CN106692125B - Antidiabetic medicine containing wild chrysanthemum extract - Google Patents
Antidiabetic medicine containing wild chrysanthemum extract Download PDFInfo
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Abstract
The invention relates to the technical field of antidiabetic drugs, in particular to an antidiabetic drug containing a wild chrysanthemum extract. The wild chrysanthemum flower extract in the antidiabetic medicine contains the sesquiterpene compounds disclosed by the invention, the sesquiterpene compounds can obviously inhibit the accumulation of triglyceride and total fat in the differentiation process of 3T3-L1 preadipocytes, and the inhibition effect is better than that of a positive control compound berberine; the alpha and beta (LXR alpha and LXR beta) of liver X receptors can be obviously excited, and the excitation effect is stronger than that of the existing LXR agonist tool molecule GW 3965; particularly, the compound 4 has obviously better agonistic effect than GW 3965. In addition, the sesquiterpene compounds can also regulate the mRNA expression level of transcription factors SREBP-1c, PPAR gamma and CEBP delta which are important for cholesterol metabolism. Therefore, the antidiabetic medicine containing the wild chrysanthemum extract has good hypoglycemic effect and good development prospect in the aspect of treating diabetes.
Description
Technical Field
The invention relates to the technical field of antidiabetic drugs, and in particular relates to an antidiabetic drug containing a wild chrysanthemum extract.
Background
Diabetes is a systemic disease characterized by chronic hyperglycemic symptoms, the major risk of which is the resulting complications, most commonly such as: foot diseases, nephropathy, eye diseases, encephalopathy, heart diseases, dermatosis, etc. The cause of type 1 diabetes is the destruction of beta cells by the autoimmune system, resulting in the inability to produce insulin. The main cause of type 2 diabetes is that the cells no longer bind to insulin, resulting in the body's inability to utilize insulin. More than 95% of diabetes belongs to type 2 diabetes. Diabetes is second only to cancer and cardiovascular disease. It is estimated that by 2030, there will be 3.66 billion diabetics worldwide. The incidence of Asians (10-12%) is higher than that of Europe (4-5%). According to the international diabetes union (IDF) data, in 2013, diabetes patients in China are in the world. Therefore, diabetes is a great harm to the health of the people in China.
The LXR agonist can obviously reduce the expression of gluconeogenic enzymes such as phosphoenolpyruvate carboxykinase and glucose-6-phosphatase in the liver of a mouse, and promote the utilization of hepatic glucose. The study report indicates that LXR agonist T0901317 treatment can greatly reduce plasma glucose levels in diabetic rats. The LXR agonist acts on the insulin-resistant zucker rat to improve the insulin sensitivity of the insulin-resistant zucker rat, has an inhibition effect on gluconeogenic genes, and obviously improves insulin secretion and plasma concentration of insulin. LXR agonists also promote glucose uptake in adipose and muscle cells and induce expression of the major glucose transporters GLUT1 and GLUT 4. In addition, the LXR agonist can regulate the cholesterol level by regulating the absorption, synthesis, metabolism and transport of cholesterol, and reduce the risks of lipid metabolism disorder and hyperlipidemia accompanied by diabetes. Therefore, LXR is a popular target for the research of antidiabetic drugs in recent years, and the development of novel LXR agonists as potential drugs for the treatment of diabetes has been the focus of leading pharmaceutical enterprises such as weishi, kowa, feverine and the like.
The existing LXR agonist mainly comprises natural oxidative sterol and tertiary amine compound (T0901317 and GW3965) derivatives, and the molecular weight is between 300-650. However, these compounds have not been able to go through clinical trials due to poor agonistic activity, low biological activity, or they have been stopped clinical trials due to human toxicity and side effects. Attempts to treat hyperlipidemia with LXR agonists have not been successful, and efforts to structurally modify the existing LXR agonist lead compounds have been elusive.
The traditional Chinese medicine is the summary of practical experience of people working in China for thousands of years in struggle with diseases, and the search of a lead compound from a traditional Chinese medicine resource treasury is one of the advantages of the development of the medicine in China. Wild chrysanthemum belongs to chrysanthemum subspecies of chrysanthemum of Compositae, is wild in wild lands such as hillside grassland, field and roadside, and is widely used in traditional Chinese medicine prescriptions and tea drinks due to the biological effects of anti-inflammation, antibiosis and cardiovascular protection. The structure of wild chrysanthemum alcohol was identified from wild chrysanthemum in German spring in 1987 (research on Chemical components of wild chrysanthemum flower. pharmaceutical Journal, 1987, 22(11):837-840.), sesquiterpene, which is an active component of wild chrysanthemum flower for inhibiting NO production, was identified in M.Yoshikawa in 2000 (Chemical & pharmaceutical bulletin, 2000, 48(5):651-656.), flavonoid glycoside derivative, which is an active component of wild chrysanthemum flower for inhibiting aldehyde ketone reductase, was identified in 2002 (Chemical & pharmaceutical bulletin, 2002, 50(7):972-975.), and anti-inflammatory activity of wild chrysanthemum crude extract was reported in 2005 in W.Cheng (Journal of ethical biology, 2005 (1-3): 334-337.). So far, the active ingredients of wild chrysanthemum have not been completely developed and utilized.
