CN116947941A - Sesquiterpene glycoside compound and preparation method and application thereof - Google Patents

Sesquiterpene glycoside compound and preparation method and application thereof Download PDF

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CN116947941A
CN116947941A CN202310600666.XA CN202310600666A CN116947941A CN 116947941 A CN116947941 A CN 116947941A CN 202310600666 A CN202310600666 A CN 202310600666A CN 116947941 A CN116947941 A CN 116947941A
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glycoside compound
sesquiterpene glycoside
sesquiterpene
ligularia
lingualis
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王慧
王思瑶
邹坤
柏杰
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China Three Gorges University CTGU
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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
    • C07H15/18—Acyclic radicals, substituted by carbocyclic rings
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
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    • C07H1/08—Separation; Purification from natural products
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Abstract

The invention discloses a sesquiterpene glycoside compound and a preparation method and application thereof, wherein 1 new sesquiterpene glycoside ligulariatinside A is separated and identified from the n-butanol extraction part of a 95% ethanol extract at the root of ligularia lingualis, and the molecular formula is determined as C by a plurality of chromatographic methods and spectrum methods such as macroporous resin column chromatography, normal-reverse phase silica gel column chromatography, high performance liquid chromatography and the like 21 H 34 O 7 The structure is (3)S,4R,5R,7R)‑11‑hydroxy‑11,12‑dihydronootkatone‑11‑O‑βD-glucopyranoside, which enriches the diversity of ligularia lingualis chemical components. The bacterial activity inhibition experiment shows that the novel sesquiterpene glycoside has a certain inhibition effect on Vibrio anguillarumThe activity is prepared, the Minimum Inhibitory Concentration (MIC) value is 50 mug/mL, and a foundation is laid for the research and development of novel antibacterial drugs.

Description

Sesquiterpene glycoside compound and preparation method and application thereof
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a sesquiterpene glycoside compound and a preparation method and application thereof.
Technical Field
About 130 plants of Ligularia (Ligularia) belonging to Asteraceae (Asteraceae) are distributed throughout the world, and only 2 plants are distributed in Europe. There are 111 kinds in our country, and most kinds are concentrated in southwest hilly areas. Ligularia lingualis Ligularia veitchiana (hemsl.) Greenm is a plant of ligularia genus of Compositae, and is mainly distributed in northwest of Yunnan, sichuan, guizhou, northwest of Hubei, southwest of Gansu, southwest of Shanxi and the like, and is grown on river sides, hillsides and under forests at an altitude of 1-3 m; the ligularia lingualis root has long history of medication, has the effects of moistening lung and descending qi, clearing heat and detoxicating, resisting bacteria and diminishing inflammation, resolving phlegm and relieving cough, promoting blood circulation and relieving pain and the like, and is mainly used for treating influenza, cough, ulcer, phthisis and other symptoms in folks.
Sesquiterpenes are groups of compounds consisting of 3 isoprene units and containing 15 carbon atoms. Sesquiterpenes are widely found in plants, microorganisms, marine organisms and certain insects, and many have important biological functions and physiological activities, such as sesquiterpene lactones have antibacterial, antitumor, antiviral, cytotoxic, immunosuppressive, phytotoxic, insect hormone, insect feeding agents and other activities, and some have nervous system activities. Chinese patent CN105535152B discloses an application of folium Eriobotryae total sesquiterpene glycoside extract, which can remarkably improve the fractional lipid metabolism of type 2 diabetes, relieve the fractional liver cell degeneration necrosis of type 2 diabetes, reduce lipid deposition, prevent the formation of non-alcoholic fatty liver, and can be used as a medicament or health care product for treating hyperlipidemia and type 2 diabetes non-alcoholic fatty liver. Chinese patent CN113041253a discloses the use of a sesquiterpene glycoside monomer compound in preparing lipid-lowering prescription. Xu Jianlong et al identified several eremophilane-type sesquiterpenes in ligularia lingualis and found that they did not show cytotoxic effects on both gastric cancer cells HGC-27 and cervical cancer cells Caski.
