Application of dracocephalum heterophyllum benth ethanol extract in preparation of medicine for treating and / or preventing bile acid metabolism abnormality

Abstract

The invention discloses application of a dracocephalum heterophyllum ethanol extract in preparation of a medicine for treating and / or preventing bile acid metabolism abnormality. The ethanol extract improves metabolic abnormality of bile acid by regulating expression of bile acid metabolism related proteins such as FXR, MRP2, BSEP, NTCP1, CYP7A1 and the like. The dracocephalum heterophyllum benth ethanol extract disclosed by the invention can be used for treating related diseases caused by bile acid abnormality, such as cholestatic liver injury and the like, and a new natural medicine choice is provided for treating the related diseases caused by bile acid abnormality.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technologies, and particularly to the use of an ethanol extract of Dracocephalum heterophyllum in the preparation of a drug for treating and / or preventing abnormal bile acid metabolism. Background Art

[0002] Bile acids are the main end products of cholesterol metabolism in the liver. After being synthesized in the liver, they are excreted into the intestine through the bile duct and participate in physiological processes such as lipid digestion, energy metabolism, and signal transduction. Under normal circumstances, the synthesis, secretion, and excretion of bile acids are in dynamic balance. Once this balance is disrupted, it will lead to abnormal bile acid metabolism, manifested as the accumulation of bile acids in the liver, an increase in the level of bile acids in the blood, and an increase in the proportion of toxic bile acids. If this metabolic disorder persists, it can further cause cholestasis (obstruction of bile flow), and ultimately develop into cholestatic liver injury, which is characterized by hepatocyte necrosis, inflammatory infiltration, and fibrosis, and even progresses to cirrhosis or liver failure.

[0003] Among the existing treatment methods, ursodeoxycholic acid (UDCA) improves cholestasis by replacing toxic bile acids and enhancing the efflux effect mediated by BSEP, but has limited regulatory effects on bile acid synthesis, and some patients have drug resistance; FXR agonists (such as obeticholic acid) can inhibit CYP7A1 to reduce bile acid synthesis and upregulate the expression of BSEP / MRP2, but long-term use may interfere with lipid metabolism and increase cardiovascular risks. In addition, the above drugs all focus on a single link of bile acid excretion or synthesis and are difficult to comprehensively correct the disorder of the bile acid metabolic network.

[0004] In recent years, natural drugs have attracted attention due to their multi-target synergistic effects. However, existing studies are mostly limited to downstream effects such as anti-inflammatory or antioxidant effects, lacking systematic regulation of the entire bile acid synthesis-transport-excretion pathway, especially direct intervention on key targets such as CYP7A1, BSEP, and NTCP. Therefore, developing a natural drug that takes the regulation of bile acid metabolism as the core and has the functions of inhibiting synthesis, promoting excretion, and reducing the toxicity of bile acids is expected to fundamentally block the pathological process of cholestasis and its secondary liver injury. Summary of the Invention

[0005] Dracocephalum heterophyllum, also known as "white flowered schizonepeta", is the dried aerial part of the herbaceous plant Dracocephalum heterophyllum Benth. of the genus Dracocephalum in the Lamiaceae family. Its Tibetan name is "Jizi Qingbao". It is currently included in the "Drug Standards of the Ministry of Health" in 1995 edition. The whole plant can be used as medicine and is a commonly used medicinal material in ethnic areas.

[0006] To solve the above problems, the present invention intends to provide an ethanol extract of Dracocephalum heterophyllum Benth., which is a natural medicine with high safety and can regulate abnormal bile acid metabolism by regulating related expressed proteins such as FXR, MRP2, BSEP, NTCP1, etc.

[0007] The present invention provides the use of an ethanol extract of Dracocephalum heterophyllum Benth. in the preparation of a drug for treating and / or preventing abnormal bile acid metabolism.

[0008] For the extraction method of the present invention, conventional plant extraction methods can be used, such as, but not limited to, heating extraction, percolation extraction, ultrasonic extraction, microwave extraction, maceration extraction, supercritical extraction, etc.

[0009] After extraction, other conventional preparation steps are also included, such as filtration, concentration, centrifugation, drying, evaporation, etc.