Disclosure of Invention
Aiming at the problems that the conventional LXR agonist cannot go through clinical tests due to poor agonistic activity and low biological activity and the LXR agonist is still unsuccessful in trying to treat diabetes, the invention provides the antidiabetic medicine which contains the wild chrysanthemum flower extract as the active ingredient and has the activity superior to that of the LXR agonist GW 395.
In order to achieve the purpose, the invention adopts the following technical scheme.
An antidiabetic drug containing a wild chrysanthemum extract comprises the wild chrysanthemum extract and/or a pharmaceutically acceptable carrier, wherein the wild chrysanthemum extract contains sesquiterpenes compounds with the following structures:
the structural formula of the sesquiterpene compound contained in the wild chrysanthemum flower extract is (compound 1):
the structural formula of the sesquiterpene compound contained in the wild chrysanthemum flower extract is (compound 2):
the structural formula of the sesquiterpene compound contained in the wild chrysanthemum flower extract is (compound 3):
the structural formula of the sesquiterpene compound contained in the wild chrysanthemum flower extract is (compound 4):
compared with the prior art, the invention has the beneficial effects that: the wild chrysanthemum extract in the antidiabetic medicine contains the sesquiterpene compounds disclosed above, the sesquiterpene compounds can obviously inhibit the accumulation of triglyceride and total fat in the differentiation process of 3T3-L1 preadipocytes, and the inhibition effect is better than that of a positive control compound berberine; the alpha and beta (LXR alpha and LXR beta) of liver X receptors can be obviously excited, and the excitation effect is stronger than that of the existing LXR agonist tool molecule GW 3965; particularly, the compound 4 has obviously better agonistic effect than GW 3965. In addition, the sesquiterpene compounds can also regulate the mRNA expression level of transcription factors SREBP-1c, PPAR gamma and CEBP delta which are important for cholesterol metabolism. Therefore, the antidiabetic medicine containing the wild chrysanthemum extract has good hypoglycemic effect and good development prospect in the aspect of treating diabetes.
Drawings
FIG. 1 is data of the activity of compounds 1, 2, 4 isolated from wild chrysanthemum extract in example 1 to inhibit total fat accumulation;
FIG. 2 is data of the triglyceride accumulation-inhibiting activity of compounds 1-4 isolated from wild chrysanthemum flower extract in example 1;
FIG. 3 is data showing the activity of Compound 4 isolated from the wild chrysanthemum flower extract in example 1 in activating LXR α and LXR β;
FIG. 4 is data on the activity (PPAR γ) of compound 3 and compound 4 isolated from the extract of wild chrysanthemum indicum in example 1 to up-regulate mRNA expression of a key transcription factor in the process of cholesterol regulation;
FIG. 5 is activity data of compound 3 and compound 4 isolated from wild chrysanthemum flower extract up-regulating mRNA expression of a key transcription factor in cholesterol regulation process (SREBP-1c) in example 1;
FIG. 6 is data of the activity of compound 3 and compound 4 isolated from wild chrysanthemum flower extract in example 1 to up-regulate mRNA expression of a key transcription factor in cholesterol regulation (CEBP. delta.).
Detailed Description
In order to more fully understand the technical contents of the present invention, the technical solutions of the present invention will be further described and illustrated with reference to the following specific embodiments.
Example 1
The embodiment provides an antidiabetic medicine containing wild chrysanthemum extract, which consists of the wild chrysanthemum extract and/or a pharmaceutically acceptable carrier. The wild chrysanthemum extract contains sesquiterpene compounds with the following structures:
the wild chrysanthemum extract is prepared by the following method. In this example, the sesquiterpene compounds in the extract of wild chrysanthemum flower were analyzed for their physiological activities.
The preparation method of the wild chrysanthemum extract comprises the following steps:
(1) extraction of
Weighing 7.0Kg of dried wild chrysanthemum flower, soaking the dried wild chrysanthemum flower in 10L of ethanol (95%) at normal temperature and pressure for 24h, then filtering, and collecting the filtrate. Extracting the wild chrysanthemum flower for three times repeatedly, combining the filtrates obtained by the three extractions, and then putting the filtrates into a water bath for normal pressure concentration to obtain 2.0Kg of extract.
