TWI428137B - Treatment of Metabolic Diseases by Lactarius and Its Derivatives - Google Patents

Description

Use of Phytolactone A and its derivatives for the treatment of metabolic diseases

The invention relates to the use of a Phyllostachysin A and a derivative thereof, in particular to the use of a Phyllostachysin A and a derivative thereof for treating metabolic diseases.

Tithonia diversifolia is a perennial herb of the genus Asteraceae, and is also known as the king's sunflower, sunflower, and small sunflower. Native to Mexico, it is now found in Asia, Africa, America and Australia. In Taiwan, starting from November every year, from the flat to the low-altitude mountain roads or abandoned land, slopes, highway slopes, it is easy to see this huge golden chrysanthemum. In addition, Wuzhijinying has the functions of clearing away heat and detoxifying, reducing swelling and relieving pain, and relieving heat and diuresis. Its whole grass tastes extremely bitter, and it is also one of the important constituent raw materials of commercially available bitter tea. It has also been confirmed to have anticancer effect, especially melanin. Cancer, skin cancer, etc.

Diabetes is a series of clinical syndromes caused by absolute or relative deficiency of insulin in the body. The main clinical manifestations of diabetes are polydipsia, polyuria, polyphagia, and weight loss ("three more than one less"), as well as high blood sugar and glucose in the urine (normal urine should not contain glucose). If diabetes is not adequately controlled, it can cause acute complications such as hypoglycemia, ketoacidosis (DKA), and nonketotic hyperosmolar coma. Serious long-term complications include: cardiovascular disease, chronic renal failure (also known as diabetic nephropathy, the main cause of hemodialysis in adults in developing countries), retinopathy (also known as diabetic eye disease, can cause blindness, is a developing country Major diseases that cause blindness in non-aged adults), neuropathy and microvascular disease. Among them, microvascular disease may lead to erectile dysfunction (impotence) and the wound is difficult to heal. Wounds that are difficult to heal can cause gangrene (commonly known as "diabetic foot"), which in turn leads to amputation.

Hypercholesterolemia is a high blood cholesterol level that can easily cause atherosclerosis and cause coronary heart disease.

Hyperglycemia and hyperlipidemia are two of the commonly known "three highs". Hyperlipidemia can be manifested as hypercholesterolemia, triglyceride or both. With the rapid development of Taiwan's economy, the increasingly static lifestyle of the people, the Westernized diet and the aging population, the chances of suffering from cardiovascular disease have increased significantly, and according to the statistics of the Department of Health, among the top ten causes of death among Chinese people, metabolic syndrome [ Refers to a comprehensive phenomenon of abdominal obesity, high blood pressure, high blood lipids and high blood sugar. The related mortality rate is as high as 35.7%. Among them, "three highs" is the main risk factor, which causes systemic blood vessels to harden and block, resulting in many Complications, such as coronary artery disease (severe causes myocardial infarction), cerebrovascular disease (severe stroke), peripheral arterial disease (severe cause amputation), kidney disease (severe cause dialysis), eye lesions (severe Leading to blindness, and problems that are unimaginable to the average person, causing disability and disability of patients, and also burdening families and society. According to another survey, more than 60% of the elderly in the country suffer from "three highs", which means that more than half of adults over 40 years of age suffer from hyperglycemia, hypertension and hyperlipidemia. The health concerns of modern people.

Nuclear receptors are a class of intracellular transcription factors. Members of the nuclear receptor superfamily play an important role in cell growth, development, differentiation and metabolism. Since the nuclear receptors are located inside the cell, their hormones (Hormone) are fat-soluble, so that the hormone can enter the cell through the cell membrane composed of fat. The study of nuclear receptors began in the 1970s, when the first nuclear receptors were extracted and separated in the late 1970s. The nuclear receptor is activated upon binding to the hormone, and the activated nuclear receptor complex is responsible for directing transcription of the target gene promoter.

The following members of the nuclear receptor family are transcription factors that can be activated by small molecule receptors or agonists. When an agonist binds to a nuclear receptor, it will alter the transcription of the target gene. Capability, and this change involves the regulation of some of the upstream genes of the target gene (such as the nuclear receptor response element), a mechanism called transcriptional activation that produces the effects of regulating diabetes, lowering blood sugar, and lowering cholesterol.

The peroxisome proliferator-activated receptor (PPAR) is a member of the nuclear receptor superfamily. There are three subtypes, α, β/δ, and γ, which are encoded by different genes and confirmed in humans. Initially, PPARs have been shown to play an important role in regulating lipid metabolism and homeostasis of blood glucose. Currently, PPAR alpha agonists such as fibrates are used to treat atherosclerosis because of its ability to lower cholesterol. PPAR gamma agonists, such as thiazolidinediones, are used in anti-diabetic treatment because of their pharmacological effects to improve insulin resistance in diabetic patients. Clinically, PPARγ agonists are commonly used as troglitazone (trade name: rezulin), rosiglitazone (trade name: avandia), piric acid (pioglitazone, trade name: actos), etc.; It is worth noting that troglitazone has caused lethal hepatotoxicity and was therefore banned from use two months after its listing in the UK (October 1997). In addition, thiazolidinedione derivatives have been ordered and fully recycled and banned in the United States. Due to the adverse cardiovascular side effects of PPAR gamma agonists, pharmaceutical companies have begun to develop PPAR alpha / gamma dual agonists in clinical trials for the treatment of diabetes.

