A sesquiterpenoid compound, and a preparation method and application thereof

Abstract

The application discloses a sesquiterpenoid compound and a preparation method and application thereof, and belongs to the field of medicines.The structural formula of the application is The separation and preparation method of the compound of the application is that the ethyl acetate part of the calyx of kadsura coccinea is divided into sections by a normal phase silica gel column, elution is carried out with CH2Cl2:MeOH (50:1-0:1), the obtained part is further eluted by normal phase CH2Cl2-MeOH (40:1-0:1) and reversed phase C18 with MeOH:H2O (30:1, 40:1, 50:1, 60:1, 80:1), and then separation and purification are carried out through gel and HPLC.The compound of the application can be used for preparing antioxidant medicines or as a lead compound for development of antioxidant medicines.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology; specifically, it relates to a sesquiterpene compound with a glycoside attached, its preparation method, and its uses. Background Technology

[0002] With the development of free radical medicine and free radical biology, free radical-induced lipid peroxidation damage has become a new pathogenic factor, attracting significant attention from biologists and medical professionals both domestically and internationally. Studies have shown that the occurrence and development of many diseases are related to free radical reactions, particularly free radical-induced lipid peroxidation. It is known that reactive oxygen species (ROS) can directly or indirectly damage DNA, leading to DNA strand breaks and chromosome breaks. Research on ROS and the structure, function, and diseases of biological membranes has progressed rapidly in recent years, achieving breakthroughs not only in theoretical aspects but also in biotechnology and medical applications. One of the important topics in modern life sciences is elucidating the phenomenon of aging. Furthermore, cancer, myocardial infarction, organ damage, and metabolic diseases remain to be conquered, and these problems are all related to lipid peroxidation. Therefore, the search for highly effective and low-toxicity antioxidant drugs is particularly important.

[0003] Physalis alkekengi L. var. franchetii (Mast.) Makino, a plant in the Solanaceae family, is a dried persistent calyx or persistent calyx with fruit. It has the effects of clearing heat and detoxifying, relieving sore throat and resolving phlegm, and promoting urination. It is bitter and cold in nature and enters the lung meridian. It is also known as Red Lantern, Red Lantern Fruit, and Golden Lantern, and is widely distributed in my country, Russia, Japan, the Korean Peninsula, and other parts of Eurasia. In my country, it is mainly distributed in Northeast China. Previous studies by botanists have mainly focused on its chemical composition, particularly the uniquely structured physalin compounds. Summary of the Invention

[0004] This invention provides a sesquiterpene compound and its preparation method. The sesquiterpene compound has antioxidant activity and potential medicinal value.

[0005] This invention isolates a sesquiterpene compound linked to a glycoside from the persistent calyx of the plant *Physalis alkekengi*. Using L-ascorbic acid as a positive control, the sesquiterpene compound of this invention exhibits certain antioxidant activity. This compound can be used to prepare antioxidant drugs or as a lead compound for the development of antioxidant drugs.

[0006] The chemical structure of the sesquiterpenoid compound of the present invention is as follows: Specifically, it is prepared according to the following steps:

[0007] Step 1: Dry the calyx of the Phyllanthus urinaria and crush it. Extract it with 95% (volume) ethanol at least 3 times. Combine the extracts and concentrate them to obtain the extract.

[0008] Step 2: Add distilled water to the extract obtained in Step 1 to suspend it, and then extract it sequentially with petroleum ether, dichloromethane and ethyl acetate.

[0009] Step 3: Take the ethyl acetate extract, mix it with silica gel at a mass ratio of 1:1.2, and perform gradient elution with CH2Cl2:MeOH at a ratio of 50:1→30:1→20:1→15:1→12:1→8:1→5:1→3:1→0:1.

[0010] Step 4: The CH2Cl2−MeOH eluents from Step 3 (volume ratios of 20:1 and 30:1) are then passed through a medium-pressure ODS column, eluted sequentially with 30% (volume) MeOH:H2O, 40% (volume) MeOH:H2O, 50% (volume) MeOH:H2O, 60% (volume) MeOH:H2O, and 80% (volume) MeOH:H2O. The 40% methanol:water eluent is then passed through a Sephadex LH-20 column and eluted with pure MeOH. Each 20 mL unit is collected and analyzed by TLC. The collected solutions of the same phases are combined and divided into five fractions, which are concentrated to dryness. The second fraction is further separated by semi-preparative HPLC using a 50:50 MeOH:H2O mobile phase at a flow rate of 2 mL / min and a detection wavelength of 210 nm. The injection volume is 80 μL per sample. R =32 min, and the sesquiterpenoid compound was obtained;

[0011] In step four, a Waters high-performance liquid chromatograph, a UV2489 ultraviolet detector, and an RP C-18 column were used for semi-preparative HPLC separation.