[0010] In some specific embodiments of the present invention, the preparation method of the ethanol extract of Dracocephalum heterophyllum Benth. comprises: mixing Dracocephalum heterophyllum Benth. with an ethanol solution, and subjecting to reduced pressure, concentration, and drying to obtain the ethanol extract of Dracocephalum heterophyllum Benth.

[0011] In the present invention, the ethanol content in the ethanol solution is selected from 10~90% v / v, further selected from 60~90% v / v, and can be, for example, ethanol at concentrations such as 60%, 65%, 70%, 75%, 80%, 85%, 90%, etc.

[0012] In some specific embodiments of the present invention, the ethanol extract of Dracocephalum heterophyllum Benth. is an extract with 60 - 90% ethanol.

[0013] In some specific embodiments of the present invention, the concentration of the ethanol solution is 75%.

[0014] In some specific embodiments of the present invention, the drug is an FXR agonist.

[0015] In some specific embodiments of the present invention, the drug is a drug that regulates the expression of at least one protein among FXR (farnesoid X receptor), MRP2 (multidrug resistance - associated protein 2), BSEP (bile salt export pump), NTCP1 (sodium - taurocholate cotransporting polypeptide 1), CYP7A1 (cholesterol 7α - hydroxylase).

[0016] The "regulation" refers to influencing the expression level or activity of target proteins (such as FXR, MRP2, BSEP, NTCP1, CYP7A1, etc.) through drug intervention, thereby regulating the synthesis, transport, or metabolic process of bile acids; the regulation includes: up-regulation (activation or enhancement) and down-regulation (inhibition or reduction). Up-regulation, for example: activating FXR to inhibit CYP7A1 (reducing bile acid synthesis), or promoting the expression of BSEP / MRP2 (enhancing bile acid excretion); down-regulation, for example: inhibiting CYP7A1 to reduce the excessive production of bile acids.

[0017] In some specific embodiments of the present invention, the drug is a drug that increases the expression of at least one of the proteins MRP2, BSEP, NTCP1, and FXR; and / or, the drug is a drug that reduces the expression level of the CYP7A1 protein.

[0018] The biosynthesis and transport of bile acids are mainly regulated by FXR, which coordinates multiple key molecules through downstream target genes. During the bile acid transport process, MRP2 is responsible for secreting cholate into bile, BSEP drives the active transport of bile acids in hepatocytes to the bile canaliculus, and NTCP1, as the main uptake protein on the basolateral membrane of hepatocytes, mediates the reuptake of bile acids in the circulation. At the level of anabolic metabolism, CYP7A1, as a rate-limiting enzyme, catalyzes the conversion of cholesterol into primary bile acids. FXR promotes bile acid excretion by activating BSEP and MRP2, and inhibits the transcription of CYP7A1, forming a negative feedback loop to maintain the dynamic balance of bile acid metabolism.

[0019] In some specific embodiments of the present invention, the drug is a drug for treating and / or preventing cholestasis.

[0020] In some specific embodiments of the present invention, the drug is a drug for treating and / or preventing cholestatic liver injury.

[0021] In some specific embodiments of the present invention, the cholestatic injury is induced by ANIT (α-naphthylisothiocyanate).

[0022] It should be noted that cholestasis and liver injury are causally related to each other. Cholestasis may lead to liver injury, and liver injury may also lead to cholestasis. However, their core mechanisms are different, so the treatment strategies vary significantly. The core mechanism of liver injury caused by cholestasis is that the obstruction of bile secretion or excretion leads to the accumulation of bile acids in the liver, directly damaging hepatocytes and bile duct epithelial cells. Therefore, for cholestatic injury, it is necessary to specifically improve bile flow and bile duct function. The core mechanism of cholestasis caused by liver injury is that extensive necrosis or fibrosis of hepatocytes destroys the microenvironment for bile production and secretion, resulting in cholestasis. In this case, the main focus is on repairing hepatocytes and regulating the primary disease. In the present invention, the ethanol extract of Dracocephalum heterophyllum Benth. relieves cholestatic liver injury by directly regulating key proteins in bile acid metabolism, reducing the abnormal accumulation of bile acids and protecting the stability of hepatocyte organelles.