The liver X receptor (LXR) is also a member of the nuclear receptor superfamily, which acts as a ligand-activated transcription factor and has a molecular mechanism similar to that of PPARs. Up to now, LXR has two subtypes, LXRα and LXRβ, which are identified in the liver, intestine, adrenal gland, central nervous system and kidney. LXR is known to act as a sensor for cholesterol (cholesterol) and glucose. Therefore, the function of LXR is mainly to regulate the metabolism of cholesterol and glucose. In the aspect of hypoglycemic effect, many genes such as phosphoenolpyruvate carboxykinase are regulated by LXRs in glucose metabolism. (phosphoenolpyruvate carboxykinase, PEPCK), glucose-6-phosphatase (G-6-Pase) and glucokinase, indicating that LXRs are also involved in glucose metabolism regulation. In addition to its role in the gluconeogenesis pathway, LXRs have also been shown to cause GLUT4 (Glucose transporter type 4) in adipose tissue in diabetic mice, which promotes glucose partitioning and improves insulin resistance. LXRs can reduce glucose concentration by down-regulating PEPCK, G-6-Pase, and GLUT4, and can also lower cholesterol by up-regulating ABCA1, PLTP, and LDLR genes.

The bile acid acceptor (FXR, NR1H4) was identified in 1995 as a member of the nuclear receptor superfamily. Cholic acid, including chenodeoxychoilc acid (CDCA), CA (cholic acid) and DCA (deoxycholic acid), has been confirmed in the literature as an endogenous ligand for FXR, in which CDCA is a bile acid synthesized by the human body. A natural activator of molecules and FXR. FXR has multiple functions in humans, and these functions are mainly involved in the bile acid mechanism including microdimers via transcription and retinoid x receptor (RXR) heterodimers to regulate the transcription of many essential genes. Partner heterodimer partner (SHP), CYP7A1 (Cholesterol 7 alpha-hydroxylase) and bile salt export pump (BSEP). Because these genes are regulated by FXR, they are involved in the action of cholic acid-related diseases, and FXR regulatory factors are considered to have therapeutic potential for cholic acid and hypercholesterolemia, including cardiovascular and lipid metabolic diseases. In addition, because FXR can adjust PEPCK and G-6-Pase down, it lowers blood glucose concentration. In addition, FXR regulatory factors are also useful for improving renal failure and sexual dysfunction in diabetic patients.

As mentioned above, P. chinensis is a common plant, and PPAR, LXR and FXR are nuclear receptors that are effective in regulating glucose and cholesterol in the body. Therefore, if a compound can be found from C. chinensis, it can be used as the three nuclear receptors. The agonist is a great boon for patients with diabetes and hyperglycemia and hypercholesterolemia.

In view of the above, the present invention provides a use of Burdock Chrysanthemum A for the preparation of a medicament for treating a metabolic disease, wherein the Titanium lactone A is a compound of the formula I, which is diabetes, hyperglycemia or High cholesterol:

Wherein A and B are each a single bond or a double bond; and R ₁ , R ₂ and R ₃ are each a hydrogen group (H), a hydroxyl group (OH) or a methoxy group (OCH ₃ ).

The compound of the formula I in the present invention is extracted from water or extracted with an organic solvent, and the organic solvent may include an alcohol such as methanol, ethanol or propanol, an ester such as ethyl acetate, an alkane such as hexane, Or a halogenated alkane such as methyl chloride or ethyl chloride, but not limited thereto, preferably an alcohol, more preferably ethanol, and the compound of the formula I is an agonist of PPARα, PPARγ, LXRs and FXR. To achieve the effects of regulating diabetes, lowering blood sugar and lowering cholesterol.

By the aforementioned compound, the present invention is applied to the regulation of diabetes, blood sugar lowering, and cholesterol lowering, and enables a medical treatment component which is further used for the treatment of diabetes, blood sugar lowering, and cholesterol lowering.

On the other hand, in the present invention, the compound of the formula I can also be used for the treatment of components of pharmaceutical compositions such as diabetes, hyperglycemia and hypercholesterolemia, thereby adjusting blood glucose and cholesterol. The aforementioned pharmaceutical compositions may include, in addition to an effective amount of a compound of formula I, a pharmaceutically acceptable carrier. The carrier may be, but is not limited to, an excipient such as water, a filler such as sucrose or starch, a binder such as a cellulose derivative, a diluent, a disintegrant, an absorption enhancer or a sweetener. . The pharmaceutical composition of the present invention can be produced according to a conventional preparation method of pharmacy, and the active ingredient dose of the compound of the formula I is mixed with one or more carriers to prepare a desired dosage form, and the dosage form can include a tablet, a powder, and a granule. , capsules or other liquid preparations, but not limited to this.

The embodiments of the present invention are further described in the following description, and the embodiments of the present invention are set forth to illustrate the present invention, and are not intended to limit the scope of the present invention. In the scope of the invention, the scope of protection of the invention is defined by the scope of the appended claims.

definition:

The term "cholesterol lowering" as used in the specification of the present invention means lowering low-density lipoprotein (LDL) or promoting high-density lipoprotein (HDL) unless otherwise specified.