At present, the ligularia lingualis contains less sesquiterpene glycoside compounds to be separated and identified, and the application of the sesquiterpene glycoside compounds is slightly researched, and particularly, the sesquiterpene glycoside has great research potential on the inhibition activity to bacteria.
Disclosure of Invention
The invention provides a sesquiterpene glycoside compound, a preparation method and application thereof, wherein a novel sesquiterpene glycoside is obtained by directional separation from ethanol extract of ligularia lingualis root, and the novel sesquiterpene glycoside compound has remarkable inhibitory effect on vibrio anguillarum through antibacterial experiment detection, and can be used for researching novel antibacterial drugs.
The technical scheme adopted by the invention is as follows:
the invention provides a sesquiterpene glycoside compound, which has the following chemical structural formula:
preferably, the sesquiterpene glycoside compound is prepared by extraction and separation from ligularia lingualis.
The invention also provides a preparation method of the sesquiterpene glycoside compound, which specifically comprises the following steps:
(1) Extracting dry ligularia lingualis root with 6 times of 95% ethanol for 2-5 times, mixing extractive solutions, concentrating under reduced pressure, and suspending with water;
(2) Extracting the suspension solution with petroleum ether, ethyl acetate and n-butanol for 2-5 times, 3-5L each time, to obtain n-butanol extract;
(3) Separating n-butanol extract by macroporous resin chromatography, eluting with ethanol-water gradient, mixing fractions Fr.20-21 (4.551 g), and separating by reverse silica gel chromatography;
(4) Eluting with methanol-water gradient, mixing the fractions Frr.R 2 -32～34(127.5mg)；
(5) Separating and purifying the product by semi-preparative HPLC to obtain the sesquiterpene glycoside compound.
Preferably, the macroporous resin chromatographic condition in the step (3) is that the macroporous resin of D101 with the column volume of 6L is used for carrying out column chromatography gradient separation at room temperature; ethanol-water gradients of 30:70, 60:40, 75:25, 95:5; the reversed phase silica gel chromatography condition is that the column chromatography gradient separation is carried out by using an Rp-C18 reversed phase silica gel column with the column volume of 420mL at room temperature.
Preferably, the gradient of methanol-water in step (4) is 30:70, 45:55, 60:40, 75:25, 90:10, 100:0.
Preferably, the mobile phase of the semi-preparative HPLC in step (5) is methanol-0.1% acetic acid water=36:64, the flow rate is 3ml·min -1 。
The invention also provides application of the sesquiterpene glycoside compound in inhibiting bacterial activity.
Preferably, the bacteria are Vibrio anguillarum.
The invention has the beneficial effects that: 1 new sesquiterpene glycoside ligulariatinside A is separated and identified from the n-butanol extraction part of the ligularia lingualis root 95% ethanol extract by using various chromatographic methods and spectroscopic methods, and the antibacterial experiment proves that the identified new sesquiterpene glycoside has a certain inhibitory activity on vibrio anguillarum, and the Minimum Inhibitory Concentration (MIC) value is 50 mug/mL.
Drawings
FIG. 1 is a chemical structural formula of the isolated and identified sesquiterpene glycosides of the present invention.
FIG. 2 shows the main HMBC, H-scheme of Compound 1 1 H COSY and NOESY correlation spectra.
FIG. 3 is a HR-ESI-MS spectrum of example 2.
FIG. 4 is a schematic diagram of example 2 1 H NMR spectrum.
FIG. 5 is a schematic diagram of example 2 13 C NMR spectrum.
FIG. 6 is a HSQC spectrum in example 2.
FIG. 7 is a schematic diagram of example 2 1 H- 1 H COSY pattern.
Fig. 8 is an HMBC spectrum in example 2.
FIG. 9 is a NOESY pattern in example 2.
Detailed Description
The technical solution of the present invention is further explained below with reference to the accompanying drawings and specific embodiments, and it should be noted that the following embodiments are only preferred embodiments of the present invention, and not limiting the present invention, and the scope of the present invention shall be defined by the claims. Modifications and substitutions made by those skilled in the art without the inventive effort fall within the scope of the invention.
