CN110898052A - Application of kaempferol in preparation of medicine for treating sepsis - Google Patents

Application of kaempferol in preparation of medicine for treating sepsis Download PDF

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CN110898052A
CN110898052A CN201911332029.9A CN201911332029A CN110898052A CN 110898052 A CN110898052 A CN 110898052A CN 201911332029 A CN201911332029 A CN 201911332029A CN 110898052 A CN110898052 A CN 110898052A
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kaempferol
lps
sepsis
medicine
treating sepsis
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姚琦
高英
罗君
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Guizhou University of Traditional Chinese Medicine
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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Abstract

The invention discloses an application of kaempferol in preparing a medicament for treating sepsis. The invention provides application of kaempferol in preparation of a medicine for treating sepsis, provides a new idea for research and development of the medicine for treating sepsis, and the kaempferol is easily separated and purified from the evodia lepta, can drive medicinal material resources to be fully developed and utilized, and further brings beneficial effects.

Description

Application of kaempferol in preparation of medicine for treating sepsis
Technical Field
The invention relates to a new application of kaempferol, in particular to an application of kaempferol in preparing a medicament for treating sepsis.
Background
Clinically, gram-negative (G)－) Severe bacterial infections often lead to the development of Systemic Inflammatory Response Syndrome (SIRS) and eventually Sepsis (Sepsis). Sepsis has a high mortality rate and seriously threatens human life. The application of a large number of broad-spectrum and high-efficiency antibiotics provides a powerful weapon for clinically treating bacterial infectious diseases, but the wide application of antibiotics leads to the large increase of drug-resistant strains. Moreover, the release of LPS while killing bacteria exacerbates the inflammatory response. Therefore, it is of great significance to find effective drugs for treating sepsis and to study their mechanisms of action.
The research reports that kaempferol has strong anti-inflammatory and antioxidant effects, but the report that kaempferol is used alone to treat sepsis does not exist.
Kaempferol, structural formula as follows:
Figure BDA0002329893140000011
alias: 3,4,5, 7-tetrahydroxyflavone with molecular formula C15H10O6Molecular weight 286.24, yellow powder compound, soluble in ethanol, methanol, dimethyl sulfoxide, etc.
The compound can be extracted and separated from Meloidogyne trigeminal (Meliopeptellifolia (champ. exbenth.) T.Hartley) of Meloidogyne of Rutaceae.
Disclosure of Invention
The invention aims to provide application of kaempferol in preparation of a medicine for treating sepsis, provides a new idea for research and development of the medicine for treating sepsis, is easy to obtain, and can drive full development and utilization of medicinal material resources.
The technical scheme of the invention is as follows: use of kaempferol in preparing medicine for treating sepsis is provided.
In the application of the kaempferol in preparing the medicament for treating sepsis, the kaempferol has the alias: 3,4,5, 7-tetrahydroxyflavone with molecular formula C15H10O6Molecular weight of 286.24, structural formula:
Figure BDA0002329893140000021
in the medicine for treating sepsis, raw materials of the medicine comprise kaempferol.
In the medicine for treating sepsis, the medicine is mainly prepared from kaempferol.
In the preparation method of the medicine for treating sepsis, kaempferol and auxiliary materials acceptable in the medicine are combined and processed according to a conventional method to prepare the corresponding medicine.
In the preparation method of the medicine for treating sepsis, the medicine is tablets, capsules, granules, oral liquid, pills or injections.
Compared with the prior art, the invention has the following beneficial effects:
1. the experiment of the invention proves that the kaempferol obviously inhibits the release of TNF- α and IL-6 in the supernatant of RAW264.7 cells stimulated by endotoxin (LPS) in vitro, the kaempferol obviously increases the survival rate of Cecal Ligation Perforation (CLP) or LPS sepsis mice in vivo, and obviously reduces the expression levels of TNF- α and IL-6 in the serum of LPS sepsis mice.