(2) Extraction of
Putting the extract into 5L of water, stirring to make the extract become turbid suspension, extracting with ethyl acetate to obtain extract liquor, concentrating the extract liquor to obtain concentrate, and drying the concentrate to obtain 425g of extract.
(3) Column chromatography
Dissolving the extract with methanol, adsorbing the dissolved extract in 200-300 mesh silica gel with the same weight, drying the silica gel at room temperature to obtain a chromatographic sample, performing column chromatography on the chromatographic sample by using 2.0Kg of 200-300 mesh silica gel, performing gradient elution by using petroleum ether and chloroform in sequence, and collecting chloroform effluent; the chloroform eluate was concentrated to obtain 120g of an initial precipitate.
(4) Second chromatography
Performing column chromatography on the primary eluate by using 200-mesh 300-mesh silica gel, and performing gradient elution by using a composite eluent, wherein the composite eluent consists of petroleum ether and ethyl acetate, and the used composite eluent with series of polarities is as follows: 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 20:80 (volume ratio of petroleum ether to ethyl acetate). The effluents are followed by Thin Layer Chromatography (TLC), and the effluents in which substances of the same polarity (under the TLC chromatography conditions) are dissolved are combined according to the chromatography conditions reflected by TLC, whereby fraction A, fraction B, fraction C and fraction D, which are gradually increased in polarity, are sequentially collected. Each fraction was concentrated to give 18g of extract A, 29g of extract B, 19g of extract C and 15g of extract D, respectively. Extract a, extract B, extract C and extract D constitute the wild chrysanthemum extract described in the antidiabetic agent of this example.
Further separating and purifying the extract A, the extract B, the extract C and the extract D respectively. Further separating and purifying to obtain the sesquiterpene compound with the following structural general formula:
the specific separation and purification steps are described in the following (5) to (8).
(5) Isolation and purification of Compound 1
Extract a was purified by column chromatography using MCI column with methanol/water gradient elution using the following series of polar methanol/water eluates: 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20 and 90:10 (volume ratio of methanol to water), collecting effluent, and concentrating the effluent to obtain extract A1.
The extract A1 was subjected to column chromatography using MCI column, followed by gradient elution with petroleum ether/ethyl acetate, chloroform/methanol, using the following series of polar petroleum ether/ethyl acetate eluents: 90:10, 80:20, 70:30 (volume ratio of petroleum ether to ethyl acetate), the series of polar chloroform/methanol eluents used were as follows: 95:5 and 80:20 (volume ratio of chloroform to methanol), collecting the effluent, and concentrating the effluent to obtain an extract A2.
Subjecting the extract A2 to reverse phase silica gel column chromatography, gradient eluting with methanol/water at a volume ratio of 70:30 and 60:40, collecting eluate, and concentrating to obtain compound 1, wherein compound 1 is white powder.
Measuring mass spectrum, nuclear magnetic resonance hydrogen spectrum and nuclear magnetic resonance carbon spectrum of compound 1 respectively, and determining that the molecular weight of compound 1 is 592.3 and the molecular formula is C35H44O8. The mass spectrum, nuclear magnetic resonance hydrogen spectrum, nuclear magnetic resonance carbon spectrum of compound 1 are consistent with the corresponding spectra of compound 2 in document 1, and thus the structural formula of compound 1 is: (document 1: Chrysanolide A, unprecedented sequiterpenoid primer from the flowers of Chrysanthemum L.RSC Advances,2013,3(26):10168.)
