CN110563794B - Myrtle triterpene lactone A as well as extraction method and application thereof - Google Patents
Myrtle triterpene lactone A as well as extraction method and application thereof Download PDFInfo
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- PZXJOHSZQAEJFE-UHFFFAOYSA-N dihydrobetulinic acid Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC5(C(O)=O)CCC(C(C)C)C5C4CCC3C21C PZXJOHSZQAEJFE-UHFFFAOYSA-N 0.000 description 1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
- C07J71/0005—Oxygen-containing hetero ring
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Diabetes (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Obesity (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Endocrinology (AREA)
- Hematology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Emergency Medicine (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Plant Substances (AREA)
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Abstract
The invention provides a myrtle triterpenoid lactone A and a preparation method thereofThe extraction method and the application are that a new compound myrtle triterpenoid lactone A is obtained by separating and extracting leaves of myrtle plants, and the structural formula is shown in the specification
(ii) a And found to exhibit a potential α -glucosidase inhibitory activity.
Description
Technical Field
The invention relates to the technical field of medicinal chemistry, in particular to myrtle triterpene lactone A and an extraction method and application thereof.
Background
Myrtle, belonging to the family myrtaceae, grows in the south and southeast asia, especially in the south china, such as the guangdong, the south of a lake and the south of the guangxi. The extract of the plant is reported to have various biological activities, some have antibacterial and anti-hepatitis effects, and the extract of the myrtle leaves also show anti-inflammatory effects. It has been reported previously that the chemical components of myrtle mainly contain triterpenes and phloroglucinol compounds, but the research on the active components and biological activity of myrtle is still relatively small, and intensive research on the active components of myrtle is very necessary to further search for the active components of the plant.
Disclosure of Invention
The invention provides myrtle triterpene lactone A as well as an extraction method and application thereof, wherein a new compound myrtle triterpene lactone A is separated and extracted from leaves of myrtle plants, and the new compound myrtle triterpene lactone A shows potential alpha-glucosidase inhibition activity.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the invention provides myrtle triterpene lactone A with a chemical formula I:
the invention also provides an extraction method of the myrtle triterpenoid lactone A with the chemical formula I, which comprises the following steps:
s1, leaching dried myrtle leaf powder with an ethanol solution with the volume concentration of 70-95% until an extracting solution is colorless, combining obtained leaching solutions, and concentrating under reduced pressure to obtain an extract;
s2, dispersing the extract with water, extracting with ethyl acetate, and recovering the solvent under reduced pressure to obtain an ethyl acetate extract;
s3, separating the ethyl acetate part by adopting silica gel column chromatography, and performing chloroform-methanol gradient elution, wherein the elution concentrations are as follows in sequence according to the volume ratio: 1:0, 9:1, 8:2, 2:1, 1:1, 0:1, and combining the same fractions by thin layer chromatography to obtain six components Fr.A-Fr.F;
s4, taking the component Fr.B for further MCI column chromatographic separation, wherein the eluent is 70-90% of methanol solution, and separating to obtain a Fr.B1 component;
s5, separating the component Fr.B1 by silica gel column chromatography, and performing chloroform-methanol gradient elution, wherein the elution concentrations are as follows in sequence by volume ratio: 50:1, 20:1 and 0:1, and detecting by thin layer chromatography to obtain 10 components Fr.B1-1-Fr.B1-10;
s6, preparing Fr.B1-2, performing preparative liquid chromatography, performing gradient elution with methanol-water, sequentially performing separation by volume ratio of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100%, and sequentially performing flow rate of 1-10.0mL/min to obtain 8 components Fr.B 1-2-1-Fr.B 1-2-8;
s7, subjecting Fr.B1-2-3 to high performance liquid chromatography, and eluting with acetonitrile-water at a volume ratio of 32:68 and a flow rate of 1.0mL/min to obtain the myrtle triterpene lactone A with the chemical formula I.
The invention provides application of myrtle triterpenoid lactone A in preparation of a medicament for treating diabetes mellitus, which is shown in a chemical formula I.