Instrument and materials: bruker AV 400 Nuclear magnetic resonance spectrometer (Bruker, switzerland); FT-ICR MS/MRMS, model SolariX (Bruce Dalton, germany); semi-preparative chromatography column (Daisogel-10 μ -100A,250mm x 10mm,10 μm); analytical chromatographic column (DAISO, 250 mm. Times.4.6 mm,5 μm); AL204-IC electronic balance (meltrele-tolido instruments limited, shanghai); cryogenic freeze dryer (Labconco, USA); d101 macroporous resin (16-60 mesh, samsung Anhui resin Co., ltd.); normal phase silica gel (300-400 mesh, qingdao ocean chemical Co., ltd.); rp-C18 reverse phase silica gel (100-200 mesh, japanese YMC Co.); chromatographic silica gel plate GF 254 (Qingdao ocean chemical plant); chromatographic pure methanol and chromatographic pure acetonitrile (TEDIA company, usa); deuterated reagents (us Cambridge Isotope Laboratories, inc.); other analytically pure reagents (Tianjin chemical reagent Co., ltd.); monosaccharide standard D-glucose (product number 47249, mass fraction > 99.5%, sigma Co.).
Ligularia lingualis roots used in the following examples were collected from the Shennong's frame in the university of Hubei in 2017, identified by doctor Wang Yubing of the university of three gorges, as ligularia lingualis Ligularia veitchiana (hemsl.) Greenm, and plant specimens (LV 2017001) were stored in the university of three gorges natural products research and utilization Hubei province key laboratory.
Example 1 extraction and isolation of Compounds
(1) Taking 9.6kg of dried ligularia lingualis root, crushing, adding 6 times of 95% ethanol solution, performing cold soaking extraction at room temperature, and repeating for 3 times;
(2) Mixing the 3 times of extracting solutions, concentrating under reduced pressure to 0.5L, and adding 1L of water to obtain suspension;
(3) Extracting with 3L petroleum ether, 3L ethyl acetate and 3L n-butanol respectively for 3 times, and mixing n-butanol extractive solutions to obtain n-butanol extractive part;
(4) Taking 90.0g of n-butanol extraction part to carry out macroporous resin column chromatography separation, wherein the condition is that D101 macroporous adsorption resin with the column volume of 6L is used for carrying out column chromatography gradient separation at room temperature to obtain resin separation liquid;
(5) Gradient elution is carried out on the resin separating liquid by ethanol-water to obtain 79 fractions Fr.1-79; combining Fr.20-21 (4.551 g) and separating by reverse phase silica gel column chromatography to obtain reverse phase separation liquid, wherein the gradient of ethanol-water is 30:70, 60:40, 75:25 and 95:5, and the chromatographic condition is that the column chromatography gradient separation is carried out by using an Rp-C18 reverse phase silica gel column with the column volume of 420mL at room temperature;
(6) Subjecting the reversed-phase separating liquid to gradient elution by methanol-water to obtain 101 fractions Frr.1-101; frr.R2-32-34 (127.5 mg) was combined to give an eluent with a methanol-water gradient of 30:70, 45:55, 60:40, 75:25, 90:10, 100:0;
(7) The eluate was subjected to semi-preparative HPLC (methanol-0.1% acetic acid water=36:64, 3ml·min -1 ) Isolation and purification gave Compound 1 (3.0 mg, t) R ＝40min)。
Example 2 structural identification of Compounds
Compound 1: a colorless oil of the product was obtained,UV(MeOH)λ max :194.0 249.4nm. HR-ESI-MS gives an excimer ion peak m/z 399.2377[ M+H ]] + (theoretical value 399.2376) (FIG. 3), molecular formula C was determined 21 H 34 O 7 The calculated unsaturation was 5.
