CN108586563B - Ursolic triterpenoid saponin compounds with E-ring cleavage and preparation method thereof - Google Patents
Ursolic triterpenoid saponin compounds with E-ring cleavage and preparation method thereof Download PDFInfo
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- CN108586563B CN108586563B CN201810660038.XA CN201810660038A CN108586563B CN 108586563 B CN108586563 B CN 108586563B CN 201810660038 A CN201810660038 A CN 201810660038A CN 108586563 B CN108586563 B CN 108586563B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms
- C07J63/008—Expansion of ring D by one atom, e.g. D homo steroids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/20—Carbocyclic rings
- C07H15/24—Condensed ring systems having three or more rings
- C07H15/256—Polyterpene radicals
Abstract
The invention provides E-ring cracked ursane-type triterpenoid saponin compounds, the structure of which is shown in formula (I). The compounds provided by the invention have the effect of protecting myocardial cells, can be used for preparing medicaments for preventing and treating acute myocardial infarction, and provide new medicament selection for preventing and treating myocardial infarction.
Description
Technical Field
The invention relates to steroids, in particular to E-ring cracked ursane type triterpenoid saponin compounds and a preparation method thereof.
Background
Ursane-type Triterpenoid saponins are more distributed in plant world, and more than 250 ursane-type Triterpenoid saponins are found in nature during 1996 to 2012 reported by literature, (Dinda B, Debnath S, Mohanta BC, Haragaya Y. Naturally Occurring Triterpenoid saponins, chemistry Biodiversity.2010,2327-2580.Hill RA, Connolly JD. Triterpenoid ids. Nat. Prod. Rep.2015,32: 273-327).
Disclosure of Invention
The invention aims to provide E-ring cracked ursane-type triterpenoid saponin compounds and a preparation method thereof.
In order to achieve the purpose, the technical scheme adopted by the invention is that E-ring cracked ursane-type triterpenoid saponin compounds have the molecular structures shown as the following formula (I):
the molecular formula C of the E-ring cracked ursane-type triterpenoid saponin compound47H74O17，HR-ESI-MS m/z[M+COOH]955.4910 with the chemical name of 3 β - [ α -L-rhodopyranosyl- (1 → 2) - β -D-glucopyranosyl- (1 → 2) - β -D-glucopyranosyl]-19-oxo-18,19-secoursa-11,13(18)-dien-28-oic acid。
The ursolic acid type triterpenoid saponin compound with cracked E ring can be prepared by a chemical synthesis method. The synthetic steps and process conditions can be determined by one skilled in the art by knowledge in the art.
The invention also provides a preparation method of the E-ring dehisced ursane-type triterpenoid saponin compound, and a method for separating the E-ring dehisced ursane-type triterpenoid saponin compound from pubescent holly root (Ilex pubescens Hook et Arn.).
The preparation method comprises the following steps:
(1) reflux-extracting radix Ilicis Pubescentis with water solution of polar solvent, and concentrating the extractive solution to obtain extract;
(2) dissolving the extract obtained in the step (1) in water, passing through a macroporous resin column, performing gradient elution by using an alcoholic solution of C1-C4, collecting a characteristic fraction , and concentrating to obtain a thick extract;
(3) passing the thick extract obtained in the step (2) through a silica gel column, carrying out gradient elution by using an organic solvent, and collecting a characteristic fraction II;
(4) concentrating the eluent obtained in the step (3), and then passing the concentrated eluent through an ODS column, wherein the volume ratio of a polar solvent: eluting with water as eluent, collecting eluate, concentrating, separating with Sephadex LH-20 column, eluting with pure methanol, and collecting eluate;
(5) and (4) concentrating the eluent obtained in the step (4), separating by using a preparation liquid phase, and removing the solvent to obtain white powdery crystals.
Preferably, the gradient elution method in step (2) is to sequentially elute with 30% ethanol, 60% ethanol and 95% ethanol.
Preferably, the characteristic fraction is a 60% ethanol eluate.
Preferably, the organic solvent eluted in gradient in step (3) is selected from chloroform-methanol, dichloromethane-methanol.
Preferably, the gradient elution method in the step (3) is to perform elution according to a gradient of chloroform-methanol of 100: 1-0: 1.
Preferably, the filler particle size of the silica gel column is 200-300 meshes.