2. The kaempferol can be extracted from the trifoliate bitter according to a conventional method, the obtained kaempferol is combined with auxiliary materials acceptable in medicines, and the mixture is processed according to the conventional method to prepare corresponding medicines, wherein the medicines comprise tablets, capsules, granules, oral liquid, pills, injections and the like. For example: the kaempferol can be further mixed or embedded with carriers such as cyclodextrin, lactose, polyethylene glycol, starch, sorbitol, mannitol, etc., to obtain kaempferol pharmaceutical composition with therapeutic amount (content > 90%), and the composition can be added with lubricant, humectant, suspending agent, correctant, antiseptic, etc., and made into various dosage forms such as tablet, capsule, granule, oral liquid, pill, etc.; can also be processed by kaempferol according to conventional method to obtain injection, and can be used for preparing medicine for treating sepsis or adjuvant treatment of sepsis.
Experiments prove that:
1. experiment of dynamic inhibition effect of kaempferol on LPS stimulation of TNF- α release and IL-6 release of RAW 264.7:
the inhibition effect of the compound on the release of proinflammatory cytokines TNF- α and IL-6 from RAW264.7 cells stimulated by LPS is measured by an enzyme-linked immunosorbent assay (ELISA).
The administration concentration of kaempferol is: 50. 100, 200. mu.g/ml.
Murine leukemia RAW264.7 cells, purchased from the American Special goods storage center (ATCC).
Collecting cells in logarithmic growth phase, adjusting cell suspension concentration to 1 × 106Perml, inoculated into 90mm cell culture dishes, added to each dishInto 10ml of cell suspension, 5% CO2Incubation at 37 ℃. And when the confluence degree of the cells in the culture dish reaches about 90%, simultaneously adding LPS and kaempferol with different concentrations to ensure that the final concentration of the LPS in the dish reaches 200ng/ml and the final concentration of the kaempferol reaches 50, 100 and 200 mu g/ml respectively. Meanwhile, a solvent control dish is set, 10ml of cell suspension in each dish, 3 parallel control dishes are set, and incubation is carried out at 37 ℃. Incubate for 4, 8, 12, 24h, respectively, and 0.5ml of cell supernatant was taken at each time point.
The mouse TNF- α and IL-6 detection kit is used, the kit specification is referred to for operation, and an enzyme-labeled detector is adopted to measure the light absorption value (OD) of each hole at 490nm, so as to obtain the expression levels of TNF- α and IL-6 in cell supernatant, the results are shown as follows:
TABLE 1 Effect of kaempferol on LPS stimulation of TNF- α (pg/ml) release from mouse leukemia cells RAW264.7 ((pg/ml))
Figure BDA0002329893140000041
n＝3)
Figure BDA0002329893140000042
TABLE 2 Effect of kaempferol on LPS stimulation of IL-6(pg/ml) release from murine leukemia cells RAW264.7 ((pg/ml))
Figure BDA0002329893140000043
n＝3)
Figure BDA0002329893140000044
Compared with the solvent, the solvent is added with the solvent,*is P<0.05，**Is P<0.01; in comparison with the LPS, the LPS is,#is P<0.05，##Is P<0.01。
The experimental results show that, as shown in fig. 1 and fig. 2, when the RAW264.7 cells are stimulated by LPS for 4, 8, 12 and 24 hours, the expression levels of TNF- α and IL-6 in the cell supernatant are obviously increased compared with the vehicle group.
Compared with LPS group, when 50-200 mug/ml kaempferol acts on RAW264.7 cells for 4h, the expression level of TNF- α and IL-6 in cell supernatant is obviously reduced.
Compared with LPS group, when 50-200 mug/ml kaempferol acts on RAW264.7 cells for 8h, the expression level of TNF- α and IL-6 in cell supernatant is obviously reduced.
Compared with LPS group, when 100-200 mug/ml kaempferol acts on RAW264.7 cells for 12h, the expression levels of TNF- α and IL-6 in cell supernatant are obviously reduced.
Compared with LPS group, when 100-200 mug/ml kaempferol acts on RAW264.7 cells for 24h, the expression levels of TNF- α and IL-6 in cell supernatant are obviously reduced.
In conclusion, kaempferol (50-200 mu g/ml) obviously inhibits the expression level of inflammatory cytokines TNF- α and IL-6 in cell supernatant in 4-24h of LPS stimulated RAW264.7 cells.
2. Experiment of therapeutic effect of kaempferol on CLP sepsis-induced mice
The dosage of the kaempferol drug is as follows: 50. 100 and 200 mg/kg.
ICR mice (male and female halves), 6-7 weeks old, 20.0 + -2.0 g. Adaptive to Specific Pathogen Free (SPF) grade barrier system, placed in dry clean plastic cages, fed with standard experimental animal feed, and fed with free water.