The following description will be divided into Examples 1 to 7. Example 1 is the separation of 1-dehydroxytagitinin A; tirotundin and tagitinin A from Pentaphyllum. In order to verify whether the two compounds isolated from Example 1 are effective for regulating diabetes, lowering blood sugar and lowering cholesterol, the method of cell culture, transient transfection reporter and cell viability determination is described in Example 2, and Examples 3-7 verified the transcriptional activity of the di-compounds with various promoters, and confirmed that the edemarin A and the 1-dehydroxy-chitosan A have the effects of regulating diabetes, lowering blood sugar and lowering cholesterol.

Example 1:

Isolation of 1-dehydroxy-toile-Chrysanthemum A and Titanium A from Astragalus membranaceus

Take 10 kg of five-jaw ginseng dry powder, extract with water or organic solvent, and the organic solvent may include alcohols (such as methanol, ethanol or propanol), esters (such as ethyl acetate), and alkanes (such as hexane). Or a halogenated alkane (e.g., methyl chloride, ethyl chloride), but not limited thereto, preferably an alcohol, more preferably ethanol. The extract was concentrated in vacuo to give a crude oil (12 g), which was then chromatographed on a ruthenium oxide column (70-230 mesh), using a gradient of n-hexane-ethyl acetate as an eluent, and 10 fractions were separated by polarity. liquid. The fourth layer (n-hexane-ethyl acetate was 3:2) was chromatographed on a ruthenium oxide column (230-400 mesh), followed by thin layer chromatography to give 10 mg of colorless crystals. The numerical verification of the previous literature verified that the result was 1-dehydroxytagitinin A (tirotundin), the molecular formula was C ₁₉ H ₂₈ O ₆ , the molecular weight was 352, and the structural formula was as shown in Formula II.

The NMR carbon spectrum (NMR C13 spectrum) of the compound of formula II is shown below. The black values in the figure are experimental values, and the values in parentheses are reference values; the reference values are taken from Herz W., Sharma RP J. Org. Chem ., 1975 , 40 , 3118.

The NMR proton spectrum of the compound of formula II is shown below. The black values in the figure are experimental values, and the values in parentheses are reference values; the reference values are taken from Herz W., Sharma RP J. Org. Chem ., 1975 , 40 , 3118.

From the above 10 sub-separations separated according to the polarity, the 7th liquid separation (n-hexane-ethyl acetate was 3:7) was used as a ruthenium oxide column (70-230 mesh) using n-hexane-ethyl acetate. The gradient was used as an eluent to give 12 mg of a white solid. NMR spectroscopy was used to verify the value of the previous literature. The result was Tagitinin A, and the molecular formula was C ₁₉ H ₂₈ O ₇ . The molecular weight is 368, and the structural formula is as shown in Formula III.

The NMR carbon spectrum (NMR C13 spectrum) of the compound of formula III is shown below. The black values in the figure are experimental values, and the values in parentheses are reference values; the reference values are taken from Barua NC, Sharma RP, Madhusudanan KP, Thyagaraian G., Herz W ., Murari R. J. Org. Chem. , 1979 , 44 , 1831.

The NMR proton spectrum of the compound of formula III is shown below. The black values in the figure are experimental values, and the values in parentheses are reference values; the reference values are taken from Barua NC, Sharma RP, Madhusudanan KP, Thyagaraian G., Herz W , Murari R. J. Org . Chem ., 1979 , 44 , 1831.

Example 2:

Cell culture, transient transfection reporter and cell viability assay

The human liver cancer cell line HepG2 was purchased from ATCC (Virginia). These cells were routinely cultured into monolayers of cells in Dulbecco's minimal essential medium containing 10% fetal bovine serum (Hyclone), penicillin (100 units/ml)/streptomycin (100 μg/ml) (purchased from GIBCO/BRL). Company) and cultured at 37 ° C in 5% CO ₂ /air saturated humidity. For transient transfection report analysis, HepG2 cells were seeded in triplicate in 48-well plates at a cell density of 1×10 ⁵ cells/well in phenol red free DMEM containing 10% activated charcoal treated fetal bovine serum, penicillin (100 units/ml) / Streptomycin (100 μg / ml). After 24 hours, three plastids were transfected into HepG2 cells (Qiagen) using the Superfect Transfection Kit. The following is the detection of the potency of the test compound for PPARα, PPARγ, LXRs and FXR activity, which is transfected into cells by three expression plastids:

To test the potency of the compounds of formula II and III of the present invention against PPARα activity, the cells were transfected with 2 μg of wild-type PPARα expression plastid (pCMV-PPARα, Panomics), and 6 μg of luciferase reporter. The body contains the reaction composition of PPARα, pSULT2A1-luc, and 1 μg normalization control, β-galactosidase reporter plastid (pCMV-β, Clontech). The SULT2A1-luc plastid construction method is in accordance with Fang HL, Strom SC, Cai H., Falany CN, Kocarek TA, Runge-Morris M. Mol . Pharmacol ., 2005 , 67 , 1257.

