CN106692125A - Anti-diabetic drug containing wild chrysanthemum flower extract - Google Patents
Anti-diabetic drug containing wild chrysanthemum flower extract Download PDFInfo
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Abstract
The invention specifically relates to an anti-diabetic drug containing wild chrysanthemum flower extract, belonging to the technical field of anti-diabetic drugs. The wild chrysanthemum flower extract in the anti-diabetic drug contains sesquiterpenoids disclosed in the invention; the sesquiterpenoids can substantially inhibit accumulation of triglyceride and total fat in the differentiation process of 3T3-L1 preadipocytes, and the inhibitory effect is superior to the inhibitory effect of a positive control compound, i.e., berberine; and the sesquiterpenoids can also obviously activate liver X receptors alpha and beta (LXR alpha and LXR beta), wherein the activation effect of the sesquiterpenoids is better than the activation effect of a conventional LXR agonist tool molecule GW3965, and in particular, a compound 4 has obviously better activation effect compared with GW3965. Moreover, the sesquiterpenoids can adjust the mRNA expression of transcription factors SREBP-1c, PPAR gamma and CEBP delta of vital importance to metabolism of cholesterol. Thus, the anti-diabetic drug containing the wild chrysanthemum flower extract in the invention has good hpyerglycemic effect and presents good development prospects in treatment of diabetes.
Description
Technical field
It is the present invention relates to antidiabetic medicine technical field more particularly to a kind of containing Flos Chrysanthemi Indici extract
Antidiabetic medicine.
Background technology
Diabetes are the systemic diseases being characterized with chronic hyperglycemia symptom, and its main hazard is that it is led
The complication of cause, it is most common such as：Pedopathy, ephrosis, illness in eye, encephalopathic, heart disease, skin disease etc..1
The cause of disease of patients with type Ⅰ DM is autoimmune system destruction β cells, causes insulin to produce.2 types sugar
The cause of disease for urinating disease is mainly cell no longer with insulin combination, causes body to utilize insulin.95%
Diabetes spp above is in diabetes B.Diabetes are be only second to cancer and angiocardiopathy the third-largest
Killer.According to estimates, to the year two thousand thirty, the whole world will have 3.66 hundred million diabetics.Asian morbidity
Rate (10-12%) is higher than the European incidence of disease (4-5%).According to International Diabetes Federation (IDF) data,
2013, China diabetes patient numerical digit occupied the hat in the whole world.Therefore, diabetes are to China's national health harm
Greatly.
Gluconeogenesis enzyme such as PEP carboxylic swashs during lxr agonist can significantly lower mouse liver
The expression of enzyme and G-6-Pase, promotes glycogen to utilize.Research report points out lxr agonist
T0901317 treatments can be greatly reduced diabetes rat plasma glucose levels.Lxr agonist is acted on
Its insulin sensitivity can be improved in the zucker rats of insulin resistance, and gluconeogenesis gene is produced
Inhibitory action, and significantly improve the PC of insulin secretion and insulin.Lxr agonist can also promote
Enter fat and muscle cell in glucose absorption, and induce main glucose transporter GLUT1 and
The expression of GLUT4.Additionally, lxr agonist can be by regulating and controlling the absorption of cholesterol, synthesis, metabolism
With transhipment regulation cholesterol levels, reduce diabetes mellitus with disorders of lipid metabolism, hyperlipidemia risk.
Therefore, LXR is the popular target of Recent study antidiabetic medicine, develops new lxr agonist
It is always the emphasis direction that the tap medicine such as Webster, China of section, Pfizer is looked forward to as the potential drug for the treatment of diabetes.
Existing lxr agonist mainly includes natural oxidisability sterol and tertiary amine compounds
The derivative of (T0901317 and GW3965), molecular weight is between 300-650.However, at present
Untill, these compounds cannot come into clinical test because agonist activity is poor, bioactivity is low, or by
Clinical test is stopped in human body toxic and side effect.Lxr agonist treatment hyperlipidemia trial not yet into
Work(, and unable to do what one wishes is seemed to the effort that existing lxr agonist lead compound carries out structure of modification.