Specific test data are as follows:
HRESI-MS m/z 591.2936[M-H]-。
1H-NMR(400MHz，CDCl3)δ:6.18(1H，dd，J＝5.84，1.00Hz，H-3`)，6.16(1H，dd，J＝5.84，1.00Hz，H-3``)，6.03(1H，d，J＝3.64Hz，13`-Ha)，5.30(1H，d，J＝3.28Hz，13`-Hb)，5.88(1H，d，J＝5.48Hz，H-2`)，5.55(1H，ddd，J＝3.68，4.24，2.04Hz，H-8)，5.49(1H，m，J＝1.36Hz，H-3)，4.08(1H，t，J＝9.72Hz，H-6`)，3.96(1H，t，J＝9.80Hz，H-6)，3.54(1H，t，J＝8.65Hz，H-7)，2.95(1H，dd，J＝6.20，3.04Hz，H-7`)，2.74(1H，m，J＝7.96Hz，H-5)，2.56(1H，dt，J＝10.68，7.60Hz，H-1)，2.36(1Hd，J＝12.12Hz，H-13a)，1.49(1H，s，H-13b)，2.26(1H，d，J＝4.54Hz，H-9a)，1.97(1H，d，H-9b)，2.18(1H，m，H-8`a)，1.46(1H，m，H-8`b)，2.24-2.04(2H，m，H-2)，1.95(1H，m，H-5`)，1.80(2H，m，H-9`)，1.97(3H，d，J＝7.36Hz，3``-CH3)，1.89(3H，s，2``-CH3-)，1.89(3H，s，H-15)，1.46(3H，s，H-14`)，1.29(3H，s，H-15`)，1.21(3H，s，H-14)。
13C-NMR(101MHz，CDCl3)δ:54.6(C-1)，33.5(C-2)，125.8(C-3)，144.8(C-4)，54.9(C-5)，79.0(C-6)，47.8(C-7)，70.1(C-8)，38.3(C-9)，73.6(C-10)，59.0(C-11)，178.3(C-12)，37.4(C-13)，33.7(C-14)，18.5(C-15)，64.8(C-1`)，133.8(C-2`)，140.6(C-3`)，57.4(C-4`)，65.9(C-5`)，79.4(C-6`)，43.1(C-7`)，23.7(C-8`)，34.9(C-9`)，72.9(C-10`)，141.3(C-11`)，170.2(C-12`)，118.4(C-13`)，15.5(C-14`)，29.9(C-15`)，166.5(C-1``)，126.6(C-2``)，143.4(C-3``)，16.3(C-4``)，20.6(C-5``)。
(6) isolation and purification of Compound 2
Extract B was first subjected to column chromatography using 300g of silica gel, and eluted with a chloroform/ethyl acetate gradient using the following polar series of chloroform/ethyl acetate eluents: 100:0, 90:10, 80:20 and 70:30 (volume ratio of chloroform to ethyl acetate), collecting the effluent, and concentrating the effluent to obtain an extract B1. The extract B1 is subjected to column chromatography by SephadexLH-20 (methanol/chloroform, volume ratio 50:50), Rp-18 (gradient elution, volume ratio of methanol/water eluent with series of polarities: 70:30, 80:20, 90:10, 100:0) and preparative HPLC (methanol/water, volume ratio 60:40) in sequence, and compound 2 can be isolated and obtained, and the compound 2 is white solid.
Measuring mass spectrum, nuclear magnetic resonance hydrogen spectrum and nuclear magnetic resonance carbon spectrum of compound 2 respectively, and determining that the molecular weight of compound 2 is 306 and the molecular formula isC17H22O5. The mass spectrum, nuclear magnetic resonance hydrogen spectrum, nuclear magnetic resonance carbon spectrum of compound 2 are consistent with the corresponding spectra of compound 12 in document 2, and thus the structural formula of compound 2 is: (document 2: Cumamrin A in Chrysanthemum boreale Makino Preparation, X-ray Crystal Structure and13C-and 1H-NMR Study of Cumambrin A[J]Kor.J.Pharmacogn,1996,27:207-211.)
specific test data are as follows:
EI-MSm/z＝307.2([M+H]+)。
1H-NMR(400MHz，CDCl3)δ:6.18(1H，d，J＝3.44Hz，H-13)，5.50(1H，d，J＝2.96Hz，H-13)，5.50(1H，d，J＝2.96Hz，H-3)，5.16(1H，ddd，J＝7.04，5.72，1.12Hz，H-8)，3.99(1H，m，H-6)，3.89(1H，tt，J＝9.36，3.12Hz，H-7)，2.76(1H，dd，J＝10.84，4.12Hz，H-5)，2.58(1H，dd，J＝18.44，8.20Hz，H-1)，2.31(1H，dd，J＝16.68，5.80Hz，H-9)，1.84(1H，d，J＝16.68Hz，H-9)，2.27-2.18(1H，m，H-2)，2.13-2.03(1H，m，H-2)，1.90(3H，s，H-14)，1.24(3H，s，H-15)，2.16(3H，s，OAc)。
13C-NMR(101MHz，CDCl3)δ:54.4(C-1)，33.7(C-2)，125.6(C-3)，138.6(C-4)，54.5(C-5)，80.4(C-6)，46.6(C-7)，73.9(C-8)，39.0(C-9)，73.6(C-10)，143.9(C-11)，169.6(C-12)，121.5(C-13)，33.7(C-14)，18.0(C-15)，170.3(C-16)，21.6(C-17)。
(7) isolation and purification of Compound 3
Performing column chromatography on the extract C by an MCI column, and performing gradient elution by using methanol/water, wherein methanol/water eluent with the following polarity series is used: 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10 (volume ratio of methanol to water), collecting the effluent, and concentrating the effluent to obtain the extract C1. The extract C1 is sequentially subjected to column chromatography using silica gel column (gradient elution, volume ratio of chloroform/methanol eluent with series polarity: 95:5, 85:15, 70:30) and Sephadex LH-20 (methanol) to obtain compound 3, wherein the compound 3 is white solid.