[0012] Further specifying, in step one, during the extraction process, the solid-liquid ratio is 1:(3-10), the temperature is controlled at 60℃, and each extraction lasts for 8 hours.

[0013] To further specify, in step one, the concentration is carried out at 35°C and 0.3 MPa until it is just able to flow out, followed by vacuum concentration.

[0014] Further specifying, in step two, the mass ratio of extract to distilled water is 1:1, the volume ratio of dichloromethane to water is 1:1, and the volume ratio of ethyl acetate to water is 1:1.

[0015] Further specifying step two, petroleum ether is added to the suspension for extraction, the mixture is allowed to stand for separation, the petroleum ether layer is extracted and concentrated, and the above steps are repeated multiple times until the PE layer becomes light in color and transparent. The resulting extracts are then combined.

[0016] Further specifying step two, dichloromethane is slowly added to the suspension from which petroleum ether has been removed. After capping the bottle, it is shaken to mix, allowed to stand for separation, and the dichloromethane layer is extracted and concentrated. This extraction is repeated multiple times until the dichloromethane layer becomes lighter in color and transparent. The resulting extracts are then combined.

[0017] To further specify, in step two, ethyl acetate is slowly added to the suspension from which dichloromethane has been removed, shaken and mixed, allowed to stand to separate into layers, the ethyl acetate layer is extracted and concentrated, and the above steps are repeated several times until the ethyl acetate layer becomes light in color and nearly colorless and transparent, and the resulting extracts are combined.

[0018] To further specify, in step three, the mesh size of the silicone is 100-200 mesh.

[0019] The application of the above-mentioned sesquiterpenoids in the preparation of antioxidant drugs.

[0020] This invention provides the first extraction and separation technique, structural identification method, and antioxidant applications of a sesquiterpene compound from Physalis alkekengi L. var. franchetii (Mast.) Makino., a plant of the Solanaceae family.

[0021] To further understand the features and technical content of this invention, please refer to the following detailed description and accompanying drawings. However, the accompanying drawings are for reference and illustration only and are not intended to limit the invention. Attached Figure Description

[0022] Figure 1 This is the structural formula of the sesquiterpene compound of the present invention;

[0023] Figure 2 It is a sesquiterpene compound of the present invention. 1 H- 1 H COSY and key HMBC related diagrams;

[0024] Figure 3 The NOSEY correlation diagram is the key to the sesquiterpenoid compounds of this invention;

[0025] Figure 4 is a GC analysis diagram of the sesquiterpenoid compounds and D-glucose derivatization of the present invention, where A (t=51.0158) is D-glucose and B (t=50.9990) is a sesquiterpenoid compound.

[0026] Figure 5 The IC of the sesquiterpenoid compounds of this invention 50 picture;

[0027] Figure 6 is a line graph showing the reducing properties of sesquiterpenoids and vitamin C in this invention;

[0028] Figure 7 It is a sesquiterpene compound of the present invention. 1 H NMR spectrum;

[0029] Figure 8 It is a sesquiterpene compound of the present invention. 13 C NMR spectrum;

[0030] Figure 9 These are the DEPT spectra of the sesquiterpenoid compounds of this invention;

[0031] Figure 10 This is the HMQC spectrum of the sesquiterpenoid compounds of this invention;

[0032] Figure 11 It is a sesquiterpene compound of the present invention. 1 H- 1 H COSY spectrum;

[0033] Figure 12 This is the HMBC spectrum of the sesquiterpenoid compounds of this invention;

[0034] Figure 13 This is the NOESY spectrum of the sesquiterpenoid compounds of this invention;

[0035] Figure 14 This is the HRESIMS spectrum of the sesquiterpenoid compounds of this invention. Detailed Implementation

[0036] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0037] Specific Implementation Method 1: The chemical structure of the sesquiterpene compound in this embodiment is as follows:

[0038] Specifically, it is prepared according to the following steps:

[0039] Step 1: Dry and crush the calyx of the Phyllanthus urinaria, then extract it with 95% (volume) ethanol at least 3 times. During the extraction, the solid-liquid ratio is 1:10, the temperature is controlled at 60℃, and each extraction lasts for 8 hours. Combine the extracts and concentrate them at 35℃ and 0.3 MPa until they can just flow out. Then concentrate under reduced pressure to obtain the extract.