13 C NMR(400MHz,DMSO-d 6 ) The spectrum combined with DEPT-135 spectrum shows that the compound contains 4 CH 3 5 CH 2 8 CH and 3 quaternary carbons.
Bonding of 1 H NMR (FIG. 4), 13 As can be seen from the C NMR (FIG. 5) and HSQC (FIG. 6) spectra, the compound contains 1 ketocarbonyl group (. Delta.) C 197.8 1 methine [ delta ] with an olefinic hydrogen proton attached H 5.70(s),δ C 124.3]Methine [ delta ] of 5 oxygen atoms H 4.27(1H,d,J＝7.6Hz),δ C 97.1；δ H 3.13(1H,t,J＝8.4Hz),δ C 77.0；δ H 3.02(1H,m),δ C 76.5；δ H 2.89(1H,t,J＝8.4Hz),δ C 73.8；δ H 3.00(1H,t,J＝8.4Hz),δ C 70.4]2 methines with fatty hydrogen protons [δ H 1.45(1H,m),δ C 40.5；δ H 2.20(1H,m),δ C 34.1]1 methylene [ delta ] of oxygen H 3.62(1H,brd,J＝11.2Hz),3.38(1H,overlapped),δ C 61.3]4 methylene [ delta ] groups with aliphatic hydrogen protons attached H 2.20(2H,m),δ C 41.9；δ H 1.88(1H,d,J＝13.2Hz),1.18(1H,t,J＝13.2Hz),δ C 33.6；δ H 2.45(1H,m),2.16(1H,m),δ C 28.3；δ H 1.68(2H,m),δ C 25.2]And 4 methyl [ delta ] H 1.10(3H,s),δ C 24.5；δ H 1.09(3H,s),δ C 22.3；δ H 1.00(3H,s),δ C 19.0；δ H 0.91(3H,d,J＝4.8Hz),δ C 15.2]。
The nuclear magnetic data of the compound is found to be similar to the data reported in the literature for (3S, 4R,5R, 7R) -3, 11-dihydro-11, 12-dihydroootkatone-11-O-beta-D-glucopyranoside, except that one OH is less at the 3-position. This speculation is by 1 H- 1 H in the H COSY Spectrum (FIG. 7) 2 -3(δ H 2.20)/H-4(δ H 2.20 And H-4 (delta) H 2.20)/H 3 -14(δ H 0.91 For example), H in HMBC spectra (FIG. 8) 2 -3(δ H 2.20)/C-2(δ C 197.8)、H-4(δ H 2.20)/C-2(δ C 197.8)、H 3 -14(δ H 0.91)/C-3(δ C 41.9 And H) 3 -14(δ H 0.91)/C-4(δ C 34.1 As evidenced by the correlation (see fig. 2).
In addition, HMBC spectra (FIG. 8) show H-1 (delta) H 5.70 And C-5 (delta) C 39.6)/C-9(δ C 28.3)，H 3 -15(δ H 1.00 And C-6 (delta) C 33.6)/C-9(δ C 28.3)，H-6(δ H 1.88 And C-8 (delta) C 25.2)/C-10(δ C 175.4)，H 3 -12(δ H 1.09 And C-7 (delta) C 40.5)/C-11(δ C 77.9)，H 3 -13(δ H 1.10 And C-7 (delta) C 40.5)/C-11(δ C 77.9 HMBC remote correlation (see fig. 2).
1 H- 1 H COSY spectrum (graph)7) It shows that H-6 (delta) H 1.88)/H-7(δ H 1.45)，H-7(δ H 1.45)/H-8(δ H 1.68)，H-8(δ H 1.68)/H-9(δ H 2.45 Presence of (a) 1 H- 1 H COSY-related.
In NOESY spectra (FIG. 9), H was observed 3 -15(δ H 1.00 With H) 3 -14(δ H 0.91)、H-9(δ H 2.16 And H-6 (delta) H 1.88 NOE-related to H-7 (delta) H 1.45 No NOE correlation, indicating H 3 -15(δ H 1.00)/H 3 -14(δ H 0.91)/H-9(δ H 2.16)/H-6(δ H 1.88 On the same side, H-7 (delta) H 1.45 On the opposite side).