Preferably, the characteristic fraction II is an eluent of 75: 25 chloroform-methanol.
Preferably, the eluent in the step (4) is 60: 40 parts of methanol: and (3) water.
Preferably, the aqueous solution of the polar solvent in the step (1) is selected from the group consisting of an aqueous ethanol solution, an aqueous methanol solution, and an aqueous acetonitrile solution.
Preferably, the aqueous solution of the polar solvent in the step (1) is 70% ethanol.
Preferably, the reflux extraction method in step (1) is reflux extraction with 12, 10 and 8 times of 70% ethanol for 2 h.
Preferably, the mobile phase for the preparation of the liquid phase separation is methanol: 35 parts of water, and collecting fractions with the retention time of 89.3 min.
Preferably, the macroporous resin column is a D-101 type macroporous resin column.
The invention has the beneficial effects that E-ring cracked ursane-type triterpenoid saponin compounds and the preparation method thereof are provided, the method is simple in process, E-ring cracked ursane-type triterpenoid saponin compounds extracted by the preparation method have the effect of protecting myocardial cells, can be used for preparing medicines for preventing and treating acute myocardial infarction, and provides a new medicine selection for preventing and treating myocardial infarction.
Drawings
FIG. 1 is a structural formula of an E-ring cracked ursane-type triterpenoid saponin compound.
FIG. 2 is a 1H-NMR chart (400MHz) of example 2.
FIG. 3 is a 13C-NMR chart (100MHz) of example 2.
FIG. 4 is a DEPT diagram of example 2.
FIG. 5 is a COSY NMR chart of example 2.
FIG. 6 is an HSQC NMR chart of example 2.
FIG. 7 is an HMBC NMR chart of example 2.
FIG. 8 is a ROESY NMR chart of example 2.
Detailed Description
Example 1
A preparation method of ursane-type triterpenoid saponin compounds with E-ring cleavage.
(1) Sequentially extracting radix Ilicis Pubescentis with 12, 10 and 8 times of 70% ethanol under reflux for 2 hr, mixing the ethanol extractive solutions, filtering, recovering ethanol, and concentrating under reduced pressure until no ethanol smell exists to obtain total extract 2.2 kg;
(2) dissolving the total extract in water, passing through D-101 type macroporous resin column, and sequentially eluting with 30% ethanol, 60% ethanol and 95% ethanol; collecting 60% ethanol eluate, recovering solvent under reduced pressure to obtain 566g of thick extract, passing through 200-300 mesh silica gel column, and eluting with chloroform-methanol as solvent at gradient ratio of chloroform-methanol of 100: 1-0: 1; collecting chloroform-methanol 75: 25 eluate, concentrating, passing through ODS column, eluting with methanol: water 60: and 40, eluting with an eluent, collecting the eluent, concentrating the eluent, passing the concentrated eluent through a Sephadex LH-20 column, collecting methanol eluent, concentrating the methanol eluent, separating by using a prepared liquid phase, wherein a mobile phase is methanol: 35 parts of water, collecting chromatographic peaks with the retention time of 89.3min, and concentrating to obtain 22.4mg of white powdery crystals.
Example 2
Identification of E-ring cleaved triterpene saponin compounds prepared by the preparation method of example 1.
The obtained white powdery crystal Liebermann-Burchard reaction is positive, and finally shows purple red, which indicates that the crystal is a triterpenoid.