The invention further provides application of the myrtle triterpenoid lactone A with the chemical formula I in preparation of a glucosidase inhibitor. Further, the glucosidase is an alpha-glucosidase.
The invention separates a novel triterpene from the leaves of myrtle plants: the structure of the myrtle triterpene lactone (rhodomoside A) is identified as 2 alpha, 3 beta, 23 alpha, 29-tetrahydroxylearan-11, 13(18) -dien-28,19 beta-olide.
Experiments prove that the myrtle triterpenoid lactone A has stronger inhibition effect on alpha-glucosidase and IC50The value is 0.213 +/-0.016 mg/mL, and the alpha-glucosidase has better inhibitory activity, so that the alpha-glucosidase can be applied to preparation of a medicament for treating diabetes and preparation of a glucosidase inhibitor, and more choices are provided for preparation of medicaments for treating diabetes.
Drawings
FIG. 1a is an HMBC spectrum of myrtle triterpene lactone A; FIG. 1b is a COSY-ROESY co-decomposition spectrum of myrtle triterpene lactone A.
FIG. 2a is a mass concentration-enzyme activity curve for Compound 1 and acarbose; FIG. 2b is a graph of the reaction time-enzyme activity of Compound 1 and acarbose.
FIG. 3 shows the triterpene lactone A of Myrtus communis1H NMR(Recorded in pyridine-d5) Spectra.
FIG. 4 shows the triterpene lactone A of Myrtus communis13C NMR(Recorded in pyridine-d5) Spectra.
FIG. 5 shows HSQC (Recorded in pyridine-d) of myrobalan triterpene A5) Spectra.
FIG. 6 shows HMBC (Recorded in pyridine-d) of myrobalan A5) Spectra.
FIG. 7 shows the triterpene lactone A of Myrtus communis1H-1H COSY(Recorded in pyridine-d5) Spectra.
FIG. 8 shows the ROESY (Recorded in pyridine-d) of the triterpene lactone A of Myrtaceae5) Spectra.
FIG. 9 is an ESI spectrum of myrtle triterpene lactone A.
FIG. 10 is the HREIMS spectrum of myrtle triterpene lactone A.
Fig. 11 is an o.r. spectrum of myrtle triterpene lactone a.
FIG. 12 is a UV spectrum of myrtle triterpene lactone A.
FIG. 13 is an IR spectrum of myrtle triterpene lactone A.
Detailed Description
The present invention is further illustrated by the following specific examples, but the scope of the present invention is not limited to the following examples.
1. Apparatus and materials
Infrared spectrometer (Bruker TENSOR 27); ultraviolet spectrophotometer (shimadzu UV2401 PC); quadrupole liquid mass spectrometer (LC-MS8030, shimadzu, japan); liquid chromatography-mass spectrometry (Exavite, Thermo Fisher Scientific); nuclear magnetic resonance scanner (Bruker drx-500 mhz); nuclear magnetic resonance scanner (avance III 600 mhz); combiflash RF (RF200, Teledyne Isco, inc., USA); high performance liquid chromatography (Agilent LC1260 definition, Agilent); a rotary evaporator (RE-52A, Shanghai Yangrong Biochemical Instrument factory); electronic balance (BS400S, beijing sidoris ltd); silica gel for chromatography (Qingdao ocean factory, 100-; silica gel GF254 precoated glass is adopted for thin layer chromatography (Qingdao ocean chemical industry Co., Ltd.); chromatography columns (RP-18, Merk); the MCI filling material is MCI-gel CHP-20P (Mitsubishi Japan); RP column (Zorbax SB-C18,5 μm, 9.4X 250mm, Agilent, USA); chloroform, petroleum ether, ethyl acetate, methanol (AR, all available from seilon technologies ltd, china); alpha-glycosidases (G0660-750UN, SIGMA, Germany); glucosidase inhibitors (109A032, Solambio technologies, Inc. of Beijing); 4-PNPG (N0493, Tokyo chemical industries, Ltd.).