Chinese medicine is that the practical experience that Chinese working people combats the disease for thousands of years is summarized, from Chinese medicine money
It is one of advantage of China's drug development that lead compound is found in the treasure-house of source.Chrysanthemum indicum belongs to composite family chrysanthemum
Kind of chrysanthemum indicum subspecies, it is wild in the wildly band such as patana, Tian Bian, roadside, because there is anti-inflammatory, resisting
Bacterium and protect cardiovascular biological effect and be widely used in prescriptions of traditional Chinese medicine, tea-drinking.1987 in moral
Spring identified from chrysanthemum indicum chrysanthemol structure (the research Acta Pharmaceutica Sinicas of chrysanthemum indicum chemical composition,
1987,22 (11):837-840.), M.Yoshikawa in 2000 identifies chrysanthemum indicum suppression NO generations
Active component sesquiterpene (Chemical＆pharmaceutical bulletin, 2000,48 (5):
651-656.), H.Matsuda in 2002 identifies the active component Huang that chrysanthemum indicum suppresses aldehyde ketone reductase
Ketoside derivative (Chemical＆pharmaceutical bulletin, 2002,50 (7):972-975.),
W.Cheng reports anti-inflammatory activity (the Journal of of chrysanthemum indicum crude extract within 2005
Ethnopharmacology, 2005,101 (1-3):334-337.).So far, the active component of chrysanthemum indicum is also
It is fairly well-developed far away and utilizes.
The content of the invention
The present invention cannot come into for existing lxr agonist because agonist activity is poor, bioactivity is low
Clinical test, the trial successful problem not yet of lxr agonist treatment diabetes, there is provided it is a kind of effectively into
It is divided into the antidiabetic medicine of Flos Chrysanthemi Indici extract and activity better than lxr agonist GW395.
To achieve the above object, the present invention uses following technical scheme.
A kind of antidiabetic medicine containing Flos Chrysanthemi Indici extract, including Flos Chrysanthemi Indici extract and/or pharmaceutically may be used
The carrier of receiving, the sesquiterpenoids containing following structure in the Flos Chrysanthemi Indici extract：
The structural formula of contained sesquiterpenoids is (compound 1) in the Flos Chrysanthemi Indici extract：
The structural formula of contained sesquiterpenoids is (compound 2) in the Flos Chrysanthemi Indici extract：
The structural formula of contained sesquiterpenoids is (compound 3) in the Flos Chrysanthemi Indici extract：
The structural formula of contained sesquiterpenoids is (compound 4) in the Flos Chrysanthemi Indici extract：
Compared with prior art, the beneficial effects of the invention are as follows：Open country in antidiabetic medicine of the invention
Chrysanthemum extract contains sesquiterpenoids chemical combination disclosed above, and these sesquiterpenoids can be significantly inhibited
The accumulation of triglycerides and total fat in 3T3-L1 PECTORAL LIMB SKELETON atomizations, inhibition is better than sun
Property control compound jamaicin；Can also substantially exciting Liver X receptor alpha and β (LXR α and LXR β),
Agonistic effects are better than existing lxr agonist tool molecule GW3965；Especially compound 4, it swashs
Dynamic effect is substantially better than GW3965.Additionally, these sesquiterpenoids can also be adjusted to cholesterol generation
Thank to the mrna expression amount of vital transcription factor SREBP-1c, PPAR γ and CEBP δ.
Therefore, the antidiabetic medicine containing disclosed Flos Chrysanthemi Indici extract has good blood sugar decreasing effect,
There is good development prospect in terms of diabetes are treated.