Compound 1 is acid hydrolyzed and then is compared with a standard substance D-glucose by HPLC to obtain that the compound 1 contains 1 glucose residue and combines with a sugar end group proton signal H-1' (delta) H 4.27 The coupling constant was 7.6Hz, suggesting that the glucose end carbon configuration was the beta configuration.
In the HMBC spectra (FIG. 8), H-1' (delta) can be seen H 4.27)/C-11(δ C 77.9 Related, suggesting that the glycosyl is attached to the C-11 position of the aglycone core.
In combination with the above spectral analysis, the structure of compound 1 was determined to be (3S, 4R,5R, 7R) -11-hydroxy-11, 12-dihydroootkatone-11-O-. Beta. -D-glucopyranoside. No relevant report was found by searching through SciFinder Scholar network, which shows that the compound 1 is 1 new sesquiterpene glycoside compound and is named ligulariatinside A (figure 1).
TABLE 1 Nuclear magnetic data for Compound 1 (400/100 MHz, DMSO-d 6 )
In vitro antibacterial Activity test of the Compounds of example 3
The antibacterial activity of the objective compound against Vibrio anguillarum was measured by a microdilution method using the purified compound 1 extracted in example 1, and the specific method is as follows:
(1) Preparation of the culture Medium
LB solid medium: 10.0g of peptone, 5.0g of beef extract, 35.0g of NaCl and 18.0g of agar powder, adding water for dissolution, then fixing the volume to 1000mL, putting the mixture into a conical flask, sealing the conical flask, sterilizing the mixture at 121 ℃ for 20min, placing the mixture into an ultra-clean workbench for cooling to about 50 ℃, performing plate pouring operation, and naturally cooling a culture dish to room temperature to obtain the LB solid culture medium.
LB liquid medium: 10.0g peptone, 5.0g beef extract and 35.0g NaCl are added with water to be dissolved, the volume is fixed to 1000mL, the mixture is divided into conical flasks, the conical flasks are sealed by sealing films, and sterilization is carried out for 20min at 121 ℃, thus obtaining the LB liquid medium.
(2) Preparation of bacterial suspension of test strain
Activation of strains: mu.L of the strain liquid was taken in LB solid medium, streaked with an inoculating loop "Z" and cultured in an incubator at 28 ℃.
Bacterial suspension: inoculating 2-3 loops of strain to 200mL of LB culture medium under a sterile environment, sealing, transferring to a constant temperature culture oscillator, and culturing for 24h at 28 ℃ for later use.
Diluting the bacterial suspension: under the aseptic environment, 10 mu L of bacterial suspension is sucked by a liquid-transfering gun, added into 100mL of LB liquid medium, gently shaken and prepared for use.
(3) Preparation of sample to be tested
Accurately weighing 0.0998mg of a suitable compound, and respectively dissolving with DMSO to prepare 10mg/mL of mother liquor; the negative control is DMSO; the positive reference substance is DMSO solution containing ciprofloxacin hydrochloride, and the concentration is 10mg/mL; after each sample to be tested is completely dissolved, the sample to be tested is subjected to ultrasonic treatment in an ultrasonic machine for 10min, and is placed at a constant temperature of 4 ℃ for refrigeration.
(4) Antibacterial activity primary screen
In an ultra-clean workbench, transferring 4 mu L of 10mg/mL of sample solution to be tested by using a pipetting gun, adding 196 mu L of diluted bacterial suspension into a 96-well plate, enabling the concentration of each well sample to be 200 mu g/mL, operating each group in parallel for 3 times, sealing a 96-well plate by negative control and positive control according to the same method, placing the 96-well plate in a constant temperature incubator at 28 ℃ for culturing for 18-24 hours, visually observing the turbidity condition of each well, and enabling samples without visible turbidity to have certain antibacterial activity, thus being capable of further rescreening.