2. Plant material
The myrtle leaves are collected in Pinna county, Guangxi and identified as the myrtle leaves by the auxiliary professor Huangdeqing of the pharmaceutical institute of the Guilin medical institute. The sample voucher (serial number: 2016082501) was deposited at the institute of Chinese medicine and Natural products, academy of medicine, Guilin medical school.
Example 1
Extracting the myrtle triterpenoid lactone A:
s1, extracting 20kg of dried myrtle leaf powder by using an ethanol solution with the volume concentration of 95% until an extracting solution is colorless, combining obtained leaching solutions, and concentrating under reduced pressure to obtain an extract;
s2, dispersing the extract with water, extracting with ethyl acetate, and recovering the solvent under reduced pressure to obtain an ethyl acetate extract;
s3, separating the ethyl acetate part by adopting silica gel (100-200 meshes) column chromatography, and carrying out chloroform-methanol gradient elution, wherein the elution concentrations are as follows in sequence according to the volume ratio: 1:0, 9:1, 8:2, 2:1, 1:1, 0:1, and combining the same fractions by thin layer chromatography to obtain six components Fr.A-Fr.F;
s4, taking the component Fr.B (342g) and further carrying out MCI column chromatography, wherein the eluent is 90% methanol solution, and separating to obtain a Fr.B1 component (187 g);
s5, taking the component Fr.B1, separating by adopting silica gel (100-: 50:1, 20:1 and 0:1, and detecting by thin layer chromatography to obtain 10 components Fr.B1-1-Fr.B1-10;
s6, preparing Fr.B1-2, performing preparative liquid chromatography, performing gradient elution with methanol-water, sequentially separating by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% in volume ratio to obtain 8 components Fr.B 1-2-1-Fr.B 1-2-8;
s7, subjecting Fr.B1-2-3 to high performance liquid chromatography, eluting with acetonitrile-water at a volume ratio of 32:68 and a flow rate of 1.0mL/min to obtain myrtle triterpene lactone A (4.1mg) (compound 1) with a chemical formula I;
s8, subjecting Fr.B1-2-5(864.9mg) to semi-preparative high performance liquid chromatography, eluting with methanol-water at a volume ratio of 70:30 and a flow rate of 3.0mL/min to obtain compounds 2-4(37.8mg, 404.3mg and 4.1mg) in sequence;
s9, taking the component Fr.A (147g), separating by adopting silica gel (100-200 meshes) column chromatography, carrying out gradient elution by using petroleum ether-ethyl acetate at the volume ratio of 1:0, 20:1, 10:1, 5:1 and 0:1, and combining the same components by thin-layer chromatography detection to obtain 8 components (Fr.A1-Fr.A 8); subjecting component Fr.A5(31.0g) to preparative liquid chromatography, separating with pure methanol (flow rate of 10.0mL/min) and semi-preparative liquid chromatography, and separating with methanol-water (V/V74: 26, flow rate of 3.0mL/min) to obtain Fr.A5-1 and Fr.A5-2; subjecting component Fr.A5-1(67.4mg) to semi-preparative high performance liquid chromatography, and separating with methanol-water (V/V65: 35, flow rate of 3.0mL/min) to obtain compound 5(26.2mg) and compound 6(8.9 mg); component Fr.A5-2(17.1mg) was separated by HPLC using acetonitrile-water (V/V55: 45, flow rate 1.0mL/min) to give compound 7(15.5 mg).
Example 2
Identification of myrtle triterpene lactone A:
example 1A novel triterpene (Compound 1) and six known triterpenes (Compounds 2-7) were isolated. Compared with the literature, the known compounds 2-7 are identified as 2 alpha, 3 beta, 19 alpha, 23-tetrahydroxyurs-12-en-28-oic acid (2), asiatic acid (3), actinidic acid (4), 28-nortup-20 (29) -en-3 beta-hydroxy-17 beta-hydroperoxide de (5), 28-nortup-20 (29) -ene-3 beta, 17 beta-diol (6), betulinic acid (7).