Measuring mass spectrum, nuclear magnetic resonance hydrogen spectrum and nuclear magnetic resonance carbon spectrum of compound 3 respectively, and determining that the molecular weight of compound 3 is 246.1 and the molecular formula is C15H18O3. The mass spectrum, nuclear magnetic resonance hydrogen spectrum, nuclear magnetic resonance carbon spectrum of compound 3 are consistent with the corresponding spectra of compound 1 in document 3, and thus the structural formula of compound 3 is: (document 3: formatting analysis of achillinand leukodin [ J ]]J Nat Prod,1988,51(2):22-228.)
Specific test data are as follows:
EI-MSm/z＝247.1([M+H]+)。
1H-NMR(400MHz，CDCl3)δ:6.14(1H，q，J＝1.32，H-3)，3.59(1H，td，J＝11.20，10.08，1.36Hz，H-6)，3.39(1H，d，J＝10.04Hz，H-5)，2.41(3H，s，H-14)，2.41(1H，m，H-9a)，2.24(1H，ddd，J＝13.64，6.72，1.52Hz，H-9b)，2.33(1H，dd，J＝3.88，1.44Hz，H-11)，2.27(3H，s，H-15)，1.97(1H，m，H-8a)，1.94(1H，m，J＝12.44，2.72Hz，H-7)，1.34(1H，dd，J＝12.12，1.64Hz，H-8b)，1.25(3H，dd，J＝6.84，1.60Hz，H-13)。
13C-NMR(101MHz，CDCl3)δ:196.0(C-2)，177.6(C-12)，170.0(C-4)，152.2(C-10)，135.6(C-3)，132.0(C-1)，84.3(C-6)，56.5(C-7)，52.7(C-5)，41.2(C-11)，37.7(C-9)，26.1(C-8)，21.7(C-14)，19.9(C-15)，12.4(C-13)。
(8) isolation and purification of Compound 4
Performing column chromatography (methanol) on the extract D by using a Sephadex LH-20 column, collecting effluent, and concentrating the effluent to obtain the extract D1.
Extract D1 was purified by column chromatography using MCI column with methanol/water gradient elution to remove pigments using the following series of polar methanol/water eluates: 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10 and 100:0 (volume ratio of methanol to water), collecting effluent, and concentrating the effluent to obtain the extract D2.
The extract D2 is sequentially subjected to column chromatography by using silica gel (gradient elution, the volume ratio of chloroform/ethyl acetate eluent with series polarity is 90: 10-30: 70), Rp-18 (gradient elution, the volume ratio of methanol/water eluent with series polarity is 60: 40-90: 10), Toyopearl HW-40 (methanol), silica gel (gradient elution, the volume ratio of chloroform/methanol eluent with series polarity is 100:0, 90:10, 80:20), Sephadex LH-20 (methanol/chloroform, volume ratio is 50:50), Rp-8 (gradient elution, the volume ratio of methanol/water eluent with series polarity is 60:40, 70:30, 80:20, 90:10), preparative HPLC (methanol/water, volume ratio is 65:35), compound 4 was obtained as a white solid.
Measuring mass spectrum, nuclear magnetic resonance hydrogen spectrum and nuclear magnetic resonance carbon spectrum of compound 4 respectively, wherein the data obtained from the measurement shows that the molecular weight of compound 4 is 798.4, and the molecular formula is C47H58O11. The mass spectrum, nuclear magnetic resonance hydrogen spectrum, and nuclear magnetic resonance carbon spectrum of compound 4 are consistent with the corresponding spectra of compound 3 in document 1, and thus the structural formula of compound 4 is: (document 1: Chrysanolide A, unprecedented sequiterpenoid primer from the flowers of Chrysanthemum L.RSC Advances,2013,3(26):10168.)