The potentiation of the compounds of formula II and formula III of the invention for PPAR gamma activity is tested to verify whether the compounds of formula II and formula III have efficacy in the treatment of diabetes. The cells were transfected with 2 μg of wild-type PPARγ-expressing plastids (pCMV-PPARγ, Panomics), and 6 μg of luciferase-reporting plastid containing PPARγ response regulator, the regulatory regulator of PPARγ These include pPPRE-luc (Panomics), pABCA1-luc or pSHP-luc, 1 microgram of normalization control, and β-galactosidase reporter plastid (pCMV-β, Clontech). The PPRE sequence is specific for the PPAR-RXR heterodimer, so if the PPAR-RXR heterodimer binds to the PPRE sequence, transcription will be initiated. The ABCA1-luc plastid construction method is based on Pullinger CR, Hakamata H., Duchateau PN, EngC., Aouizerat BE, Cho MH, Fielding CJ, Kane JP Biochem. Biophys. Res. Commun ., 2000 , 271 , 451; SHP- The luc plastid construction method is in accordance with Lin HR, Abraham DJ Bioorg. Med. Chem. Lett. 2006 , 16 , 4178.

To test the potency of the compounds of formula II and formula III of the present invention for LXRs activity, to verify whether the compounds of formula II and formula III have the effect of lowering blood glucose concentration, the cells are transfected with 2 micrograms of wild-type LXRα or β-expressing plastids (pCMV). -LXRα or pCMV-LXRβ, GeneCopoeia), and 6 μg of luciferase reporter plastid containing the LXR reaction regulator element, including prCYP7A1-luc, pPEPCK-luc or pABCA1-luc and 1 μg Normalization control, β-galactosidase reporter plastid (pCMV-β, Clontech). The rCYP7A1-luc plastid construction method is in accordance with Chen W., Owsley E., Yang Y., Stroup D., Chiang JY J. Lipid Res ., 2001 , 42 , 1402; PEPCK-luc plastid construction method according to Wang XL , Herzog B., Waltner-Law M., Hall RK, Shiota M., Granner DK J. Biol. Chem ., 2004 , 279 , 34191; ABCA1-luc plastid construction method according to Pullinger CR, Hakamata H., Duchateau PN, Eng C., Aouizerat BE, Cho MH, Fielding CJ, Kane JP Biochem. Biophys. Res. Commun. , 2000 , 271 , 451.

To test the potency of the compounds of formula II and formula III of the present invention for FXR activity to verify whether the compounds of formula II and formula III have cholesterol lowering effect, and to express plastids in cells of 2 micrograms of wild-type FXR (pCMV-FXR, GeneCopoeia) Company), and 6 micrograms of luciferase reporter plastid containing FXR reaction element, pSHP-luc and 1 microgram of normalized control, β-galactosidase reporter plastid (pCMV-β, Clontech). The SHP-luc plastid construction method is in accordance with Lin HR, Abraham DJ Bioorg. Med. Chem. Lett. 2006 , 16 , 4178.

After transfection, HepG2 cells were treated with different concentrations of the compound of formula II and formula III and a positive control vehicle (dimethyl hydrazine DMSO) in phenol red free medium. After further incubation for 24 hours, the cells were washed with PBS and then lysed with a lysate to produce a cell lysate. This cell lysate is further used to determine the transcriptional activity of PPARs, LXRs or FXR and the normalization of the transfection rate of β-galactosidase. For the luciferase activity assay, 20 microliters of cell lysate and 100 microliters of luciferase assay buffer (Promega) were added to one well of a 96 well plate. Fluorescence was detected using a luminescent microplate reader (Synergy 2). For β-galactosidase activity assay, 20 μl of lysate and 180 μl of β-galactosidase assay buffer (Clontech) were added to one well of a 96-well plate. The luminescence intensity of β-galactosidase activity was measured using Synergy 2 . The activity of the normalized luciferase was calculated by the formula, and the activity of the luciferase was normalized = luciferase activity / β-galactosidase activity. For the agonistic effect of the compounds of formula II and formula III, the normalized luciferase activity value is further converted to the relative normalized luciferase activity by using the carrier (DMSO) as a standard value of 1. Experiments for each compound were tested at least in duplicate or in triplicate.

Example 3:

Effects of 1-dehydroxyl-Chrysanthemum A (T: tirotundin) and Titanium lactone A (TA:tagitinin A) on the transcriptional activation activity of PPAR α-dependent SULT2A1 gene promoter

Human hydroxysteroid sulfotransferase (SULT2A1) plays an important role in hepatic cholesterol balance by catalyzing the sulfonation of exogenous carcinogens, hydroxysteroids and bile acids. The SULT2A1 enzyme is mainly expressed in the liver, but also in other metabolically active tissues such as the intestine and adrenal gland. PPAR alpha has been shown to activate transcription of the human SULT2A1 gene via a PPRE region (aggtg AAAGgtaa) located in the distal portion of the 5 flanking region of the human SULT2A1 gene (-5949 to -5929). To assess the agonistic activity of 1-dehydroxy-chitosan A and piperient A for PPARα, HepG2 cells were used for transient transfection report analysis. The results are shown in the first panel. For the PPAR α-dependent SULT2A1 gene promoter, 1-dehydroxy-chitosan A and Phytolactone A exhibit similar transcriptional activation activities, and at 10 μM, the two The compound has 2.3 times the carrier activity. At 100 nM, the two compounds still had 1.3 times the vector activity.