By the reaction of compound 11H and13careful study of the C nmr spectroscopic data found it to be very similar to termichelolide, which was reported to be isolated from myrobalan. the main difference between termicebolide and Compound 1 is that one hydrogen of the methyl group at position 29 of termicebolide is replaced by a hydroxyl group, which becomes the Oxymethylene hydroxyl group, δ, of Compound 1HThis was confirmed by the correlation of 3.95(d, J ═ 10.3Hz,1H, H-29) and 3.58(d, J ═ 10.3Hz,1H, H-29) with HMBC at C-19, C-20, C-21, C-30 (fig. 1). The correlation of H-19 and H-29 in ROESY shows that H-29 is in the alpha configuration. The structure of compound 1 was therefore identified as 2 α,3 β,23 α, 29-tetrahydroxylearan-11, 13(18) -dien-28,19 β -olide and was named rhodotomeside a.
Rhodotomoside A(1)
A yellow powder, and a white pigment,
UV(MeOH),λmax(logε)253(4.05)nm；IR(KBr)νmax 3427,3031,2930,2857,1761,1711,1630,1570,1456,1384,1309,1260,1201,1167,1050,995,861cm-1；1H NMR(Pyridine-d5,600MHz)δH:6.25(1H,dd,J＝10.1,2.7Hz,H-12),5.87(1H,d,J＝10.1Hz,H-11),5.58(1H,s,H-19),4.38-4.32(1H,m,H-2),4.28(1H,d,J＝5.6Hz,H-3),4.26(1H,d,J＝10.5Hz,Ha-23),3.95(1H,d,J＝10.3Hz,Ha-29),3.74(1H,d,J＝10.5Hz,Hb-23),3.58(1H,d,J＝10.3Hz,Hb-29),2.59(1H,dd,J＝12.3,4.2Hz,Ha-1),2.45(overlapping,Ha-16),2.35(1H,s,H-9),1.89(1H,m,H-5),1.82(overlapping,Ha-6),1.80(overlapping,Ha-22),1.76(overlapping,Ha-15),1.63(overlapping,Hb-22),1.59(overlapping,Ha-21),1.55(overlapping,Hb-15),1.53(overlapping,Ha-7),1.48(overlapping,Hb-16),1.44(1H,dd,J＝12.3,11.5Hz,Hb-1),1.42(overlapping,Hb-6),1.35(overlapping,Hb-21),1.34(overlapping,Hb-7),1.17(3H,s,H-30),1.11(3H,s,H-25),1.07(3H,s,H-24),0.95(3H,s,H-27),0.76(3H,s,H-26).13C NMR data(Pyridine-d5,150MHz)δH:41.1(C-20),68.0(C-29),77.9(C-3),47.5(C-5),53.3(C-9),132.8(C-18),80.4(C-19),44.7(C-17),40.8(C-14),41.3(C-8),34.2(C-22),43.7(C-4),47.7(C-1),135.1(C-13),37.9(C-10),24.8(C-16),33.0(C-7),28.6(C-21),14.1(C-24),25.8(C-15),68.8(C-2),123.8(C-12),130.0(C-11),18.9(C-30),18.2(C-6),177.9(C-28),20.0(C-25),17.1(C-26),65.8(C-23),19.1(C-27).ESI-MS m/z 523[M+Na]+；HR-ESI-MS(positive ion mode)m/z 523.3037[M+Na]+(calcd for C30H44O6na,523.3036) (see figures 3-13 for each of the above data).