Specific test data are as follows:
EI-MS m/z＝799.40([M+H]+)。
1H-NMR(400MHz，CDCl3)δ:6.25(1H，d，J＝5.36Hz，H-3`)，6.06(1H，d，J＝3.16Hz，H-3``)，5.99(1H，d，J＝5.24Hz，H-13``a)，5.32(1H，d，J＝5.60Hz，H-13``b)，5.88(1H，d，J＝5.40Hz，H-2``)，5.85(1H，d，J＝5.48Hz，H-2`)，5.46(1H，s，H-8)，5.34(1H，m，J＝2.76Hz，H-3)，4.14(1H，t，J＝9.60Hz，H-6`)，4.00(1H，t，J＝9.60Hz，H-6``)，3.88(1H，t，J＝9.76Hz，H-6)，3.48(1H，dd，J＝18.28，9.08Hz，H-7)，3.18(1H，d，J＝18.84，9.56Hz，H-7`)，2.81(1H，m，J＝17.12，9.20Hz，H-5``)，2.73(1H，m，J＝17.68，8.20Hz，H-5)，2.53(1H，dd，J＝17.68，7.88Hz，H-5`)，2.38(1H，d，J＝11.80Hz，H-7``)，2.11(2H，s，H-9)，1.84(3H，s，H-OAc)，1.84(3H，s，H-15)，1.37(3H，s，H-14`)，1.32(3H，s，H-15``)，1.29(3H，s，H-15`)，1.25(3H，s，H-14``)，1.19(3H，s，H-14)。
13C-NMR(101MH，CDCl3)δ:179.4(C-12)，178.7(C-12``)，170.9(C-OAc)，170.6(C-12`)，144.1(C-4)，141.3(C-11``)，141.1(C-3`)，139.9(C-3``)，134.3(C-2`)，133.6(C-2``)，125.5(C-3)，118.7(C-13``)，80.1(C-6`)，78.8(C-6``)，77.4(C-6)，73.4(C-10)，73.1(C-10`)，71.8(C-10``)，70.9(C-8)，66.3(C-5`)，65.8(C-5``)，64.5(C-1`)，64.3(C-1``)，60.4(C-11)，57.7(C-11`)，56.8(C-4`)，56.7(C-4``)，54.2(C-1)，54.0(C-5)，47.1(C-7)，43.9(C-7`)，43.5(C-7``)，38.4(C-9)，36.8(C-13)，36.8(C-13`)，35.5(C-9`)，35.0(C-9``)，33.6(C-14)，33.3(C-2)，29.8(C-14`)，29.4(C-14``)，23.5(C-8`)，22.3(C-8``)，22.2(C-OAc)，18.2(C-15)，15.5(C-15`)，15.3(C-15`)。
as can be seen from the above results of further separation and purification of extract a, extract B, extract C and extract D, the wild chrysanthemum flower extract of this example contained compound 1, compound 2, compound 3 and compound 4 as described above.
The drug effect-related experiments of the compounds 1 to 4 were as follows.
Test 1: activity assay of Compounds 1-4 for inhibiting triglyceride accumulation and total fat accumulation.
(1) Cell culture
3T3-L1 (mouse embryo fibroblasts, preadipocytes) complete medium (DMEM medium with 10% fetal calf serum, 100U/mL penicillin, 100. mu.g/mL streptomycin) was used at 37 ℃ with a volume fraction of 5% CO2Cultured in an incubator.
(2) Pharmaceutical intervention
The test compound is prepared into 10mM mother solution by DMSO, and is diluted into working solution with different concentrations during testing. To correct for the effect of the mother liquor solvent DMSO, the blank was supplemented with 0.1% DMSO.
3T3-L1 was inoculated into 48-well plates and cultured until complete contact (day 0), and the medium was changed to differentiation medium I or differentiation medium I added with the specified concentration of the test compound was cultured for 3 days (day 3). Formula of differentiation medium I: complete medium was supplemented with 2. mu.g/mL insulin, 100ng/mL dexamethasone, 0.5mM 3-isobutyl-1-methylxanthine and 10ng/mL biotin.
The differentiation medium and the test compound were allowed to act for 3 days, and the medium was changed to differentiation medium II or differentiation medium II containing the test compound at a specified concentration was added and the culture was continued for 3 days (day 6). Formula of differentiation medium II: insulin was added at 2. mu.g/mL to the complete medium.
On day 6, cells were harvested for triglyceride or oil red O staining analysis.
(3) Triglyceride testing
Treated 3T3-L1 cells, ice PBS (0.2M NaCl, 10mM Na)2HPO4，3mM KCl，2mM KH2PO4pH 7.4), ultrasonically breaking the cells, and detecting the absorbance at 546nm by using a triglyceride kit and a multifunctional microplate reader so as to calculate the content of triglyceride in the cell breaking solution. Results are expressed as a percentage of triglyceride content in the blank cells.
(4) Oil red O dyeing
3T3-L1 cells were fixed in 10% (V/V) formalin solution at room temperature for 1 hour, stained with freshly prepared oil red O working solution at 60 ℃ for 30 minutes, washed twice with distilled water, and total fat stained red by oil red O, observed and photographed in an inverted microscope equipped with a CCD digital camera. The magnification factor and the light intensity are unchanged in the shooting process.