Example 4:

Transcriptional activation activity of 1-dehydroxyl- chitolide A (T: tirotundin) and T. lactis A (transactivation) PPAR γ-dependent PPRE promoter

First, to confirm the effect of 1-dehydroxy-chitosan A and piperazine A on PPARγ, we used a reporter plastid in the transient transfection report analysis, which was included in front of the luciferase reporter gene. There is a repeating PPAR gamma response regulator element. The results are shown in the second panel. At a concentration of 10 μM, 1-dehydroxy-chitosan A promotes the transcriptional activation of the PPARγ-dependent PPRE promoter to 2-fold vector activity, and the chitosan A shows 1-Dehydroxyl-Chrysanthemum A slightly lower activity.

Example 5:

Effects of 1-dehydroxyl-Chrysanthemum A (T: tirotundin) and Titanium lactone A (TA:tagitinin A) on PPAR γ, LXR α and β-dependent ABCA1 gene promoter activity

The ATP-binding cassette transporter A1 (ABCA1) acts to regulate the cellular efflux of phospholipids and cholesterol into apoA-I containing lipoproteins, and reverses cholesterol transport to exert an important anti-atherosclerotic effect. In addition to reverse cholesterol transport, ABCA1 is also involved in the formation of nascent high-density lipoprotein particles. The transcriptional line of the ABCA1 gene is highly regulated by several nuclear receptors such as the liver X receptor (LXR) and PPAR. The PPAR gamma agonist WY14643 and rosiglitazone have been reported to increase transcriptional activation of the ABCA1 gene, and this activity is via the LXR pathway.

To assess the PPAR gamma agonist activity of 1-dehydroxy-chitosan A and Tithalin A, the ABCA1 promoter reporter plastid was used for transient transfection report analysis to assess PPARγ performance. Results As shown in the third panel, 1-dehydroxy-chitosan A and Phytolactone A at a concentration of 10 μM each showed a transcriptionally activated ABCA1 gene promoter with a 3-fold vector activity in a dose-dependent manner. . Even at a concentration of 1 μM, the two compounds each had 1.8 to 2 times the carrier activity. At 100 nM, 1-dehydroxyl-Chrysanthemum A still had a 1.4-fold vector activity, but the Phytolactone A completely lost its agonistic activity.

To evaluate the LXRα and LXRβ agonist activities of 1-dehydroxy-chitosan A and Phytolactone A, the ABCA1 promoter was used to report plastids for transient transfection report analysis to evaluate LXRα and LXRβ, respectively. Results As shown in the fourth panel, 1-dehydroxy-chitosan A and Tithalin A promoted LXRα-dependent transcriptional activation of the ABCA1 gene promoter in a dose-dependent manner. At a concentration of 10 μM, it was shown that the LXRα-dependent transcriptional activation activity of the ABCA1 gene promoter was twice as high as that of the vector, and the chitinyl lactone A was slightly more active than the 1-dehydroxyristolochito A.

Furthermore, for the agonism of LXRβ, as shown in the fifth panel, 1-dehydroxy-chitosan A and Tithalin A also promoted LXRβ-dependent transcriptional activation of the ABCA1 gene promoter in a dose-dependent manner. However, at a concentration of 10 μM, 1-dehydroxy-chitolide A had a 2.2-fold higher activity than the carrier activity of the chitosan A with a slightly higher transcriptional activation activity. This result supports that the compounds of the formula II and formula III of the present invention can be used as LXR α/β double agonists.

Example 6

Transcriptional activation effects of 1-dehydroxyl-Chrysanthemum A (T: tirotundin ) and Titanium lactone A (TA:tagitinin A) on PPARγ, FXR-dependent SHP gene promoter activity

SHP is a small heterodimer partner protein and is a special member of the nuclear receptor superfamily. SHP is considered an orphan nuclear receptor due to the lack of a defined natural ligand. SHP is commonly used as a transcription factor cofactor for several nuclear receptors and other transcription factors, and other transcription factors such as estrogen receptor, androgen receptor, and NF-kB. By interacting with other transcription factors, SHP regulates hepatic acid synthesis, lipid metabolism, liver fibrosis, glucose metabolism, and insulin secretion. The SHP gene expression has been shown in previous studies to be regulated by several nuclear receptors, including FXR and PPARγ. FXR is directly linked to the SHP gene promoter at position -294 to -276, and PPARγ binds to the -100 to -57 position of the SHP promoter to activate SHP gene expression. To further confirm whether 1-dehydroxyl-chitolide A and piperazine A can act as agonists of PPAR gamma and FXR, we used the human SHP gene promoter for transient transfection report analysis to confirm its The transcriptional activation effect of SHP in PPAR γ and FXR overexpression. For comparison, a drug carrier (dimethyl hydrazine DMSO) was used as a standard value as 1.

The results in the sixth panel show that the dose-dependent transcriptional activation activity of the 1-dehydroxy-chitosan A and the chiral chrysanthemum A at 10 μM for the SHP gene promoter is 3.5 times that of the vector. However, the transcriptional activation activity of 1 μM of 1-dehydroxy-toile-Chrysanthemum A decreased to 1.8-fold vector activity, while the Phytolactone A increased the transcriptional activation of the SHP promoter to 2.8-fold vector activity at the same concentration of 1 μM. It shows that 1-dehydroxy-chitosan A and Phytolactone A have the effect of PPAR γ agonist.