Brief description of the drawings
Fig. 1 suppresses total fat for the compound 1,2,4 from Flos Chrysanthemi Indici extract separate in embodiment 1
The activity data of accumulation；
Fig. 2 suppresses triglycerides product for the compound 1-4 from Flos Chrysanthemi Indici extract separate in embodiment 1
Tired activity data；
Fig. 3 be embodiment 1 in from Flos Chrysanthemi Indici extract separate compound 4 activation LXR α and
The activity data of LXR β；
Fig. 4 raises courage for the compound 3 and compound 4 that from Flos Chrysanthemi Indici extract separate in embodiment 1
The activity data (PPAR γ) of the mRNA expression of key transcription factor during sterol regulation；
Fig. 5 raises courage for the compound 3 and compound 4 that from Flos Chrysanthemi Indici extract separate in embodiment 1
The activity data (SREBP-1c) of the mRNA expression of key transcription factor during sterol regulation；
Fig. 6 raises courage for the compound 3 and compound 4 that from Flos Chrysanthemi Indici extract separate in embodiment 1
The activity data (CEBP δ) of the mRNA expression of key transcription factor during sterol regulation.
Specific embodiment
In order to more fully understand technology contents of the invention, with reference to specific embodiment to skill of the invention
Art scheme is described further and illustrates.
Embodiment 1
The present embodiment provides a kind of antidiabetic medicine containing Flos Chrysanthemi Indici extract, by Flos Chrysanthemi Indici extract and
/ or pharmaceutically acceptable carrier composition.Sesquiterpenoids chemical combination containing following structure in Flos Chrysanthemi Indici extract
Thing：
Above-described Flos Chrysanthemi Indici extract is prepared by following methods.The present embodiment is also carried to chrysanthemum indicum
The sesquiterpenoids taken in thing has carried out physiologically active analysis.
The preparation method of Flos Chrysanthemi Indici extract is comprised the following steps：
(1) extract
The dry chrysanthemum indicums of 7.0Kg are weighed, dry mother chrysanthemum is soaked with the ethanol (95%) of 10L at normal temperatures and pressures
Flower 24h, then filters, and collects filtrate.Repeat to extract chrysanthemum indicum three times, merge three filtrates of extraction,
Then filtrate is placed in normal pressure concentration in water-bath, obtains 2.0Kg extract medicinal extract.
(2) extract
Extract medicinal extract is put into 5L water, stirring makes extract medicinal extract become cloudy suspension, then
It is extracted with ethyl acetate, obtains extract, concentrated extract obtains concentrate, and concentrate obtains 425g after drying
Extract.
(3) column chromatography
It is adsorbed in etc. in the silica gel of the 200-300 mesh of weight after the dissolving of extract methyl alcohol, silica gel is in room temperature
Chromatography samples are obtained after lower drying, post then is carried out to chromatography samples with the 200-300 mesh silica gel of 2.0Kg
Chromatography, carries out gradient elution with petroleum ether, chloroform successively, collects chloroform efflux；Concentration chloroform outflow
Liquid, thing is chromatographed at the beginning of obtaining 120g.
(4) secondary chromatography
Column chromatography is carried out to just chromatography thing with 200-300 mesh silica gel, gradient elution is carried out with compound eluent,
Compound eluent is made up of petroleum ether and ethyl acetate, and the compound eluent of set of polar used is as follows：
90:10、80:20、70:30、60:40、50:50、40:60、20:80 (the bodies of petroleum ether and ethyl acetate
Product ratio).Efflux is tracked by thin-layer chromatography (TLC), according to the chromatography situation that TLC reflects, is closed
And the efflux of the material of identical polar (under the TLC chromatography conditions) is dissolved with, so as to receive successively
Collect the ascending stream part A of polarity, stream part B, stream part C and stream part D.Each stream part is concentrated respectively,
Correspondence obtains 18g extract As, 29g extract Bs, 19g extract Cs and 15g extracts D.Carry
Take thing A, extract B, extract C and extract D and constitute institute in the antidiabetic medicine of the present embodiment
The Flos Chrysanthemi Indici extract stated.
Purification is further separated to extract A, extract B, extract C, extract D respectively.