Experimental methods animals were randomly grouped by body weight, 10 animals per group. The experiment is designed into a sham operation group, a CLP + kaempferol low-dose group (50mg/kg), a CLP + kaempferol medium-dose group (100mg/kg) and a CLP + kaempferol high-dose group (200 mg/kg).
Mice were anesthetized with 2.5% sevoflurane inhalation, and after the animals were unresponsive, they were placed supine on the operating plate and iodophors disinfected the abdominal skin. A longitudinal incision about 1.0cm long is cut in the center of the abdomen, skin and subcutaneous tissues are cut layer by layer to find out a white abdominal line, the rectus abdominis and peritoneum are cut open by an ophthalmic scissors at the position, the cecum is pulled out gently, the cecum and mesenteric blood vessels are ligated by a 3-0 silk thread at a position which is about the whole cecum length 1/3-1/2 away from the tail end of the cecum, and then the cecum ligated part is punctured and ligated by a 20G needle for 2 times. Slightly squeezing to ensure that a small amount of intestinal contents overflow from the puncture hole to ensure smooth operation; the treated cecum was then returned to the abdominal cavity, and after abdominal skin was sutured, the mice were placed in a heating pad to maintain the anal temperature at 37 ± 0.5 ℃. After the abdominal cavity was opened, the animals in the sham operation group were closed and the skin was sutured without any treatment. After the mice revived, the mice had free water and had a normal diet. The animals in the sham operation group were subjected to intragastric administration of distilled water according to body weight, and the animals in each treatment group of kaempferol were subjected to intragastric administration of different doses of the drugs according to body weight for one-time intragastric administration volume: 0.2ml/20 g.
The status and survival of the mice were recorded every 8 hours and the survival rate and survival time of 168h mice were calculated. The results are shown below:
TABLE 3 Effect of kaempferol on survival and survival time of CLP-induced septic mice for 168h ((S))
Figure BDA0002329893140000061
n＝10)
Figure BDA0002329893140000062
Compared with the false operation, the operation method has the advantages that,**is P<0.01; in comparison with the CLP, the CLP is compared,#is P<0.05，##Is P<0.01。
The results show that after CLP operation for 8h, the mice are found to have symptoms of piloerection, gradually reduced activity and feeding, curly and trembling and the like. As shown in fig. 3, the death time of the mice was mainly concentrated at 48h after CLP surgery, 6 animals died within 48h, accounting for 60% of the total. All the animals in the CLP control group die within 72h, which indicates that the CLP sepsis mouse model is successfully replicated.
5,7 and 8 animals of the kaempferol low, medium and high dose groups (50-200mg/kg) survive respectively, the survival rates are respectively 50%, 70% and 80%, and compared with the CLP control group, the survival time of the mice of the three administration groups is obviously prolonged. As can be seen from the table above, kaempferol (50-200mg/kg) has obvious protective effect on CLP sepsis-induced mice.
3. Experiment for influence of kaempferol on survival rate of LPS sepsis-induced mice
The dosage of the kaempferol drug is as follows: 50. 100 and 200 mg/kg.
ICR mice (male and female halves), 6-7 weeks old, 20.0 + -2.0 g. Feeding in SPF barrier system, placing in dry and clean plastic cage, feeding with standard experimental animal feed, and feeding with free water.
Experimental methods animals were randomly grouped by body weight, 10 animals per group. The experiment was carried out using a blank control group, an LPS + kaempferol low dose group (50mg/kg), an LPS + kaempferol medium dose group (100mg/kg), and an LPS + kaempferol high dose group (200 mg/kg).
LPS (40mg/kg) was injected into the tail vein, and normal saline was administered to the blank control group by intragastric administration. After LPS injection, each administration group of kaempferol immediately dredges the stomach according to the weight of the animal and then administers different doses of the medicine once. The blank control group and the LPS injection group were subjected to intragastric administration of physiological saline according to body weight, intragastric administration volume: 0.2ml/20 g.