[0040] Step 2: Add distilled water to the extract obtained in Step 1 to suspend it, and then extract it sequentially with petroleum ether, dichloromethane and ethyl acetate.

[0041] The mass ratio of extract to distilled water is 1:1, the volume ratio of dichloromethane to water is 1:1, and the volume ratio of ethyl acetate to water is 1:1.

[0042] Add petroleum ether to the suspension for extraction, allow to stand until separation, extract the petroleum ether layer for concentration, repeat the above steps multiple times until the PE layer becomes light in color to transparent, and combine the obtained extracts.

[0043] Slowly add dichloromethane to the suspension from which petroleum ether has been removed, cap the bottle, shake well, allow to stand and separate into layers, extract the dichloromethane layer for concentration, and repeat the extraction process multiple times until the dichloromethane layer becomes lighter in color and transparent. Combine the obtained extracts.

[0044] Slowly add ethyl acetate to the suspension from which dichloromethane has been removed, shake well, allow to stand and separate into layers, extract the ethyl acetate layer and concentrate it. Repeat the above steps several times until the ethyl acetate layer becomes light in color and nearly colorless and transparent. Combine the obtained extracts.

[0045] Step 3: Take the ethyl acetate extract, mix it with 200-mesh silica gel at a mass ratio of 1:1.2, and elute with CH2Cl2:MeOH using a gradient elution of 50:1→30:1→20:1→15:1→12:1→8:1→5:1→3:1→0:1.

[0046] Step 4: The CH2Cl2−MeOH eluents from Step 3 (volume ratios of 20:1 and 30:1) were further eluted using a medium-pressure ODS column with 30% (volume) MeOH:H2O, 40% (volume) MeOH:H2O, 50% (volume) MeOH:H2O, 60% (volume) MeOH:H2O, and 80% (volume) MeOH:H2O. The 40% (volume) methanol:water eluent was then eluted with pure MeOH using a Sephadex LH-20 column. Each 20 mL unit was collected and analyzed by TLC. The collected solutions of the same phases were combined and divided into five fractions, which were concentrated to dryness. The second fraction was further separated by semi-preparative HPLC using a 50:50 MeOH:H2O mobile phase at a flow rate of 2 mL / min and a detection wavelength of 210 nm. Each injection volume was 80 μL, yielding the sesquiterpenoid compound (5.3 mg, t). R =32 min);

[0047] In step four, a Waters high-performance liquid chromatograph, a UV2489 ultraviolet detector, and an RP C-18 column were used for semi-preparative HPLC separation.

[0048] The absolute configuration of the new compound was determined using NMR and chemical reaction methods.

[0049] This invention relates to colorless sesquiterpene crystals, soluble in methanol. Spotting the sample on a thin-layer chromatography plate produces dark spots under 254 nm UV light. After development with 10% sulfuric acid-ethanol and drying, the dark spots are still visible. High-resolution mass spectrometry (HR-ESIMS) shows a quasi-molecular ion peak at m / z: 419.20456 [M + Na]. + Its molecular formula is presumed to be C 21 H 32 O7 (calcd for C) 21 H 32 O7Na 419.20402), the calculated degree of unsaturation is 6. 1 Two characteristic methyl peaks can be observed in the H-NMR spectrum. H δ: 2.01 (s, 3H, H-12), 0.99 (d, J = 7.0 Hz, 3H, H-11). Two olefin signals δ H 5.74 (s, 1H, H⁻²), 5.13 (s, 1H, H⁻¹⁵), 5.02 (s, 1H, H⁻¹⁵). Hydrogen proton signals δ on the six oxygen-bound carbons. H : 4.40 (d,J = 12.4 Hz, 1H, H-14), 4.29 (d, J = 8.7 Hz, 1H, H-1'), 4.15 (d, J = 12.4 Hz,1H, H-14), 3.87 (d, J = 11.8 Hz, 1H, H-6'), 3.66 (dd, J = 11.8, 5.4 Hz, 1H,H-6'), 3.35 (t, J=8.7 Hz, 1H, H-5'), 3.27 (m, 2H, H-4',H-3'), 3.21 (t, J =8.7 Hz, 1H, H-2').