By further separating-purifying, the sesquiterpenoids of following general structure is can obtain：
Specific following (5)-(8) of separating-purifying step are described.
(5) separating-purifying compound 1
Extract A carries out column chromatography by using MCI posts, and gradient elution is carried out with methanol/water, used
Set of polar methanol/water eluent it is as follows：20:80、30:70、40:60、50:50、60:40、70:30、
80:20、90:10 (volume ratios of methyl alcohol and water), collect efflux, and extract is obtained after concentration efflux
A1。
Extract A 1 carries out column chromatography by using MCI posts again, successively with petrol ether/ethyl acetate,
Chloroform/methanol carries out gradient elution, and the petrol ether/ethyl acetate eluent of set of polar used is as follows：
90:10、80:20、70:30 volume ratios of ethyl acetate (petroleum ethers with), the chlorine of set of polar used
Imitative/meoh eluate is as follows：95:5、80:20 (volume ratios of chloroform and methyl alcohol), collect efflux, dense
Extract A 2 is obtained after contracting efflux.
Extract A 2 carries out column chromatography by using reverse phase silica gel post again, and gradient elution is carried out with methanol/water,
The volume ratio of methanol/water eluent used is 70:30 and 60:40, efflux is collected, concentrate efflux
After can obtain compound 1, compound 1 is white powder.
Mass spectrum, proton nmr spectra, the carbon-13 nmr spectra of compound 1 are surveyed respectively, by testing institute's total
According to understanding that the molecular weight of compound 1 is 592.3, molecular formula is C35H44O8.The mass spectrum of compound 1,
Proton nmr spectra, carbon-13 nmr spectra are consistent to the corresponding collection of illustrative plates of the compound 2 in document 1, therefore
The structural formula of compound 1 is：(document 1：Chrysanolide A,an unprecedented
sesquiterpenoid trimer from the flowers of Chrysanthemum indicum L.RSC
Advances,2013,3(26):10168.)
Specific test data is 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) separating-purifying compound 2
Extract B is first passed through carries out column chromatography using the silica gel of 300g, and ladder is carried out with chloroform/ethyl acetate
Degree wash-out, the chloroform/ethyl acetate eluent of set of polar used is as follows：100:0、90:10、80:20、
70:30 (volume ratios of chloroform and ethyl acetate), collect efflux, and extract is obtained after concentration efflux
B1.Extract B 1 passes sequentially through use Sephadex LH-20 again, and (methyl alcohol/chloroform, volume ratio is 50:50)、
(gradient elution, the volume ratio of the methanol/water eluent of set of polar used is Rp-18：70:30、80:20、
90:10、100:0), (methanol/water, volume ratio is 60 to preparation HPLC:40) column chromatography is carried out, can be divided
From compound 2 is obtained, compound 2 is white solid.
Mass spectrum, proton nmr spectra, the carbon-13 nmr spectra of compound 2 are surveyed respectively, by testing institute's total
According to understanding that the molecular weight of compound 2 is 306, molecular formula is C17H22O5.The mass spectrum of compound 2,
Proton nmr spectra, carbon-13 nmr spectra are consistent to the corresponding collection of illustrative plates of the compound 12 in document 2, because
The structural formula of this compound 2 is：(document 2：Cumambrin A in Chrysanthemum boreale
Makino Preparation,X-ray Crystal Structure and 13C-and 1H-NMR Study of
Cumambrin A[J]Kor.J.Pharmacogn,1996,27:207-211.)
Specific test data is 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) separating-purifying compound 3
Extract C first passes through MCI posts and carries out column chromatography, and gradient elution, system used are carried out with methanol/water
The methanol/water eluent of row polarity is as follows：20:80、30:70、40:60、50:50、60:40、70:30、
80:20、90:10 (volume ratios of methyl alcohol and water), collect efflux, and extract is obtained after concentration efflux
C1.Extract C 1 pass sequentially through again using silicagel column (gradient elution, the chloroform of set of polar used/
The volume ratio of meoh eluate is：95:5、85:15、70:30), Sephadex LH-20 (methyl alcohol) enter
Row column chromatography can obtain compound 3, and compound 3 is white solid.