On the other hand, it was confirmed whether the compound of the present invention is a FXR agonist or not, as shown in the seventh panel, showing that the concentration of 10-M, 1-dehydroxy-chitosan A and Phytolactone A transcriptionally activates the SHP gene. The activity of the subunit is twice that of the carrier, and it is important that the activity is slightly greater than 1.8 times the activity of the carrier of CDCA. The transcriptional activation activity of the di-compound at a concentration of 1 μM was 1.5 times that of the vector. This result shows that the compounds of the formula II and the formula III of the present invention have the effect of the FXR agonist.

Example 7

Transcriptional activation effects of 1-dehydroxyl- chicortin A (T: tirotundin) and Titanium lactone A (TA:tagitinin A) on LXRα -dependent rat CYP7A1 and PEPCK gene promoter activities

LXRα up-regulates the expression of CYP7A1 in rats. CYP7A1 in rats is a microsomal cytochrome P450 isozyme and is mainly expressed in the liver. CYP7A1 plays a catalytic role in the metabolism of cholesterol to bile acids, and in the first and rate-determining steps that catalyze this pathway. In this approach, the CYP7A1 modified steroid ring includes hydroxylation at the 7α position, epimerization of the 3-β hydroxy group, and saturation of the steroid ring. For example, CYP7A1 converts cholesterol to 7α-hydroxycholesterol, and further modifies 7α-hydroxycholesterol with another p450 enzyme and produces the final bile acid. LXRα is a major subtype in the liver, and the activity of LXRα causes an accelerated conversion of cholesterol to bile acid, followed by secretion of bile acid and a decrease in the amount of cholesterol.

To evaluate the activity of 1-dehydroxy-chitosan A and Phytolactone A agonist on rat CYP7A1 transcription via LXRα, the HepG2 cell line was used for transient transfection reporter assay. . The results are shown in Figure 8, in which the basic broth (DMSO) agonist activity was set to 1, and it was shown that 1-dehydroxy-chitosan A and Titanium A could transcribe the mouse CYP7A1 gene promoter. Transactivated, and this transcriptional activation is dose dependent. When the concentration of the two compounds is 10 μM, it is used for transcriptional activation twice as much as the activity of the carrier; in addition, the LXR synthetic agonist developed by GW3965 for Glaxo SmithKline is used. The activity of the activated LXRs was very strong (EC _{50 of} LXRα = 190 nM), which showed that the transcriptional activation activity at 1 μM was three times that of the vector.

In the liver, in addition to CYP7A1, LXR alpha also regulates the expression of phosphoenolpyruvate carboxykinase (PEPCK), a rate-limiting enzyme of gluconeogenesis. Evidence suggests that LXRα activity is regulated by down-regulation including peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1), PEPCK, and glucose-6-phosphatase ( Glucose-6-phosphatase expression, G-6-Pase), inhibits the gluconeogenesis process. The synthetic LXR agonist GW3965 has been experimentally confirmed to reduce the amount of mRNA of the PEPCK gene in a mouse model. Therefore, in the present invention, we evaluated the 1-dehydroxy-chitosan A and the Phytolactone by transient transfection report analysis. The effect of A on reducing the activity of the PEPCK gene. Results As shown in the ninth panel, 1-dehydroxy-chitosan A and Phytolactone A were transrepressed with PEPCK gene promoter activity in a dose-dependent manner. At 10 μM, 1-dehydroxyl-Chrysanthemum A reduced the activity of the PEPCK gene promoter by 42% compared with the carrier activity, and the Phytolactone A decreased by 57% compared with the carrier activity. Dehydroxyl-chitolide A is slightly lower. More importantly, 1-dehydroxyl-Chrysanthemum A and Tithalin A showed a similar trans-inhibitory activity as GW3965 at a dose of 1 μM, ie 62% of the activity of the vector. At 100 nM, both inhibitory activities were greatly reduced.

Example 8

Derivatives of Compounds of Formula II~III and Activity Verification of Compounds of Formula IV~XIIII

Due to the structure-activity relationship of the compound of formula II (tirotundin) and the compound of formula III (tagitinin A), it can be seen that if R ₁ , R ₂ , R ₃ (especially R ₃ ) is OH, then The derivatives of the II~III compounds also have similar activities, that is, they have the effects of LXRs, FXR, PPARα, γ agonists or treating diabetes, lowering blood sugar and lowering cholesterol; that is, having double bonds in C1 and C2, C4 and C5 Derivatives of the compound of formula II are, for example, compounds of formula VI and formula VIII, since the double bond has little effect on the 3D stereo configuration of R ₃ =OH.

In addition, according to the literature, compounds of the formulae IV to VIII can be obtained, which should also have LXRs, FXR, PPARα, γ agonists or the efficacy of treating diabetes, lowering blood sugar and lowering cholesterol.