[0023] In some specific embodiments of the present invention, the drug is a drug that regulates at least one of the indicators of TBA (total bile acid), TBIL (total bilirubin), AST (aspartate aminotransferase), ALT (alanine aminotransferase), and ALP (alkaline phosphatase).

[0024] In the present invention, the drug is a drug that regulates at least one of the inflammatory factors of TNF-α, IL-6, IL-1β, IL-8, IL-17, IL-10, and IL-37 / IL-38.

[0025] In some specific embodiments of the present invention, the drug is a drug that reduces at least one of the inflammatory factors of TNF-α, IL-6, and IL-1β.

[0026] In the present invention, unless otherwise specified, terms such as "regulate" and "control" can be used interchangeably herein, which refer to upregulating or downregulating the expression level, activity, or function of target biomolecules (including but not limited to proteins such as FXR, MRP2, BSEP, NTCP1, CYP7A1, etc., and inflammatory factors) through drug intervention, so as to ultimately maintain or restore the normal level.

[0027] The present invention also provides a pharmaceutical composition, which comprises the above-mentioned ethanol extract of Dracocephalum heterophyllum Benth. and a pharmaceutically acceptable excipient.

[0028] In the present invention, "pharmaceutically acceptable excipients" refer to auxiliary materials used for stabilizing, loading, or delivering drugs in a preparation, excluding the active ingredient, including but not limited to diluents, solvents, disintegrants, binders, coating materials, etc. "Acceptable" means that it meets the pharmacopoeia standards, has no interaction with other components in the composition, and is safe for the human body. The pharmaceutical composition contains the ethanol extract of Dracocephalum heterophyllum Benth. described in the present invention and pharmaceutically acceptable excipients. The excipients can be in the form of solids, semi-solids, liquids, etc., depending on the preparation requirements.

[0029] In some specific embodiments of the present invention, the preparation is an oral preparation, an injection, or a topical preparation.

[0030] In some specific embodiments of the present invention, the oral preparation is an ointment, pill, oral liquid, powder, tablet, granule, or capsule.

[0031] In some specific embodiments of the present invention, the topical preparation is a solution, lotion, liniment, ointment, plaster, paste, or patch.

[0032] In some specific embodiments of the present invention, the injection is a solution, powder, or tablet.

[0033] The present invention also provides a method for treating and / or preventing cholestatic liver injury, including: administering / applying an effective amount of the ethanol extract of Dracocephalum heterophyllum Benth. to an individual; the administration / application can be through systemic administration or local administration.

[0034] In the present invention, the term "treatment" refers to prophylactic (e.g., preventive medication), curative, or palliative treatment. Specifically, treatment here refers to administering or applying the ethanol extract of Dracocephalum heterophyllum Benth. described in the present invention or a pharmaceutical composition containing this Dracocephalum heterophyllum Benth. to an individual who may have a disease, symptoms related to the disease, secondary diseases or abnormalities of the disease, or is prone to suffering from the above diseases, in order to partially or completely relieve, improve, or reduce one or more symptoms or characteristics of a specific abnormality and / or disease condition, or delay its occurrence, hinder its progression, reduce its severity and / or reduce its incidence. Treatment can also be carried out on an individual who has not yet shown signs of a disease, abnormality, and / or disease condition and / or an individual who has shown early signs, in order to reduce the risk of developing pathological changes related to the disease, abnormality, and / or disease condition.

[0035] In the present invention, "individual" refers to an individual who can accept the extract and pharmaceutical composition described in the present invention, and the individual is a mammal, including humans. Unless otherwise specified, the terms "administration" and "application" can be used interchangeably herein, and they refer to providing the ethanol extract of Dracocephalum heterophyllum Benth. or the pharmaceutical composition of the present invention to an individual in need of treatment.