Mass spectrum, proton nmr spectra, the carbon-13 nmr spectra of compound 3 are surveyed respectively, by testing institute's total
According to understanding that the molecular weight of compound 3 is 246.1, molecular formula is C15H18O3.The mass spectrum of compound 3,
Proton nmr spectra, carbon-13 nmr spectra are consistent to the corresponding collection of illustrative plates of the compound 1 in document 3, therefore
The structural formula of compound 3 is：(document 3：Conformational analysis of achillinand leukodin
[J]J Nat Prod,1988,51(2):22-228.)
Specific test data is 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) separating-purifying compound 4
Extract D is first passed through carries out column chromatography (methyl alcohol) using Sephadex LH-20 posts, collects outflow
Liquid, extract D1 is obtained after concentration efflux.
Extract D1 carries out column chromatography by using MCI posts, carries out gradient elution to remove with methanol/water
Depigmentaton, the methanol/water eluent of set of polar used is as follows：20:80、30:70、40:60、50:50、
60:40、70:30、80:20、90:10、100:0 (volume ratio of methyl alcohol and water), collects efflux, dense
Extract D2 is obtained after contracting efflux.
Extract D2 is passed sequentially through using silica gel (gradient elution, the chloroform/acetic acid of set of polar used again
The volume ratio of ethyl ester eluent is：90:10 to 30:70), Rp-18 (gradient elution, set of polar used
Methanol/water eluent volume ratio be 60:40 to 90:10), Toyopearl HW-40 (methyl alcohol), silicon
(gradient elution, the volume ratio of the chloroform/methanol eluent of set of polar used is 100 to glue:0、90:10、
80:20), (methyl alcohol/chloroform, volume ratio is 50 to Sephadex LH-20:50), Rp-8 (gradient elution,
The volume ratio of the methanol/water eluent of set of polar used is 60:40、70:30、80:20、90:10), make
(methanol/water, volume ratio is 65 to standby HPLC:35) column chromatography is carried out, compound 4, compound 4 is obtained
It is white solid.
Mass spectrum, proton nmr spectra, the carbon-13 nmr spectra of compound 4 are surveyed respectively, by testing institute's total
According to understanding that the molecular weight of compound 4 is 798.4, molecular formula is C47H58O11.The mass spectrum of compound 4,
Proton nmr spectra, carbon-13 nmr spectra are consistent to the corresponding collection of illustrative plates of the compound 3 in document 1, therefore
The structural formula of compound 4 is：(document 1：Chrysanolide A,an unprecedented
sesquiterpenoid trimer from the flowers of Chrysanthemum indicum L.RSC
Advances,2013,3(26):10168.)
Specific test data is 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`).
By more than to extract A, extract B, extract C, extract D further separating-purifying
Result it can be confirmed that in the Flos Chrysanthemi Indici extract of the present embodiment containing above-mentioned compound 1, compound 2,
Compound 3 and compound 4.
The drug effect related experiment of compound 1-4 is as follows.
Experiment 1：Compound 1-4 suppresses triglycerides accumulation, the determination of activity of total Fat Accumulation.
(1) cell culture
3T3-L1 (MEC, PECTORAL LIMB SKELETON) is using complete medium (DMEM
10% hyclone, 100U/mL penicillin, 100 μ g/mL streptomysins are added in culture medium) 37 DEG C,
Volume fraction 5%CO2Incubator in cultivate.
(2) pharmaceutical intervention
Testing compound is made into 10mM mother liquors with DMSO, and various concentrations working solution is diluted to during test.
It is the DMSO influences of correction parent solution solvents, blank addition 0.1%DMSO.
3T3-L1 is inoculated in 48 orifice plates culture to completely attaching to (the 0th day), and culture medium is changed into point
Change culture medium I or add the differential medium I of prescribed concentration testing compound to cultivate 3 days (the 3rd day).