The status and survival of the mice were recorded every 8 hours and the survival rate and survival time of 168h mice were calculated.
Results
TABLE 4 Effect of kaempferol on survival and survival time of LPS-induced septic mice 168h ((S))
Figure BDA0002329893140000071
n＝10)
Figure BDA0002329893140000072
In comparison with the blank, the number of the blank,**is P<0.01; in comparison with the LPS, the LPS is,#is P<0.05，##Is P<0.01。
The results show that after 8 hours of LPS tail vein injection, the mice are found to have the symptoms of hair erection, gradually reduced activity and food intake, diarrhea and the like. As shown in FIG. 4, the death time of the mice is mainly concentrated on 24-48h after tail vein injection of bacteria, and all animals in LPS injection groups within 72h die, indicating that the LPS sepsis mouse model is successfully replicated. The low, medium and high dosages of kaempferol respectively enable 1, 4 and 8 animals to survive, and the survival rates are respectively 10%, 40% and 80%. Compared with LPS injection control group, the survival time of the animals in the kaempferol medium and high dose groups is obviously prolonged. As can be seen from the above table, kaempferol (100-200mg/kg) has an obvious protective effect on LPS-induced sepsis mice.
4. Experiment for influence of kaempferol on expression levels of TNF- α and IL-6 in serum of mice with LPS-induced sepsis
The dosage of the kaempferol drug is as follows: 50. 100 and 200 mg/kg.
ICR mice (male and female halves), 6-7 weeks old, 20.0 + -2.0 g. Feeding in SPF barrier system, placing in dry and clean plastic cage, feeding with standard experimental animal feed, and feeding with free water.
Experimental methods animals were randomly grouped by body weight, 10 animals per group. The experiment was carried out using a blank control group, an LPS + kaempferol low dose group (50mg/kg), an LPS + kaempferol medium dose group (100mg/kg), and an LPS + kaempferol high dose group (200 mg/kg).
LPS (40mg/kg) was injected into the tail vein, and normal saline was administered to the blank control group by intragastric administration. After LPS injection, each administration group of kaempferol immediately dredges the stomach according to the weight of the animal and then administers different doses of the medicine once. The blank control group and the LPS injection group were subjected to intragastric administration of physiological saline according to body weight, intragastric administration volume: 0.2ml/20 g. After LPS injection for 8h, venous blood was obtained by an eye picking method, centrifuged at 4 ℃ for 10min at 3000 rpm, and serum was collected.
Mouse TNF- α and IL-6 detection kits are used, the kit specification is referred for operation, and an enzyme-labeled detector is adopted to measure the light absorption value (OD) of each hole at 490nm, so that the expression levels of TNF- α and IL-6 in cell supernatant are obtained.
Results
TABLE 5 Effect of kaempferol on LPS-induced serum TNF- α, IL-6 expression levels in septic mice (II)
Figure BDA0002329893140000081
n＝10)
Figure BDA0002329893140000082
In comparison with the blank, the number of the blank,**is P<0.01; in comparison with the LPS, the LPS is,#is P<0.05，##Is P<0.01。
The results show that the levels of TNF- α and IL-6 expression in serum of sepsis mice are obviously increased after LPS tail vein injection for 8h compared with the blank group as shown in figure 5 and figure 6.
After 8h of LPS tail intravenous injection, the expression levels of TNF- α and IL-6 in serum of sepsis mice induced by LPS are inhibited in a dose-dependent manner by using kaempferol in low, medium and high doses compared with an LPS injection control group.
In conclusion, kaempferol obviously inhibits the levels of TNF- α and IL-6 released by LPS stimulation in the supernatant of RAW264.7 cells, obviously increases the survival rate and survival time of mice with sepsis caused by CLP or LPS, and obviously reduces the expression levels of TNF- α and IL-6 in the serum of mice with sepsis caused by LPS, thereby generating a protective effect on the mice with sepsis.
Therefore, the invention provides the application of kaempferol in preparing the medicament for treating sepsis, and provides a new idea for the research and development of the medicament for treating sepsis. The kaempferol is easily separated and purified from the trifoliate bitter, and can drive the full development and utilization of medicinal material resources, thereby producing beneficial effects.
Drawings