Referring to FIGS. 2 to 6, IR shows hydroxyl absorption (3426 cm)-1) Carbonyl absorption (1703 cm)-1) Double bond absorption (1644 cm)-1). High resolution mass spectrometry gives the formula C47H74O17，[M+COOH]-955.4910, the unsaturation degree of the molecule is calculated to be 11 according to the molecular formula. The compound was analyzed by TLC and HPLC after acid hydrolysis and derivatization and compared with derivatives of sugar standards, demonstrating that the sugar units were D-xylose and D-glucose. With reference to table 1, see the drawings,13a total of 47 carbon signals were shown in the C-NMR spectrum, 17 of which were assigned to sugars, leaving 30 carbon signals suggesting possible triterpene aglycones. With reference to table 1, see the drawings,1the H-NMR spectrum showed 6 angular methyl signals [ delta ]H0.82,0.85,1.06,1.06,1.34,2.11]And methyl signals [ delta ] in two peaksH1.08] group of disubstituted double bonds (delta)H5.62,δC127.7,C-11；δH6.18,δC130.8, C-12) and groups of trisubstituted double bonds (. delta.)C142.5,C-13；δH5.88,δC129.6, C-18), ketocarbonyl groups (. delta.)C212.0, C-19), carboxyl groups (. delta.)C178.9, COOR-28), terminal proton signals (. delta.) of xyloseH4.91,δC106.2, CH-Xyl-1), terminal proton signal (. delta.) for glucoseH5.79,δC102.6, CH-Glc-1), and terminal proton signals (δ) of rhamnosesH6.39,δC102.3, CH-Rha-1) with coupling constants of 5.2 and 5.8, suggesting that D-xylose and D-glucose are both in β -configuration, the above information suggests that the compound may be an ursane-type triterpenoid saponin having carboxyl, ketocarbonyl and hydroxyl groups simultaneously, compared with the known compound 3 β -hydroxy-19-oxo-18,19-seco-11,13(18) -ursa-diene-28-oic acid, the chemical shifts of the two are very similar, and the difference is that the sugar unit at position 3 is different。
Referring to FIG. 7, HMBC spectra show alkene hydrogen deltaH6.18(1H, d, H-12) and δC54.8(C-9)、δC127.7(C-11)、δC142.5(C-13) and δC42.8(C-14) are all distantly related, indicating that the double bonds are between C-11, C-12 and C-13, C-18, respectively. HMBC spectra show methyl deltaH1.08(1H, d, H-30) and δC212.0(C-19)、δC47.8(C-20) and δC28.5(C-21) are all distantly related, suggesting that the ketone carbonyl is at the C-19 position. HMBC spectra show deltaH4.91(d, J ═ 5.2Hz, CH-xyl-1) and δC89.9(C-3) there was a remote correlation to determine that xylose was attached at the C-3 position.
Referring to FIG. 8, the ROESY spectrum shows that the related peaks are H-C (3)/H-C (5), H-C (5)/H-C (9), H-C (9)/H-C (27), indicating that 24-Me and 27-Me are at position α, and the related peaks are H-C (23)/H-C (25), H-C (25)/H-C (26) indicating that 3-position oxygen substitution, 23-Me, 25-Me and 26-Me are at position β.
TABLE 1 preparation of compound I1HNMR and13c NMR data (Pyridine-d)5,J＝Hz)
Example 3
The myocardial cell protection effect of the compound is verified by the influence of the compound on the survival rate of the myocardial cells by adopting H9c2 myocardial cell injury caused by hypoxia reoxygenation as a model.
Materials and methods
1. Sample 1 was derived from the preparation method shown in specific example 1. For the positive control, DZ was diazoxide.
2. Rat ventricular myocyte line H9c2 was purchased from American Standard cell Bank and stored in 100units/ml plates containing 10% FBSPenicillin G, 100 ug/ml streptomycin culture solution, at saturated humidity, 37 deg.C and 5% CO2Culturing under the condition.
3. The compound was dissolved in 5% DMSO, the stock solution was made 30mM, and the working concentration was 1. mu.M.
4. H9C2 cardiomyocytes were cultured in DMEM complete medium for 2 days, washed 2 times with KPB wash buffer and 95% N2-5％CO2KRB culture buffer solution pre-saturated for 30min was substituted for normal culture medium, and the plates were then transferred to an adjustable anoxic cassette, connected to a gas flow meter (25L/min) to allow the oxygen partial pressure in the cassette to be from 20kpa to 0kpa within 1 min. And placing the hypoxia box into a conventional incubator to perform hypoxia culture for 3 hours at 37 ℃, changing the culture medium into a DMEM complete culture medium after hypoxia is finished, and placing the cells in an normoxic environment to continue to culture for 3 hours. The experiment is provided with a normal control group, an anoxic reoxygenation injury model group and different drug intervention groups, wherein each group has 3 multiple holes. The time of administration was 1 hour before the plates were transferred to the anoxic box.
5. The MVS method comprises the following steps: after the cell culture was completed, the medium containing H9c2 cells was covered with 2-fold amount of mitochondrial viability stain per 100. mu.L of the medium, and the cells were placed in a dark constant temperature environment (5% CO)2) After incubation for 4h, the fluorescence intensity was measured with an M200 microplate reader at an excitation wavelength of 550nm and an emission wavelength of 590 nm.