Differential medium I is formulated：2 μ g/mL insulin, 100ng/mL ground plug rice are added in complete medium
Pine, 0.5mM 3-isobutyl-1-methylxanthines and 10ng/mL biotins.
Differential medium and testing compound are acted on the 3rd day, and culture medium is replaced with into differential medium II
Or add the differential medium II of prescribed concentration testing compound to continue to cultivate 3 days (the 6th day).Differentiation
Medium ii is formulated：2 μ g/mL insulin are added in complete medium.
6th day, harvesting carried out triglycerides or oil red O stain analysis.
(3) triglycerides test
3T3-L1 cells after results treatment, ice PBS (0.2M NaCl, 10mM Na2HPO4,
3mM KCl, 2mM KH2PO4, pH 7.4) wash twice, sonicated cells use glycerine
Absorbance at three ester kits and multi-function microplate reader detection 546nm, so that sweet in calculating clasmatosis liquid
Oily three ester contents.Result is represented in the form of blank cell content of triglyceride percentage.
(4) oil red O stain
3T3-L1 cells fix 1 hour with 10% (V/V) formalin solution room temperature, with freshly prepd
60 DEG C of oil red O working solutions are dyeed 30 minutes, and twice, then total fat is contaminated by oil red O for distillation washing
Into red, observe and take pictures with the inverted microscope equipped with CCD digital cameras.Amplify in shooting process
Multiple, luminous intensity are constant.
(5) result treatment
According to absorbance at triglyceride reagent box and multi-function microplate reader detection 546nm, blank pair is subtracted
It is as a result thin with negative control group than upper negative control group cell absorbance according to the background absorbance value of group
The form of born of the same parents' content of triglyceride percentage is represented.All data are represented as average value ± standard variance.
Test result is as depicted in figs. 1 and 2.As shown in Figure 2, compound 1-4 can drop in various degree
Low 3T3-L1 intracellular triglycerides accumulation, the 3T3-L1 after wherein CI-15 is processed 6 days is intracellular several
Without oil droplet accumulation, content of triglyceride is zero.As shown in Figure 1, triglyceride reducing effect is best
Compound 1, compound 2 and compound 4 substantially reduce the intracellular total fat drop contents of 3T3-L1.
Experiment 2：The determination of activity of the excitement of compound 4 LXR.
(1) cell culture
HEK293T (people source embryonic kidney cell) (is added using complete medium in DMEM culture mediums
10% hyclone, 100U/mL penicillin, 100 μ g/mL streptomysins) in 37 DEG C, volume fraction
5%CO2Incubator in cultivate.
(2) pharmaceutical intervention
HEK293T cells are laid in 96 orifice plates with the density of 2 × 104 cells in every hole, and culture is extremely
Cell growth to about 90% coverage rate, according to liposome Lipofectamine TM2000 reagent specifications,
By 6.5 μ g pGL3/ (DR-4)-c-fos-FF-luc (firefly luciferase reporter gene) plasmids, 1.3 μ g
PSG5/hLXR α (or pSG5/hLXR β, Liver X receptor alpha or β) plasmid, 1.3 μ g
(sea pansy is glimmering for pSG5/hRXR α (retinoic acid X acceptors) plasmids and 0.13 μ gpCMV/Renilla-luc
Light element enzyme reporter gene) plasmid and 10 μ LLipofectamineTM2000 liposome reagents are fully mixed,
It is stored at room temperature 30 minutes, liposome-DNA solution is added in 96 orifice plates, per the μ L of hole 5.
Culture changes complete medium into after 5 hours, continue to cultivate 5 hours.
Adding testing compound mother liquor made working concentration for 10 μM, with co-culture of cells 20 hours.Make
Firefly luciferase activity and sea pansy fluorescence are detected with multi-function microplate reader work station Flex Station 3
Plain enzymatic activity.