[0050] 13 The C-NMR spectrum showed 21 C signals, which, combined with DEPT and HSQC spectral analysis, can be attributed to two methyl groups (δ¹⁺). C 21.18, C-12; 16.09, C-11); 7 methylene groups (δ C 111.38, C-15; 43.85, C-7; 42.21, C-6; 35.39, C-9; 34.22, C-10), including two hydroxymethylene groups (δ-). C 72.69, C-14; 62.83, C-6'); 8 methines, including 5 hydroxymethyl groups (δ-methyl groups). C103.22, C-1'; 78.19, C-5'; 77.98, C-3'; 75.11, C-2'; 72.69, C-4'); 4 quaternary carbons, including 1 sp. 3 Hybridized quaternary carbon (δ C 51.59, C-4) and 3 sps 2 Hybridized quaternary carbon (δ C 202.07, C-1; 170.65, C-3; 149.10, C-13).

[0051] 1 H NMR and 13 The C NMR spectral data are shown in Table 1; HRESIMS m / z 417.1873 [M + Na] + (calcdfor C 21 H 30 O7Na 417.1884).

[0052] Table 1. Sesquiterpenoids 1 H NMR, 13 C NMR, HMBC, 1 H- 1 H COSY data

[0053] NO <![CDATA[δ H ]]> <![CDATA[δ C ]]> HMBC COSY 1 130.22 2 188.77 3 6.20, d 126.48 H-3→C-1, 5, 15 4 6.97, d 160.09 H-4→C-2, 5, 6, 10, 15 5 42.12 6 163.81 7 2.97, d2.35, t 34.39 H-7→C-5, 6, 8, 9 H-7→H-14 8 2.21, t 43.78 H-8→H-9 9 1.85, m 1.78, m 27.92 H-9→H-8 H-9→H-10 10 1.95, d1.35, dd 39.17 H-10→H-9 11 150.59 12 4.45, d4.20, d 72.21 H-12→C-1', 8, 13 H-12→H-13 13 5.22, s 5.13, s 112.77 H-13→C-8, 11, 12 H-13→H-12 14 1.91, s 10.80 H-14→C-1, 2, 6 H-14→H-7 15 1.30, s 23.93 H-15→C-4, 5, 6, 9, 10 1’ 4.30, d 103.27 H-1'→C-3', 12 H-1'→H-2' 2’ 3.20, t 75.22 H-2'→C-1', 3' H-2'→H-1' 3’ 3.26, s 78.16 4’ 3.27, s 71.88 H-4'→H-6' 5’ 3.35, s 78.35 H-5'→C-4' 6’ 3.87, d3.65, dd 63.00 H-6'→C-3', 4' H-6'→H-4'

[0054] The information provided by HMBC and COSY in Table 1 verified the previous hypothesis. 1 H- 1 H COSY data shows that H-3 and H-4 are correlated. Combined with HMBC spectral data, H-3 is correlated with C-1, C-5, and C-15; H-4 is correlated with C-2, C-5, C-6, C-10, and C-15, confirming that positions 3 and 4 form a carbon-carbon double bond. The C-H relationship is as follows: Figure 2 As shown. Analysis of the NOSEY spectrum of the compound reveals a NOE correlation between α(H) at position 7 and 14-CH3, indicating they are in the same plane. A NOE correlation also exists between β(H) and 15-CH3, and there is no NOE correlation between the two methyl groups, confirming that position 15 is a β-CH3. The spectrum also shows correlations between H-13 and H-15, and H-4 and H-15. See the attached figure for spectral information. The H-H relationships in NOSEY are as follows: Figure 3 As shown.

[0055] Acid hydrolysis of glycosidic bonds in compounds

[0056] To determine the absolute configuration of glucose in the sesquiterpenoid compounds of this embodiment, 1 mg of each sesquiterpenoid compound was added to 2 ml of trifluoroacetic acid (4 mol / L), and the mixture was reacted at 90°C for 4 hours. Subsequently, the reaction solution was evaporated to dryness, and after drying, 2 ml of water was added to dissolve it. The solution was then extracted with 1 ml of ethyl acetate each time, for a total of 4 times.

[0057] Monosaccharide derivatization: After concentrating and evaporating the aqueous layer to dryness, anhydrous pyridine (1 ml) and 2 mg of L-cysteine ​​methyl hydrochloride (L-CMEH) were added, and the mixture was reacted at 60°C for 1 hour. Then, 0.2 ml of trimethylsilimidazole was added, and the reaction was continued for another hour. After the reaction was complete, the mixture was concentrated and evaporated to dryness, 1 ml of water was added, and then extracted with 0.5 ml of n-hexane. The n-hexane layer was used for GC analysis. D-glucose standards were processed using the same method.