Example 3
Activity determination of myrtle triterpenoid lactone A
The inhibitory activity of the myrtle triterpene lactone A and acarbose (positive control) on alpha-glucosidase is determined, a 1% DMSO solution of the myrtle triterpene lactone A is used as a stock solution, the stock solution is used as a mother solution, and stock solution buffer solutions (phosphate buffer solution, pH 6.9) with different concentrations are prepared for test use. 50 μ L of different concentrations of compound (0.001, 0.01, 0.05, 0.1, 0.2, 0.5mg/mL) and 100 μ L0.5U/mL of α -glucosidase were added to a 96-well plate. Incubate at 25 ℃ for 15min, then add 50 μ L of 5mM 4-pNPG to 96-well plates, then incubate at 25 ℃ for 10 min. Absorbance was measured at a wavelength of 405nm (1, 5, 10, 15, 20 min). Alpha-glucosidase inhibition (%) - (a mark-a mark)/a mark]X 100%. The A scale is the absorbance value for the enzyme without compound/positive drug and the A sample is the absorbance value for the enzyme with compound/positive drug. IC (integrated circuit)50The results are shown in FIG. 2 for the concentration of inhibitor required to inhibit 50% of the enzyme activity.
The test results showed that the inhibition rate was gradually increased with increasing concentration at concentrations of 0.001, 0.01, 0.05, 0.1, 0.2, 0.5mg/mL, and the inhibition effect was dose-dependent, similar to acarbose, with the results shown in FIG. 2 a. The inhibition rates of the myrtacobalide A and the acarbose are 77.82 percent and 65.75 percent respectively, and the IC is50The values were 0.213. + -. 0.016 and 0.017. + -. 0.004mg/mL, respectively. In a primary enzyme kinetics experiment, the inhibitory activity of the enzyme is measured under the condition that the concentration of the myrtle triterpene lactone A is 0.5mg/mL, the reaction time is 1min, 5min, 10min, 15min and 20min respectively, and it can be seen that the myrtle triterpene lactone A can rapidly inhibit the activity of the enzyme, the inhibition rate can reach the maximum value within 1min and then is reduced, and the result is shown in figure 2 b. Thus, compound 1 exhibited potential α -glucosidase inhibitory activity.
Claims (5)
1. Myrtle triterpene lactone A of formula I:
2. a process for the extraction of the triterpene lactone a of formula I as claimed in claim 1, comprising the steps of:
s1, leaching dried myrtle leaf powder with an ethanol solution with the volume concentration of 70-95% until an extracting solution is colorless, combining obtained leaching solutions, and concentrating under reduced pressure to obtain an extract;
s2, dispersing the extract with water, extracting with ethyl acetate, and recovering the solvent under reduced pressure to obtain an ethyl acetate extract;
s3, separating the ethyl acetate part by adopting silica gel column chromatography, and performing chloroform-methanol gradient elution, wherein the elution concentrations are as follows in sequence according to the volume ratio: 1:0, 9:1, 8:2, 2:1, 1:1, 0:1, and combining the same fractions by thin layer chromatography to obtain six components Fr.A-Fr.F;
s4, taking the component Fr.B for further MCI column chromatographic separation, wherein the eluent is 70-90% of methanol solution, and separating to obtain a Fr.B1 component;
s5, separating the component Fr.B1 by silica gel column chromatography, and performing chloroform-methanol gradient elution, wherein the elution concentrations are as follows in sequence by volume ratio: the ratio of 50:1, 20:1 and 0:1, and 10 components Fr.B1-1-Fr.B1-10 are obtained by thin-layer chromatography detection;
s6, preparing Fr.B1-2, performing preparative liquid chromatography, performing gradient elution with methanol-water, sequentially performing volume ratio of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100%, and sequentially performing separation at a flow rate of 1-10.0mL/min to obtain 8 components Fr.B 1-2-1-Fr.B 1-2-8;
s7, subjecting Fr.B1-2-3 to high performance liquid chromatography, and eluting with acetonitrile-water at a volume ratio of 32:68 and a flow rate of 1.0mL/min to obtain the myrtle triterpene lactone A with the chemical formula I.
3. Use of myrtle triterpene lactone A of formula I as defined in claim 1 in the preparation of a medicament for treating diabetes.
4. Use of myrtle triterpene lactone A of formula I as claimed in claim 1 for preparing glucosidase inhibitor.
5. Use according to claim 4, characterized in that: the glucosidase is alpha-glucosidase.
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