6. Statistical analysis: the above test results are expressed as X + -S, statistical data analysis is performed on the sample combined with blank control group data, and P <0.05 represents significant difference.
TABLE 2 protection Rate of Compound 1 in H9C2 cells
The experimental results show that the compound 1 can remarkably protect the H9c2 myocardial cell injury caused by hypoxia reoxygenation, and is more obvious than a positive control drug diazoxide at point .
Example 4
The preparation of the medicine injection.
Taking 1000mg of the compound obtained by the method in the example 1, adding 1000ml of water for injection, adjusting the pH value to 7-7.5 by using sodium carbonate, stirring to dissolve the compound, sterilizing, filtering, filling and sealing, and sterilizing by flowing steam at 100 ℃ for 15 minutes to prepare 2mg/2ml injection for injection.
Example 5
And (4) preparation of a medicinal capsule.
5000mg of the compound obtained by the method of the embodiment 1 is fully mixed with auxiliary materials such as 4000mg of microcrystalline cellulose, 500mg of sodium carboxymethyl starch, 400mg of sodium dodecyl sulfate and the like, dry granulation is carried out by a rolling method, and the mixture is uniformly mixed with a proper amount of magnesium stearate and filled into 3# hollow capsules to prepare capsules with the specification of 100 mg/capsule for oral administration.
Example 6
And (4) preparing a medicine tablet.
5000mg of the compound obtained by the method of the embodiment 1 is uniformly mixed with 4000mg of starch, 200mg of cross-linked PVP and 300mg of sodium carboxymethyl starch, a 75% ethanol solution of 5% PVP is used as a binding agent to prepare a soft material, the soft material is granulated by a 18-mesh sieve, dried at 60 ℃ for 1 hour, a proper amount of talcum powder is added after 20-mesh granulation, the mixture is uniformly mixed and tabletted, and the tablet with the specification of 100 mg/tablet is prepared to be orally taken.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (5)
1, E-ring cracked ursane type triterpenoid saponin compounds, the molecular structure of which is shown as formula (I):
a process for the preparation of ursolic triterpenoid saponins with E-ring cleavage as claimed in claim 1, which comprises the following steps:
(1) reflux-extracting radix Ilicis Pubescentis with water solution of polar solvent, and concentrating the extractive solution to obtain extract;
(2) dissolving the extract obtained in the step (1) in water, passing through a macroporous resin column, performing gradient elution by sequentially using 30% ethanol, 60% ethanol and 95% ethanol, collecting 60% ethanol eluate, and concentrating to obtain a thick extract;
(3) passing the thick extract obtained in the step (2) through a silica gel column, and eluting by taking chloroform-methanol as a solvent according to a gradient of chloroform-methanol of 100: 1-0: 1; collecting chloroform-methanol eluate at ratio of 75: 25;
(4) concentrating the eluent obtained in the step (3), and then passing the concentrated eluent through an ODS column, wherein the volume ratio of methanol: water 60: eluting with eluent 40, collecting eluate, concentrating, separating with Sephadex LH-20 column, eluting with pure methanol, and collecting eluate;
(5) and (4) concentrating the eluent obtained in the step (4), separating by using a preparation liquid phase, and removing the solvent to obtain white powdery crystals.
3. The method according to claim 2, wherein the aqueous solution of the polar solvent in the step (1) is 70% ethanol.
4. The method of claim 2, wherein the mobile phase for the preparative liquid phase separation is methanol: 35 parts of water, and collecting fractions with the retention time of 89.3 min.
5. The method according to claim 2, wherein the macroporous resin column is a D-101 type macroporous resin column.
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| CN106892956A (en) * | 2015-12-17 | 2017-06-27 | 广州中医药大学 | A kind of Ursane triterpene saponin componds and its preparation method and application |
| CN106892958A (en) * | 2015-12-17 | 2017-06-27 | 广州中医药大学 | A kind of exocyclic double bond Ursane triterpene saponin componds and its preparation method and application |
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| CN106892956A (en) * | 2015-12-17 | 2017-06-27 | 广州中医药大学 | A kind of Ursane triterpene saponin componds and its preparation method and application |
| CN106892958A (en) * | 2015-12-17 | 2017-06-27 | 广州中医药大学 | A kind of exocyclic double bond Ursane triterpene saponin componds and its preparation method and application |
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