(5) Determination of Minimum Inhibitory Concentration (MIC)
Samples to be tested were diluted to 9 concentration gradients (200, 100, 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78 μg/mL) in 96-well plates using a double dilution method, and positive control drugs were diluted to 12 concentration gradients (200, 100, 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, 0.2, 0.1 μg/mL). In an ultra-clean workbench, 8 mu L of sample solution to be detected is sucked into a 96-well plate by a liquid-transfering gun, 192 mu L of diluted bacterial suspension is added, after full mixing, 100 mu L of diluted bacterial suspension is sucked into the next row by the liquid-transfering gun, 100 mu L of diluted bacterial suspension is added again, the mixture is pushed until the mixture is diluted to the target concentration, the last row is added with negative control DMSO and diluted bacterial suspension, each group of holes is repeated for two rows, and 200 mu L of sterile distilled water is added around the 96-well plate. The sealed 96-well plate was placed in a constant temperature incubator at 28℃for 24 hours, turbidity of the medium was observed, and the lowest concentration of the sample which was not visible to the naked eye as MIC was recorded.
The results show that: compound 1 can inhibit the growth of Vibrio anguillarum, and the minimum inhibitory concentration MIC value is 50 mug/mL.

Claims (10)

1. A sesquiterpene glycoside compound characterized by: the chemical structural formula of the sesquiterpene glycoside compound is as follows:
。
2. the sesquiterpene glycoside compound according to claim, wherein the sesquiterpene glycoside compound is characterized in that: the sesquiterpene glycoside compound is prepared by extracting and separating ligularia lingualis.
3. A process for producing a sesquiterpene glycoside compound according to claim 1, characterized in that: the method comprises the following steps:
(1) Taking dry ligularia lingualis root, carrying out ethanol cold extraction, combining the extracting solutions, concentrating under reduced pressure, and suspending by water;
(2) Sequentially extracting the suspension solution with petroleum ether, ethyl acetate and n-butanol to obtain n-butanol extraction part;
(3) Separating n-butanol extract by macroporous resin chromatography, eluting with ethanol-water gradient, mixing fractions Fr.20-21 (4.551 8 g), and separating by reverse silica gel chromatography;
(4) Eluting with methanol-water gradient, mixing the fractions Frr, R 2 -32~34（127.5 mg）；
(5) Separating and purifying the product by semi-preparative HPLC to obtain the sesquiterpene glycoside compound.
4. A method of preparation according to claim 3, characterized in that: the mass fraction of the ethanol in the step (1) is 95%, the dosage is 6 times of that of ligularia lingualis, and the cold extraction times are 2-5 times.
5. A method of preparation according to claim 3, characterized in that: the dosage of petroleum ether, ethyl acetate and n-butanol in the step (2) is 3-5L, and the extraction times are 2-5 times.
6. A method of preparation according to claim 3, characterized in that: performing column chromatography gradient separation by using D101 macroporous adsorption resin with column volume of 6L at room temperature under the macroporous resin chromatographic condition of the step (3); ethanol-water gradients of 30:70, 60:40, 75:25, 95:5; the reversed phase silica gel chromatography condition is that the column chromatography gradient separation is carried out by using an Rp-C18 reversed phase silica gel column with the column volume of 420mL at room temperature.
7. A method of preparation according to claim 3, characterized in that: the gradient of methanol-water in step (4) was 30:70, 45:55, 60:40, 75:25, 90:10, 100:0.
8. A method of preparation according to claim 3, characterized in that: step (5) the semi-preparative HPLCThe mobile phase is methanol-0.1% acetic acid water=36:64, the flow rate is 3 mL.min -1 。
9. Use of a sesquiterpene glycoside compound according to any one of claims 1-2 or a sesquiterpene glycoside compound prepared by the preparation method according to any one of claims 3-8 for inhibiting bacterial activity.
10. The use according to claim 9, characterized in that: the bacteria are Vibrio anguillarum.
CN202310600666.XA 2023-05-25 2023-05-25 Sesquiterpene glycoside compound and preparation method and application thereof Pending CN116947941A (en)

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