FIG. 1 is a graph showing the dynamic inhibitory effect of kaempferol in 1 on LPS stimulation of TNF- α released from RAW264.7 of mouse leukemia cells;
FIG. 2 is a graph showing the dynamic inhibitory effect of kaempferol in 1 on LPS stimulation of mouse leukemia cell RAW264.7 release IL-6;
FIG. 3 is a graph showing the effect of kaempferol 2 on the survival rate of CLP sepsis-induced mice;
FIG. 4 is a graph showing the effect of kaempferol in 3 on the survival rate of mice with LPS-induced sepsis, which is proved by experiments of the invention;
FIG. 5 is a graph showing the effect of kaempferol in 4 on the expression level of TNF- α in the serum of mice with LPS-induced sepsis, which is proved by experiments of the present invention;
FIG. 6 is a graph showing the effect of kaempferol in 4 on the expression level of IL-6 in the serum of mice with LPS-induced sepsis.
Detailed Description
The present invention is further illustrated by the following examples, which are not to be construed as limiting the invention.
Example 1. Mixing kaempferol (purchased from market) with appropriate amount of conventional adjuvants, granulating, drying, tabletting, and making into tablet. Each tablet contains 75mg of kaempferol.
The usage and dosage are as follows: the preparation is taken 2 times a day, 2-4 tablets each time, and is used for treating sepsis.
Example 2. Mixing kaempferol (purchased from market) with appropriate amount of conventional adjuvants, oven drying, sterilizing, making into hard capsule, and packaging. Each tablet contains 75mg of kaempferol.
The usage and dosage are as follows: the preparation is taken 2 times a day, 2-4 pills each time, and is used for treating sepsis.
Example 3: adding appropriate amount of common adjuvants into kaempferol (purchased from market), granulating, drying, grading, and making into granule. 5g of granules per bag, and the content of kaempferol in the granules is 15 mg/g.
The usage and dosage are as follows: the preparation is taken 2 times a day, 2-4 bags each time, and is used for treating sepsis.
Example 4: adding appropriate amount of solubilizing agent into kaempferol (purchased from market), grinding, adding small amount of water for dilution, mixing, adding correctant and antiseptic, mixing, adding water to desired amount, filtering, mixing, packaging, sterilizing, and making into oral liquid. The content of kaempferol in the oral liquid is 7.5 mg/ml.
The usage and dosage are as follows: the preparation is taken 2 times a day, 20-40 ml each time, and is used for treating sepsis.
Example 5: adding appropriate amount of common adjuvants into kaempferol (purchased from market), making into pill, drying, and making into pill. Each pill contains kaempferol 75 mg.
The usage and dosage are as follows: the preparation is taken 2 times a day, 2-4 pills each time, and is used for treating sepsis.
Example 6: kaempferol (purchased from market) is prepared into related salts, and then the injection is prepared according to related methods. The content of kaempferol in the injection is 25 mg/ml.
The usage and dosage are as follows: the preparation is injected for 2 times a day, 5-10 ml each time, and is used for treating sepsis.

Claims (6)

1. Use of kaempferol in preparing medicine for treating sepsis is provided.
2.Use of kaempferol according to claim 1 in the manufacture of a medicament for the treatment of sepsis, wherein: kaempferol is also named as: 3,4,5, 7-tetrahydroxyflavone with molecular formula C15H10O6Molecular weight of 286.24, structural formula:
Figure FDA0002329893130000011
3. a medicament for treating sepsis, which is characterized in that: the raw material of the medicine comprises kaempferol.
4. A medicament for treating sepsis, which is characterized in that: the medicine is mainly prepared from kaempferol.
5. A method for the preparation of a medicament for the treatment of sepsis according to claim 3 or 4, wherein: combining kaempferol and pharmaceutically acceptable adjuvants, and processing by conventional method to obtain corresponding medicine.
6. The method for preparing a medicament for treating sepsis according to claim 5, wherein: the medicine is tablet, capsule, granule, oral liquid, pill or injection.
CN201911332029.9A 2019-12-21 2019-12-21 Application of kaempferol in preparation of medicine for treating sepsis Pending CN110898052A (en)

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CN112656793A (en) * 2020-09-23 2021-04-16 吕奔 Application of quinoline derivative in preparation of medicine for treating sepsis

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