[0036] In the present invention, an "effective amount" refers to the amount of Dracocephalum heterophyllum Benth. ethanol extract and / or the pharmaceutical composition sufficient to produce the desired therapeutic effect. The effective amount of the medicament does not necessarily have to be capable of curing a disease or disorder, but can delay, impede or prevent the occurrence of the disease or disorder, or can alleviate the symptoms associated with the disease or disorder. The therapeutically effective amount can be divided into one, two or more doses and administered one, two or more times in a suitable dosage form over a specified period. The specific therapeutically effective amount or sufficient dose depends on various factors, such as the specific condition to be treated, the physiological condition of the individual (e.g., the individual's weight, age or gender), the mammalian or animal type being treated, the duration of treatment, the nature of concurrent treatments (if any), and the specific formulation and structure of the active ingredient used. The effective amount can be expressed in any suitable manner, for example, it can be expressed as the total weight of Dracocephalum heterophyllum Benth. ethanol extract (expressed in g, mg or μg) or as a proportion by body weight (such as mg / kg).

[0037] The beneficial effects of the present invention are as follows:

[0038] (1) The Dracocephalum heterophyllum Benth. ethanol extract provided by the present invention can comprehensively improve bile acid metabolic disorders by upregulating the expression of FXR protein, inhibiting the activity of CYP7A1, and simultaneously regulating transporters such as MRP2, BSEP, and NTCP1;

[0039] (2) The Dracocephalum heterophyllum Benth. ethanol extract can reduce the levels of serum TBA, TBIL, ALT, AST, and ALP (P < 0.01), restore the liver physiological indicators to the normal range, and inhibit the release of pro-inflammatory factors such as IL-6, IL-1β, and TNF-α, thereby alleviating the liver inflammatory response.

[0040] The following abbreviations have the following meanings:

[0041] ANIT represents α-naphthyl isothiocyanate;

[0042] FXR represents farnesoid X receptor;

[0043] MRP2 represents multidrug resistance-associated protein 2;

[0044] BSEP represents bile salt export pump;

[0045] NTCP1 represents sodium-taurocholate cotransporting polypeptide 1;

[0046] CYP7A1 represents cholesterol 7α-hydroxylase;

[0047] TBA represents total bile acid;

[0048] TBIL represents total bilirubin;

[0049] AST represents aspartate aminotransferase;

[0050] ALT is expressed as glutamic pyruvic transaminase / alanine aminotransferase;

[0051] ALP is expressed as alkaline phosphatase. Description of the Drawings

[0052] Figure 1 It is a design diagram for animal experiments;

[0053] Figure 2 It is for the morphological observation of liver tissues (a. Con group; b. Mod group; c. UDCA group; d. YL group; e. YM group; f. YH group);

[0054] Figure 3 It is for the liver function indexes in the serum of mice induced by ANIT (a. TBA; b. TBIL; c. AST; d. ALT; e. ALP);

[0055] Figure 4 It is for the inflammatory factor indexes in the liver tissues of mice induced by ANIT (a. IL-6; b. IL-1β; c. TNF-α);

[0056] Figure 5 It is for detecting the gene expression levels of FXR, CYP7A1, BSEP, FXR, and NTCP1, etc. by Western blot experiment in cholestatic mice induced by ANIT. Detailed Implementation Modes

[0057] The technical solutions of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0058] Example 1

[0059] 1 Experimental Method

[0060] Ethanol extract of Dracocephalum heterophyllum Benth.: After the whole plant of Dracocephalum heterophyllum Benth. was crushed, it was extracted with 75% ethanol and then concentrated under reduced pressure and freeze-dried to obtain the ethanol extract of Dracocephalum heterophyllum Benth.