(3) result treatment
Interpretation of result renilla luciferase activity correction firefly luciferase activity, therefore correction enzymatic activity
=firefly luciferase activity value/renilla luciferase activity value.Relative luciferase activity value=experiment
Group correction enzymatic activity value/negative control group correction enzymatic activity value.
Test result is as shown in figure 3, the activity of the excitement LXR of compound 4 α is better than existing LXR α
The active and existing LXR β of activator tool molecule GW3965, the excitement LXR of compound 4 β are exciting
GW3965 is suitable for agent tool molecule.
Experiment 3：Compound 3 and compound 4 raise the mRNA of key transcription factor on LXR paths
The determination of activity of expression quantity.
(1) cell culture
3T3-L1 (MEC, PECTORAL LIMB SKELETON) is using complete medium (DMEM
10% hyclone, 100U/mL penicillin, 100 μ g/mL streptomysins are added in culture medium) 37 DEG C,
Volume fraction 5%CO2Incubator in cultivate.
(2) pharmaceutical intervention
Testing compound is made into 10mM mother liquors with DMSO, and various concentrations working solution is diluted to during test.
It is the DMSO influences of correction parent solution solvents, blank addition 0.1%DMSO.
3T3-L1 is inoculated in 48 orifice plates culture to completely attaching to (the 0th day), and culture medium is changed into point
Change culture medium I or add the differential medium I of prescribed concentration testing compound to cultivate 24 hours the (the 1st
My god).Harvesting carries out fluorescent quantitative PCR.
(3) quantitative fluorescent PCR
The total serum IgE of 3T3-L1 cells is extracted using RNAiso Plus, by Oligo dT18 by 1 μ g
Total serum IgE reverse transcription is cDNA, by quantitative PCR technique, uses Toyobo company's Ts hunderBird
SYBR qPCR Mix reagents enter performing PCR amplification to primer to specifications.3 multiple holes of every group of sample,
To ensure the validity of experimental data.The use of β-actin is internal reference, testing goal gene is relative to internal reference
The expression of gene changes to quantify.Using 2-ΔΔCt(i.e. experimental group genes of interest is relative for method analyze data
In the change multiple of the expression quantity of control group).
The primer is：
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 treatment
Testing goal gene is quantified relative to the expression change of reference gene.Using 2-ΔΔCtMethod is analyzed
Data (i.e. change multiple of the experimental group genes of interest relative to the expression quantity of control group).
As Figure 4-Figure 6, the compound 4 under 10 μM of concentration can dramatically increase 3T3-L1 to test result
The mrna expression amount of intracellular SREBP-1c be (the treated cell SREBP-1c's of compound 4
Mrna expression amount is 8 times of blank control group), while increasing the mRNA of PPAR γ and CEBP δ
Expression quantity (is 3 times).Compound 3 can dramatically increase the mRNA tables of PPAR γ and CEBP δ
Up to amount.
Embodiment 2
The present embodiment provides a kind of antidiabetic medicine containing Flos Chrysanthemi Indici extract, contains institute in embodiment 1
The compound 1 and/or pharmaceutically acceptable carrier stated, active ingredient are compounds 1.
From the test result in embodiment 1, the anti-diabetic containing Flos Chrysanthemi Indici extract of the present embodiment
Medicine has the effect for reducing oil droplet accumulation and triglycerides accumulation in 3T3-L1 PECTORAL LIMB SKELETON atomizations
Really, therefore with good blood sugar decreasing effect, there is good development prospect in terms of diabetes are treated.
Embodiment 3
The present embodiment provides a kind of antidiabetic medicine containing Flos Chrysanthemi Indici extract, contains institute in embodiment 1
The compound 2 and/or pharmaceutically acceptable carrier stated, active ingredient are compounds 2.
From the test result in embodiment 1, the anti-diabetic containing Flos Chrysanthemi Indici extract of the present embodiment
Medicine has the effect for reducing oil droplet accumulation and triglycerides accumulation in 3T3-L1 PECTORAL LIMB SKELETON atomizations
Really, therefore with good blood sugar decreasing effect, there is good development prospect in terms of diabetes are treated.