(5) Result processing
And detecting the absorbance at 546nm according to a triglyceride kit and a multifunctional microplate reader, subtracting the background absorbance value of the blank control group, and comparing the background absorbance value with the absorbance value of the negative control group cell, wherein the result is expressed in the form of the content percentage of triglyceride in the negative control group cell. All data are expressed as mean ± standard deviation.
The test results are shown in fig. 1 and 2. As can be seen from FIG. 2, compounds 1-4 all reduced intracellular triglyceride accumulation to varying degrees in 3T3-L1, with almost no oil droplets accumulated in 3T3-L1 cells 6 days after CI-15 treatment and a triglyceride level of zero. As can be seen from FIG. 1, Compound 2 and Compound 4, which had the best triglyceride lowering effect, significantly reduced the total lipid droplet content in 3T3-L1 cells.
Test 2: determination of LXR agonistic activity of compound 4.
(1) Cell culture
HEK293T (human embryonic kidney cells) was prepared in complete medium (DMEM medium supplemented with 10% fetal bovine serum, 100U/mL penicillin, 100. mu.g/mL streptomycin) at 37 ℃ with a volume fraction of 5% CO2Cultured in an incubator.
(2) Pharmaceutical intervention
HEK293T cells were plated at a density of 2X 104 cells per well in 96-well plates and cultured until the cells grew to approximately 90% coverage, and 6.5. mu.g pGL3/(DR-4) -c-fos-FF-luc (firefly luciferase reporter) plasmid, 1.3. mu.g pSG5/hLXR α (or pSG5/hLXR β, liver X receptor α or β) plasmid, 1.3. mu.g pSG5/hRXR α (retinoic acid X receptor) plasmid and 0.13. mu.g pCMV/Renilla-luc (Renilla luciferase reporter) plasmid were plated with 10. mu.LLIPOFECTAMINE, according to Lipofectamine TM2000 protocolTM2000 liposome reagents were mixed well, left to stand at room temperature for 30 minutes, and the liposome-DNA solution was added to a 96-well plate at 5. mu.L per well.
After 5 hours of culture, the medium was changed to complete medium and culture was continued for 5 hours.
The test compound stock solution was added to a working concentration of 10. mu.M, and co-cultured with the cells for 20 hours. Firefly luciferase activity and renilla luciferase activity were detected using a multifunctional microplate reader workstation, Flex Station 3.
(3) Result processing
Results analysis firefly luciferase activity was corrected for renilla luciferase activity, so the corrected enzyme activity was the firefly luciferase activity value/renilla luciferase activity value. Relative luciferase activity value-corrected enzyme activity value of experimental group/corrected enzyme activity value of negative control group.
The test result is shown in fig. 3, the activity of the compound 4 for exciting the LXR alpha is stronger than that of the existing LXR alpha agonist tool molecule GW3965, and the activity of the compound 4 for exciting the LXR beta is equivalent to that of the existing LXR beta agonist tool molecule GW 3965.
Test 3: and (3) measuring the activity of up-regulating the mRNA expression level of a key transcription factor on the LXR pathway by using the compound 3 and the compound 4.
(1) Cell culture
3T3-L1 (mouse embryo fibroblasts, preadipocytes) complete medium (DMEM medium with 10% fetal calf serum, 100U/mL penicillin, 100. mu.g/mL streptomycin) was used at 37 ℃ with a volume fraction of 5% CO2Cultured in an incubator.
(2) Pharmaceutical intervention
The test compound is prepared into 10mM mother solution by DMSO, and is diluted into working solution with different concentrations during testing. To correct for the effect of the mother liquor solvent DMSO, the blank was supplemented with 0.1% DMSO.
3T3-L1 was inoculated into 48-well plates and cultured until complete contact (day 0), and the medium was changed to differentiation medium I or differentiation medium I added with the specified concentration of the test compound was cultured for 24 hours (day 1). And harvesting the cells for fluorescent quantitative PCR amplification.
(3) Fluorescent quantitative PCR
Total RNA from 3T3-L1 cells was extracted using RNAiSo Plus, 1. mu.g of total RNA was reverse transcribed into cDNA by Oligo dT18, and primers were PCR amplified by quantitative PCR technique using Toyobo company Thunde BirdSYBR qPCR Mix reagent as per the instructions. Each set of samples had 3 duplicate wells to ensure validity of experimental data. The expression change of the target gene relative to the reference gene is detected by using beta-actin as the reference gene for quantification. By using 2-ΔΔCtThe method analyzes data (i.e., fold change of expression amount of target gene of experimental group relative to control group).