[0061] Design of the mouse experiment plan: As Figure 1As shown, a total of 60 male C57BL / 6J mice were adaptively fed for one week. After that, the 60 mice were divided into 6 groups according to body weight, with 10 mice in each group, namely the normal group, the model group, the positive drug group, and the low-, medium-, and high-dose groups of Dracocephalum heterophyllum Benth. ethanol extract. Mice in the normal group (CON) were intragastrically administered 0.9% normal saline every day. Mice in the model group (MOD) were intragastrically administered 0.9% normal saline every day, and on the 12th day, they were intragastrically administered ANIT (80 mg / kg) for model establishment. Positive drug group (UDCA): Administered 0.3 mL of UDCA by gavage at a dose of 100 mg / kg every day, and on the 12th day, they were intragastrically administered ANIT (80 mg / kg) for model establishment. Low-dose group of Dracocephalum heterophyllum Benth. (YL): Administered Dracocephalum heterophyllum Benth. ethanol extract by gavage at a dose of 100 mg / kg every day, and on the 12th day, they were intragastrically administered ANIT (80 mg / kg) for model establishment; Medium-dose group of Dracocephalum heterophyllum Benth. (YM): Administered Dracocephalum heterophyllum Benth. ethanol extract by gavage at a dose of 200 mg / kg every day, and on the 12th day, they were intragastrically administered ANIT (80 mg / kg) for model establishment; High-dose group of Dracocephalum heterophyllum Benth. (YH): Administered Dracocephalum heterophyllum Benth. ethanol extract by gavage at a dose of 400 mg / kg every day, and on the 12th day, they were intragastrically administered ANIT (80 mg / kg) for model establishment. On the evening of the 14th day, the mice were fasted but allowed to drink water, and were sacrificed on the 15th day.

[0062] Sample collection and treatment: After obtaining whole blood by puncturing the orbital venous plexus, it was left standing at room temperature for 1 h to separate the serum, centrifuged at 4000 rpm for 15 min at 4 °C, and the supernatant was taken as the serum, which was aliquoted and stored at -80 °C for later use. After blood collection, the mice were euthanized by cervical dislocation. After dissection, the liver tissue was dissected out, rinsed with normal saline and wiped dry with filter paper, and photographed to observe the bile and liver morphology. The liver tissue was divided into 3 parts, placed in 1.5 mL EP tubes, then placed in liquid nitrogen and transferred to an -80 °C refrigerator for storage, for subsequent determination of biochemical indexes and WB.

[0063] Determination of biochemical indexes in liver tissue: After taking out the liver tissue frozen at -80 °C and slowly thawing it at 4 °C, the liver tissue was weighed and mixed with PBS of corresponding volume (weight-to-volume ratio of 1 g: 9 mL) and homogenized thoroughly on ice. Then the homogenate was centrifuged at 12000 rpm for 15 min at 4 °C, and the supernatant was taken for analysis and detection of biochemical indexes in the liver.

[0064] Western blot detection of related protein expression: Cut 15-20 mg of liver tissue, add protein lysate and PMSF according to the ratio (1 g: 9 mL), homogenize, place at 4°C for 30 min, add an equal volume of PBS for dilution, and centrifuge at 4°C, 12,000 rpm for 15 min. Take the supernatant. Determine the protein concentration by BCA protein quantification. After adding the protein sample according to the concentration calculated by the normalization method, make up the volume with PBS, add loading buffer, and perform protein denaturation in a metal bath at 100°C for 10 min for later Western blot analysis.

[0065] 2 Result Analysis

[0066] (1) Effects of ethanol extract of Dracocephalum heterophyllum Benth. on the liver and gallbladder morphology of ANIT-induced cholestatic mice

[0067] As Figure 2 shown, macroscopically, the livers of mice in the normal group were normal, the gallbladders were light yellow and small. After ANIT treatment, obvious liver injury occurred in the liver, showing white as a whole, the gallbladders were swollen and dark green. After administration, the livers were ruddy and the color and morphology of the gallbladders gradually tended to be normal, indicating that the ethanol extract of Dracocephalum heterophyllum Benth. could effectively improve the cholestatic characteristics induced by ANIT.

[0068] (2) Effects of Dracocephalum heterophyllum Benth. on liver function of ANIT-induced cholestatic mice

[0069] As Figure 3 shown, compared with the normal group, the levels of TBA and TBIL in the serum of the model group were extremely significantly increased (P < 0.01). Compared with the model group, the levels of TBA and TBIL in the serum of the positive drug group and the medium- and high-dose groups of Dracocephalum heterophyllum Benth. were extremely significantly decreased (P < 0.01), proving that the drug promoted the reuptake of bile acids by the liver and alleviated ANIT-induced cholestasis. Compared with the normal group of mice, the activities of AST, ALT, and ALP in the livers of the model group of mice were extremely significantly increased (P < 0.01), indicating serious liver injury and a certain degree of cholestasis. Compared with the model group, the activities of AST, ALT, and ALP in the livers of the positive drug and the medium- and high-dose groups of Dracocephalum heterophyllum Benth. were significantly decreased (P < 0.01), proving that Dracocephalum heterophyllum Benth. could reduce the activities of AST, ALT, and ALP in the liver, thereby alleviating the damage of liver cells and achieving the effect of protecting the liver.