[0058] GC detection conditions: HP-5 column (30m × 0.25mm × 0.25μm); FID detector; initial temperature 60°C, hold for 3 min; ramp to 200°C at 4°C / min; ramp to 280°C at 10°C / min, hold for 1 min; injection port temperature 250°C; carrier gas: high-purity nitrogen; split ratio 5:1; injection volume 1 μl; detector temperature 300°C. Using D-glucose standard silanized derivatives as a control, the GC detection showed the standard retention time to be 51.0158 min, and the new compound retention time to be 50.9990 min. The absolute configuration of the sugar in the sesquiterpenoid compounds of this example is D-glucose (Figures 4 and 5).

[0059] It showed a certain ability to scavenge DPPH free radicals. With L-ascorbic acid as a positive control, the specific data are shown in Table 2.

[0060] Table 2

[0061] Sample <![CDATA[IC 50 (µM)]]> New compound 33.69±6.65 Vc 7.24±0.83

[0062] As shown in Table 2, sesquiterpenoids have a certain DPPH scavenging ability, indicating that they have antioxidant activity.

Claims

1. A sesquiterpene compound, characterized in that, The structural formula of the sesquiterpene compound is as follows: 。 2. The method for preparing sesquiterpenoid compounds according to claim 1, characterized in that... The preparation method is carried out according to the following steps: Step 1: Dry the calyx of the Phyllanthus urinaria and crush it. Extract it with 95% ethanol at least 3 times, combine the extracts, and concentrate them to obtain the extract. Step 2: Add distilled water to the extract obtained in Step 1 to suspend it, and then extract it sequentially with petroleum ether, dichloromethane and ethyl acetate. Step 3: Take the ethyl acetate extract, mix it with silica gel at a mass ratio of 1:1.2, and perform gradient elution with CH2Cl2:MeOH at a ratio of 50:1→30:1→20:1→15:1→12:1→8:1→5:1→3:1→0:

1. Step 4: The CH2Cl2:MeOH eluents from Step 3 (volume ratios of 20:1 and 30:1) are then passed through a medium-pressure ODS column and eluted sequentially with 30% (v / v) MeOH:H2O, 40% (v / v) MeOH:H2O, 50% (v / v) MeOH:H2O, 60% (v / v) MeOH:H2O, and 80% (v / v) MeOH:H2O. The 40% (v / v) MeOH:H2O eluent is then passed through a Sephadex LH-20 column and eluted with pure MeOH. Each 20 mL unit is collected and analyzed by TLC. The collected solutions of the same phases are combined and divided into five fractions, which are concentrated to dryness. The second fraction is further separated by semi-preparative HPLC using a 50:50 (v / v) MeOH:H2O mobile phase at a flow rate of 2 mL / min and a detection wavelength of 210 nm. Each injection volume is 80 μL. R =36 min, and the sesquiterpenoid compound was obtained; In step four, a Waters high-performance liquid chromatograph, a UV2489 ultraviolet detector, and an RP C-18 column were used for semi-preparative HPLC separation.

3. The preparation method according to claim 2, characterized in that, In step one, during the extraction process, the solid-liquid ratio is 1:(3-10), the temperature is controlled at 60℃, and each extraction lasts for 8 hours. In step one, the concentration is carried out at 35℃ and 0.3 MPa until the solution is just able to flow out, and then the concentration is carried out under reduced pressure.

4. The preparation method according to claim 2, characterized in that, In step two, the mass ratio of extract to distilled water is 1:1, the volume ratio of dichloromethane to water is 1:1, and the volume ratio of ethyl acetate to water is 1:

1.

5. The preparation method according to claim 2, characterized in that, In step two, petroleum ether is added to the suspension for extraction. The mixture is allowed to stand until it separates into layers. The petroleum ether layer is extracted and concentrated. The above steps are repeated multiple times until the PE layer becomes lighter in color and transparent. The resulting extracts are then combined.

6. The preparation method according to claim 2, characterized in that, In step two, dichloromethane is slowly added to the suspension from which petroleum ether has been removed. After capping the bottle, it is shaken and allowed to stand to separate into layers. The dichloromethane layer is extracted and concentrated. The extraction is repeated several times until the dichloromethane layer becomes lighter in color and transparent. The resulting extracts are then combined.

7. The preparation method according to claim 2, characterized in that, In step two, ethyl acetate is slowly added to the suspension from which dichloromethane has been removed, shaken to mix, allowed to stand to separate into layers, and the ethyl acetate layer is extracted and concentrated. The above steps are repeated several times until the ethyl acetate layer becomes light in color and nearly colorless and transparent. The resulting extracts are then combined.

8. The preparation method according to claim 2, characterized in that, In step three, the silicone mesh size is 100-200 mesh.

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