The primers used were:
β-actin(sense:5’-TGGAATCCTGTGGCATCCATGAAA-3’；antisense:5’-TAAAACGCAGCTCAGTAACAGTCC-3’)、
SREBP-1c(sense:5’-CAGCTCAGAGCCGTGGTGA-3’；antisense:5’-TGTGTGCACTTCGTAGGGTC-3’)、
PPAR-γ(sense:5’-TGCTGTTATGGGTGAAACTCTG-3’；antisense:5’-GAAATCAACTGTGGTAAAGGGC-3’)、
CEBPδ(sense:5’-AGCCCAACTTGGACGCCAG-3’；antisense:5’-TCGTCGTACATGGCAGGAGT-3’)。
(4) result processing
Detecting the expression change of the target gene relative to the reference gene to quantify. By using 2-ΔΔCtThe method analyzes data (i.e., fold change of expression amount of target gene of experimental group relative to control group).
As shown in FIGS. 4-6, compound 4 at a concentration of 10. mu.M significantly increased the mRNA expression level of SREBP-1c in 3T3-L1 cells (the mRNA expression level of SREBP-1c in compound 4-treated cells was 8-fold higher than that in the blank control group), while the mRNA expression levels of PPAR γ and CEBP δ were increased (3-fold both). The compound 3 can obviously increase the mRNA expression level of PPAR gamma and CEBP delta.
Example 2
This example provides an anti-diabetic agent containing a wild chrysanthemum extract, which contains compound 1 described in example 1 and/or a pharmaceutically acceptable carrier, and the active ingredient is compound 1.
The test results in example 1 show that the antidiabetic drug containing the wild chrysanthemum extract has the effects of reducing the accumulation of oil droplets and the accumulation of triglyceride in the differentiation process of 3T3-L1 preadipocytes, so that the antidiabetic drug has a good hypoglycemic effect and has a good development prospect in the aspect of treating diabetes.
Example 3
This example provides an anti-diabetic agent containing a wild chrysanthemum extract, which contains compound 2 described in example 1 and/or a pharmaceutically acceptable carrier, and the active ingredient is compound 2.
The test results in example 1 show that the antidiabetic drug containing the wild chrysanthemum extract has the effects of reducing the accumulation of oil droplets and the accumulation of triglyceride in the differentiation process of 3T3-L1 preadipocytes, so that the antidiabetic drug has a good hypoglycemic effect and has a good development prospect in the aspect of treating diabetes.
Example 4
This example provides an anti-diabetic agent containing a wild chrysanthemum extract, which contains compound 3 described in example 1 and/or a pharmaceutically acceptable carrier, and the active ingredient is compound 3.
From the test results in example 1, it can be seen that the anti-diabetic agent containing the wild chrysanthemum extract of this example has the effects of reducing the accumulation of oil droplets and the accumulation of triglycerides during the differentiation of 3T3-L1 preadipocytes, and increasing the expression levels of mrnas of PPAR γ, SREBP-1c, and CEBP δ, so that the anti-diabetic agent has a good blood glucose reducing effect and a good development prospect in the aspect of treating diabetes.
Example 5
This example provides an anti-diabetic agent containing a wild chrysanthemum extract, which contains compound 4 described in example 1 and/or a pharmaceutically acceptable carrier, and the active ingredient is compound 4.
From the test results in example 1, it can be seen that the anti-diabetic agent containing the wild chrysanthemum extract of this example has the effects of reducing the accumulation of oil droplets and the accumulation of triglycerides during the differentiation of 3T3-L1 preadipocytes, and increasing the expression levels of mrnas of PPAR γ, SREBP-1c, and CEBP δ, so that the anti-diabetic agent has a good blood glucose reducing effect and a good development prospect in the aspect of treating diabetes.
The technical contents of the present invention are further illustrated by the examples, so as to facilitate the understanding of the reader, but the embodiments of the present invention are not limited thereto, and any technical extension or re-creation based on the present invention is protected by the present invention.
Claims (3)
1. The application of sesquiterpenes extracted from wild chrysanthemum flowers in preparing antidiabetic drugs is characterized in that the sesquiterpenes have the structural formula:
2. the application of sesquiterpenes extracted from wild chrysanthemum flowers in preparing antidiabetic drugs is characterized in that the sesquiterpenes have the structural formula:
3. the application of sesquiterpenes extracted from wild chrysanthemum flowers in preparing antidiabetic drugs is characterized in that the sesquiterpenes have the structural formula:
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| Chrysanolide A, an unprecedented sesquiterpenoid trimer from the flowers of Chrysanthemum indicum L;Qiong Gu等;《RSC Advances》;20130513;第3卷;第10168-10172页 * |
| 清热解毒药对2 型糖尿病小鼠血糖血脂的影响;秦崇涛 等;《福建中医药大学学报》;20140630;第24卷(第3期);第55-56页 * |
| 野菊花活性成分及应用的研究进展;陈凯 等;《安徽化工》;20140228;第40卷(第1期);第10-13页 * |
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