[0070] (3) Improvement effect of ethanol extract of Dracocephalum heterophyllum Benth. on inflammation in ANIT-induced cholestatic mice

[0071] As Figure 4As shown, compared with the normal group, the levels of IL-6, IL-1β, and TNF-α in the livers of mice in the model group increased extremely significantly (P<0.01), suggesting that ANIT-induced modeling can trigger the occurrence of inflammatory reactions. Compared with the model group, the levels of IL-6, IL-1β, and TNF-α in mice in the positive drug group and the medium- and high-dose Dracocephalum heterophyllum groups decreased significantly (P<0.05 or P<0.01). The above results indicate that the ethanol extract of Dracocephalum heterophyllum has a significant anti-inflammatory effect and can significantly inhibit the occurrence of inflammatory reactions in the livers of cholestatic mice.

[0072] (4)Effect of ethanol extract of Dracocephalum heterophyllum on the expression of related proteins in the liver of ANIT-induced cholestatic mice

[0073] As Figure 5 shown, compared with the normal group, the expression level of FXR protein in the livers of mice induced by ANIT decreased (P<0.05). After treatment with the positive drug and the medium- and high-dose ethanol extracts of Dracocephalum heterophyllum, the expression level of FXR protein could return to normal. The protein expressions of the downstream transporters MRP2, BSEP, and NTCP1 of FXR decreased significantly after ANIT treatment (P<0.05 or P<0.01). Compared with the model group, both the positive drug and the ethanol extract of Dracocephalum heterophyllum could increase the expression levels of related proteins to a certain extent, and the improvement effect of Dracocephalum heterophyllum showed a dose-dependent effect, with the best improvement effect in the high-dose group (P<0.01). The protein expression level of CYP7A1, a downstream regulatory factor of FXR biosynthesis, increased extremely significantly after ANIT treatment (P<0.05). Both the positive drug and the ethanol extract of Dracocephalum heterophyllum could decrease the protein expression level of CYP7A1 to a certain extent, and the improvement effect of Dracocephalum heterophyllum showed a dose-dependent effect, with the best improvement effect in the high-dose group (P<0.01).

[0074] The above research results show that the ethanol extract of Dracocephalum heterophyllum is an effective agonist of FXR, and can effectively alleviate cholestasis and improve liver injury by improving the expression of FXR and related proteins in its downstream pathway.

[0075] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Use of ethanol extract of Dracocephalum heterophyllum Benth. in the preparation of a drug for treating and / or preventing abnormal bile acid metabolism.

2. The use according to claim 1, wherein The ethanol extract of Dracocephalum heterophyllum Benth. is an extract with 60-90% ethanol.

3. The use according to claim 1, wherein The drug is an FXR agonist.

4. The use according to claim 1, characterized in that, The drug is a drug for regulating the expression of at least one protein among FXR, MRP2, BSEP, NTCP1, and CYP7A1.

5. The use according to claim 1, characterized in that, The drug is a drug for increasing the expression of at least one protein among MRP2, BSEP, NTCP1, and FXR; and / or, the drug is a drug for reducing the expression level of CYP7A1 protein.

6. The use according to claim 1, wherein The drug is a drug for treating and / or preventing cholestasis.

7. The use according to claim 1, wherein The drug is a drug for treating and / or preventing cholestatic liver injury.

8. The use according to claim 1, characterized in that, The cholestatic injury is induced by ANIT.

9. The use according to claim 1, wherein The drug is a drug for regulating at least one index among TBA, TBIL, AST, ALT, and ALP.

10. The use according to claim 1, characterized in that, The drug is a drug for regulating at least one inflammatory factor among TNF-α, IL-6, IL-1β, IL-8, IL-17, IL-10, and IL-37 / IL-38; further, the drug is a drug for reducing at least one inflammatory factor among TNF-α, IL-6, and IL-1β.

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