight, concentrating to 1/5 of the original volume with rotary evaporator at a weight ratio of Folium Alternantherae to ethanol of 1:5; loading on HPD-100 type macroporous resin column pretreated by 95% ethanol; loading 4300ml of total volume, eluting with 3500ml of distilled water, washing the column with 70% ethanol, collecting 70% ethanol column-passing fraction to obtain first column-passing sample, and purifying the first obtained sample for the second time according to the above method; evaporating to dryness to obtain extract powder of total flavonoids of herba Trifolii Pratentis.
(2) The preparation process of the capsule comprises the following steps: adding appropriate amount of conventional adjuvants into the prepared MPTF, mixing, oven drying, sterilizing, making into hard capsule, and packaging to obtain capsule. Each tablet contains 75mg of total flavonoids of trigeminal bitter.
The usage amount is as follows: the preparation is administered 2 times daily, 2 capsules each time, and can be used for treating sepsis-related encephalopathy.
Example 3:
(1) Extraction of the extract powder of the total flavonoids of the trigeminal sowthistle: pulverizing 1.12kg of Folium Alternantherae, sieving, soaking in 95% ethanol overnight, concentrating to 1/4 of the original volume with rotary evaporator at a weight ratio of Folium Alternantherae to ethanol of 1:10; loading on HPD-100 type macroporous resin column pretreated by 95% ethanol; loading 4300ml of total volume, eluting with 3500ml of distilled water, washing the column with 70% ethanol, collecting 70% ethanol column-passing fraction to obtain first column-passing sample, and purifying the first obtained sample for the second time according to the above method; evaporating to dryness to obtain extract powder of total flavonoids of herba Trifolii Pratentis.
(2) The preparation process of the granule comprises the following steps: adding appropriate amount of common adjuvants into the prepared total flavonoids, granulating, drying, grading, and making into granule. 2 g/bag of granule, wherein the content of total flavonoids in the granule is 75mg/g.
The usage amount is as follows: the preparation is administered 2 times daily, 2 bags each time, and can be used for treating sepsis related encephalopathy.
Example 4:
(1) Extraction of the extract powder of the total flavonoids of the trigeminal sowthistle: pulverizing 1.12kg of Folium Alternantherae, sieving, soaking in 95% ethanol overnight, concentrating to 1/5 of the original volume with rotary evaporator at a weight ratio of Folium Alternantherae to ethanol of about 1:8; loading on HPD-100 type macroporous resin column pretreated by 95% ethanol; loading 4300ml of total volume, eluting with 3500ml of distilled water, washing the column with 70% ethanol, collecting 70% ethanol column-passing fraction to obtain first column-passing sample, and purifying the first obtained sample for the second time according to the above method; evaporating to dryness to obtain extract powder of total flavonoids of herba Trifolii Pratentis.
(2) The preparation process of the oral liquid comprises the following steps: adding appropriate amount of solubilizer into the prepared total flavonoids, grinding, adding small amount of water for dilution, mixing, adding correctant and antiseptic, mixing, adding water to specified amount, filtering, mixing, packaging, and sterilizing to obtain oral liquid. The total flavone content of the trigeminal bitter in the oral liquid is 5mg/ml.
The usage amount is as follows: the preparation is administered 2 times daily, about 30ml each time, and can be used for treating sepsis-related encephalopathy.
Example 5:
(1) Extraction of the extract powder of the total flavonoids of the trigeminal sowthistle: pulverizing 1.12kg of Folium Alternantherae, sieving, soaking in 95% ethanol overnight, concentrating to 1/4 of the original volume with rotary evaporator at a weight ratio of Folium Alternantherae to ethanol of about 1:7; loading on HPD-100 type macroporous resin column pretreated by 95% ethanol; loading 4300ml of total volume, eluting with 3500ml of distilled water, washing the column with 70% ethanol, collecting 70% ethanol column-passing fraction to obtain first column-passing sample, and purifying the first obtained sample for the second time according to the above method; evaporating to dryness to obtain extract powder of total flavonoids of herba Trifolii Pratentis.
(2) The preparation process of the pill comprises the following steps: adding appropriate amount of common adjuvants into the prepared total flavonoids, making into pill, drying, and making into pill. Each pill contains 75mg of total flavonoids of Tripterygium wilfordii.
The usage amount is as follows: the preparation is administered 2 times daily, 2 pills each time, and can be used for treating sepsis related encephalopathy.
Claims (3)
1. An application of radix Tripterygii Wilfordii extract in preparing medicine for treating sepsis related encephalopathy is provided;
the extract of the trigeminal bitter is total flavone of the trigeminal bitter;
the preparation method of the total flavonoids of the trigeminal picrorhiza comprises the following steps:
(1) Pulverizing folium Folium Alternantherae, sieving, soaking in 95% ethanol overnight, concentrating under reduced pressure with rotary evaporator to obtain product A;
(2) Loading the product A on a HPD-100 type macroporous resin column pretreated by 95% ethanol, loading a sample volume 4300mL, eluting with 3500mL distilled water, then flushing the column with 4500mL 70% ethanol, and collecting 70% ethanol column-passing fraction to obtain product B;
(3) And (3) repeating the step (2) for one time, collecting 70% ethanol, passing through a column, and evaporating to dryness to obtain the total flavonoids extract powder of the trigeminal picrorhiza.
2. The use of trigeminal bitter total flavonoids according to claim 1 in the manufacture of a medicament for treating sepsis-related encephalopathy, characterized in that: in the step (1), the weight ratio of the trigeminal bitter to the 95% ethanol is about 1:5-10, and the filtrate is concentrated to 1/5-1/4 of the original volume under reduced pressure.
3. The use of trigeminal bitter total flavonoids according to claim 1 in the manufacture of a medicament for treating sepsis-related encephalopathy, characterized in that: in the step (1), the pressure of the rotary evaporator is reduced to 400-600 mmHg.
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Non-Patent Citations (4)
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| 三丫苦茎总提取物及分段样品抗菌活性研究;高英等;《云南中医学院学报》;第第40卷卷(第第3期期);28-32页 * |
| 三叉苦质量控制及体外抗炎、抗氧化作用初步研究;甘杰华;《中国优秀硕士学位论文全文数据库医药卫生科技辑》(第2019/02期);66页第2段，73页第1-2段 * |
| 三桠苦活性成分的研究;刁远明;《中国优秀硕士学位论文全文数据库医药卫生科技辑》(第2005/06期);19页第1段，19-20页流程图1-3，43页最后1段 * |
| 甘杰华.三叉苦质量控制及体外抗炎、抗氧化作用初步研究.《中国优秀硕士学位论文全文数据库医药卫生科技辑》.2019,(第2019/02期),66页第2段，73页第1-2段. * |
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