Embodiment 4
The present embodiment provides a kind of antidiabetic medicine containing Flos Chrysanthemi Indici extract, contains institute in embodiment 1
The compound 3 and/or pharmaceutically acceptable carrier stated, active ingredient are compounds 3.
From the test result in embodiment 1, the anti-diabetic containing Flos Chrysanthemi Indici extract of the present embodiment
Medicine has the effect for reducing oil droplet accumulation and triglycerides accumulation in 3T3-L1 PECTORAL LIMB SKELETON atomizations
Really, and improve PPAR γ, SREBP-1c, CEBP δ mrna expression amount effect, therefore tool
There is good blood sugar decreasing effect, have good development prospect in terms of diabetes are treated.
Embodiment 5
The present embodiment provides a kind of antidiabetic medicine containing Flos Chrysanthemi Indici extract, contains institute in embodiment 1
The compound 4 and/or pharmaceutically acceptable carrier stated, active ingredient are compounds 4.
From the test result in embodiment 1, the anti-diabetic containing Flos Chrysanthemi Indici extract of the present embodiment
Medicine has the effect for reducing oil droplet accumulation and triglycerides accumulation in 3T3-L1 PECTORAL LIMB SKELETON atomizations
Really, and improve PPAR γ, SREBP-1c, CEBP δ mrna expression amount effect, therefore tool
There is good blood sugar decreasing effect, have good development prospect in terms of diabetes are treated.
The above only further illustrates technology contents of the invention with embodiment, more holds in order to reader
It is readily understood, but do not represent embodiments of the present invention and be only limitted to this, any technology done according to the present invention is prolonged
Stretch or recreate, by protection of the invention.
Claims (5)
1. a kind of antidiabetic medicine containing Flos Chrysanthemi Indici extract, including Flos Chrysanthemi Indici extract and/or pharmacy
Upper acceptable carrier, it is characterised in that the sequiterpene containing following structure in the Flos Chrysanthemi Indici extract
Class compound：
2. a kind of antidiabetic medicine containing Flos Chrysanthemi Indici extract, its feature according to claim 1
It is that the structural formula of the sesquiterpenoids is：
3. a kind of antidiabetic medicine containing Flos Chrysanthemi Indici extract, its feature according to claim 1
It is that the structural formula of the sesquiterpenoids is：
4. a kind of antidiabetic medicine containing Flos Chrysanthemi Indici extract, its feature according to claim 1
It is that the structural formula of the sesquiterpenoids is：
5. a kind of antidiabetic medicine containing Flos Chrysanthemi Indici extract, its feature according to claim 1
It is that the structural formula of the sesquiterpenoids is：
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110343116A (en) * | 2018-12-21 | 2019-10-18 | 中山大学 | A kind of Flos Chrysanthemi Indici extract and preparation method thereof and the application in preparation treatment medicine for nasopharyngeal |
| CN111603463A (en) * | 2020-06-28 | 2020-09-01 | 河北中医学院 | Application of dehydrodeacetyloxymatrine or deacetylmatricin in preparing blood sugar and blood lipid lowering medicine |
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| KR20030068608A (en) * | 2002-02-15 | 2003-08-25 | 경상남도 | Pharmaceutical Composition Comprising Cumambrin A for Lowering Level of Blood Glucose |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110343116A (en) * | 2018-12-21 | 2019-10-18 | 中山大学 | A kind of Flos Chrysanthemi Indici extract and preparation method thereof and the application in preparation treatment medicine for nasopharyngeal |
| CN110343116B (en) * | 2018-12-21 | 2022-02-22 | 中山大学 | Wild chrysanthemum extract, preparation method thereof and application thereof in preparation of medicine for treating nasopharyngeal carcinoma |
| CN111603463A (en) * | 2020-06-28 | 2020-09-01 | 河北中医学院 | Application of dehydrodeacetyloxymatrine or deacetylmatricin in preparing blood sugar and blood lipid lowering medicine |
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