Extraction references for the above compounds include:

1. Herz W., Sharma RP J. Org . Chem ., 1975 , 40 , 3118.

2. Barua NC, Sharma RP, Madhusudanan KP, Thyagaraian G., Herz W., Murari R. J. Org . Chem ., 1979 , 44 , 1831.

3. Garcia A., Delgado g. J. Mex . Chem . Soc ., 2006 , 50 , 180.

4. Kuo YH, Chen CH J Nat Prod ., 1998 , 61 , 827.

5. Pal R., Kuishreshta D.K., Rastogi R.P.,J . Pharm .Sci . , 1976 , 65 , 918.

The structural simulation data of the above compounds of formula IV~IX are as follows:

Here, a computer simulation method was used to confirm that the compounds of the formula IV~VIIIVIII are similar in three-dimensional structure, so that PPARα, γ, LXRα, β and FXR have similar binding ability to formula II and formula III. The energy-optimized compounds of formula IV~VIIIVIII were separately dosed into PPARα (protein data bank, PDB ID: 3FEI), γ (PDB ID: 3G9E), LXRα (PDB) using the Ligandfit program of Discovery Studio (Accelry). ID: 3IPQ), β (PDB ID: 2ACL) or FXR (PDB ID: 1OT7) in the ligand binding pocket X-ray crystal and the PLP value is used to determine the binding ability of the compound to interact with the protein. The PLP value is Calculate the sum of the hydrophilic interaction and the hydrophobic interaction generated by the interaction between the compound and the protein. The higher the PLP value, the stronger the binding ability between the compound and the protein. The PLP values of IV-type VIIII compounds against PPARα, γ, LXRα, β and FXR are known to be related to Phytolactone A (PLP: PPARα=72.57, PPARγ=72.48, FXR=69.39, LXRα=78.01, LXRβ= 78.42) These nuclear receptors have similar binding ability, so the IV~VIIII compounds have the activity of PPARs, LXRs and FXR agonists.

Example 9

Reaction of a compound of formula VI with VIII to produce a compound of formula II

It has been found experimentally that the compounds of formula VI and VIII can be produced via reaction method 1 or reaction method 2:

Reaction method 1:

Hydrogenation of a compound of formula VI or VIII to produce a compound of formula II: first place 50 mg (50 mg) of 10% Pd-C in a round bottom bottle and add 50 ml (ml) of methanol, then add the appropriate amount (40 mg; 40 mg) of the compound of formula VI or VIII was dissolved in the round bottom flask described above, followed by introduction of hydrogen into the round bottom flask of the reaction and hydrogenation at room temperature for 10 to 30 minutes. After completion of the reaction, the solution in the round bottom flask was filtered to obtain a pure solution, and the solution was concentrated under reduced pressure to obtain a crude oil, which was then separated and purified by a chromatography column to obtain a product of the formula II.

Reaction method 2:

Hydrogenation of a compound of formula VI or VIII to produce a compound of formula II: 10 mmol (10 mmol) of polyethylene glycol (400 MW) was first placed in a round bottom flask and 0.05 mM (0.05 mmol) was added. PtO ₂ , then add 1 mM (1 mmol) of the compound of formula VI or VIII to the round bottom flask described above, then introduce hydrogen into the round bottom flask and carry out the hydrogenation reaction at room temperature for 10 to 30 minutes. . After the reaction, 50 ml (ml) of diethyl ether or ethyl acetate was added to a round bottom bottle to extract the product and the product was left to the organic layer (ie, diethyl ether or ethyl acetate layer), and the extracted organic layer was extracted after repeated extraction four times. The solutions are combined to obtain the product organic solution, and then the solution is washed with water and brine solution, then dehydrated with Na ₂ SO _{4 and} concentrated under reduced pressure to obtain a crude oil, and finally by chromatography column method. The separation and purification are carried out to give the product of formula II.

As can be seen from the above examples, the compound of the formula I isolated from the genus Pseudostellariae can be proved to be an active ingredient of the anti-diabetic action of P. chinensis by the PPAR γ route. Importantly, the compounds of formula I are useful as PPAR alpha/gamma dual agonists and do not have the adverse cardiovascular side effects of rosiglitazone. In addition to the PPAR α/γ dual agonist, the compound of formula I can be further used as an LXRs and FXR agonist. Since the synthetic agonist of PPAR γ and FXR has been used in anti-diabetic treatment or in clinical evaluation, the compound of formula I is an active ingredient of anti-diabetes, hypoglycemic and cholesterol of Wuzhi Jinying. Basically, since PPAR α, γ, LXRs and FXR have multiple medicinal beneficial effects for patients with diabetes and hyperglycemia and hypercholesterolemia, the compounds of the formula I of the present invention are anti-diabetic and hyperglycemic in herbal medicines, The treatment of hypercholesterolemia is effective.

On the other hand, in the present invention, the compound of the formula I can also be used for the treatment of components of pharmaceutical compositions such as diabetes, hyperglycemia and hypercholesterolemia, thereby adjusting blood glucose and cholesterol. The aforementioned pharmaceutical compositions may include, in addition to an effective amount of a compound of formula I, a pharmaceutically acceptable carrier. The carrier may be, but is not limited to, an excipient such as water, a filler such as sucrose or starch, a binder such as a cellulose derivative, a diluent, a disintegrant, an absorption enhancer or a sweetener. . The pharmaceutical composition of the present invention can be produced according to a conventional preparation method of pharmacy, and the active ingredient dose of the compound of the formula I is mixed with one or more carriers to prepare a desired dosage form, and the dosage form can include a tablet, a powder, and a granule. , capsules or other liquid preparations, but not limited to this.

The first panel is the detection of the transcriptional activation activity of the 1-hydroxyl-chitolide A and the chiral chrysanthemum A in the PPAR α-dependent SULT2A1 gene promoter (T represents 1-dehydroxy-chitosan lactone) A, TA stands for Titanium A).

The second panel is the results of the activity assay of 1-dehydroxy-chitosan A and Phytolactone A on the transcription-activated PPAR γ-dependent PPRE promoter (T represents 1-dehydroxyl Ester A, TA stands for Tithrone A).

The third panel is the result of the detection of the transcriptional activation of PPAR γ-dependent ABCA1 gene promoter activity by 1-dehydroxy-chitosan A and Phytolactone A (T represents 1-dehydroxyl Ester A, TA stands for Tithrone A).

The fourth panel is the result of the detection of the transcriptional activation of the LXRα-dependent ABCA1 gene promoter by 1-dehydroxy-chitosan A and Phytolactone A (T represents 1-dehydroxy-chitosan lactone) A, TA stands for Titanium A).

The fifth panel is the result of the detection of 1-dehydroxy-chitosan A and Phytolactone A on the transcription-activated LXRβ-dependent ABCA1 gene promoter activity (T represents 1-dehydroxy-chitosan lactone) A, TA stands for Titanium A).

The sixth panel is the result of the detection of the transcriptional activation of PPAR γ-dependent SHP gene promoter activity by 1-dehydroxy-chitosan A and Phytolactone A (T represents 1-dehydroxyl Ester A, TA stands for Tithrone A).

The seventh panel is the result of the detection of the activity of the transcription-activated FXR-dependent SHP gene promoter of 1-dehydroxy-chitosan A and Phytolactone A (T represents 1-dehydroxy-chitosan lactone A, TA stands for Titanium A).

The eighth figure is the detection result of 1-dehydroxy-chitosan A and Phytolactone A in transcriptionally activated LXRα-dependent rat CYP7A1 gene promoter activity (T represents 1-dehydroxyl Ester A, TA stands for Tithrone A).

The ninth figure is the detection result of the transcriptional activation of LXRα-dependent rat PEPCK gene promoter in 1-dehydroxy-chitosan A and Phytolactone A (T represents 1-dehydroxyl Ester A, TA stands for Tithrone A).

Claims (11)

Use of a compound of formula I for the manufacture of a medicament for the treatment of metabolic diseases: Wherein, when R ₁ is a hydrogen group (H), R ₂ and R ₃ are a hydroxyl group (OH), and A is a single bond, B may be a single bond or a double bond; when R ₁ and R ₂ are a hydrogen group ( H), when R ₃ is a hydroxyl group (OH), A and B may each be a single bond or a double bond; however, when B is a single bond, A must be a single bond; when R ₁ and R ₂ are a hydrogen group (H) And R ₃ is a methoxy group (OCH ₃ ), when B is a double bond, A may be a single bond or a double bond; and when R ₁ and R ₃ are a hydroxyl group (OH), and R ₂ is a hydrogen group (H) When, both A and B are single bonds. The use of claim 1, wherein the compound of formula I is a compound of formula II: The use according to claim 1, wherein the compound of formula I is a compound of formula III: The use according to any one of claims 1 to 3, wherein the metabolic disease is diabetes, hyperglycemia, hypercholesterolemia, or renal failure and sexual dysfunction in a diabetic patient. The use according to any one of claims 1 to 3, wherein the compound of the formula I is obtained by extracting a liquid of a pentaphyllum from water or an organic solvent, wherein the organic solvent is selected from the group consisting of alcohols and esters. a group of alkane and alkane. The use according to any one of claims 1 to 3, wherein the compound of the formula I is used as an agonist of PPARα to achieve a cholesterol lowering effect. The use according to any one of claims 1 to 3, wherein the compound of the formula I is used as an agonist of PPARγ to achieve the effect of treating diabetes. The use according to any one of claims 1 to 3, wherein the compound of the formula I is used as an agonist of LXRs to achieve the effects of lowering blood sugar and lowering cholesterol. The use according to any one of claims 1 to 3, wherein the compound of the formula I is used as an agonist of FXR for the treatment of diabetes, lowering cholesterol, and improving renal failure and sexual dysfunction in diabetic patients. . The use according to claim 1, wherein when A is a single bond and B is a double bond, or when A and B are double bonds, the compound of formula I can treat diabetes and hyperglycemia via hydrogenation reaction. Use of symptoms and high cholesterol. A use of a five-pronged gold extract for the preparation of a medicament for the treatment of a metabolic disease, wherein the five-pronged extract comprises a compound of formula I: Wherein, when R ₁ is a hydrogen group (H), R ₂ and R ₃ are a hydroxyl group (OH), and A is a single bond, B may be a single bond or a double bond; when R ₁ and R ₂ are a hydrogen group ( H), when R ₃ is a hydroxyl group (OH), A and B may each be a single bond or a double bond, but when B is a single bond, A must be a single bond; when R ₁ and R ₂ are a hydrogen group (H) And R ₃ is a methoxy group (OCH ₃ ), when B is a double bond, A may be a single bond or a double bond; and when R ₁ and R ₃ are a hydroxyl group (OH), and R ₂ is a hydrogen group (H) When, both A and B are single bonds.