Sesquiterpenoid compounds from Lycium bark and their preparation method and application

Abstract

The present invention belongs to the fields of traditional Chinese medicine extraction and separation, phytochemistry, and medicine, and discloses sesquiterpenoid compounds from the bark of Lycium bark. These sesquiterpenoid compounds have anti-neuroinflammatory activity and have promising application prospects in the preparation of anti-neuroinflammatory drugs. The present invention also discloses the use of these sesquiterpenoid compounds in the preparation of drugs for treating neuroinflammatory conditions.

Description

Technical Field

[0001] The invention belongs to the fields of traditional Chinese medicine extraction and separation, phytochemistry and medicine, and relates to sesquiterpenoid compounds separated from the bark of Lycium bark of the Solanaceae plant, as well as a preparation method and application thereof. Background Art

[0002] As a chronic metabolic disease, diabetes is characterized by high morbidity and many complications. The development of diabetes is often accompanied by inflammation, and neuropathy is its most important chronic complication. If neuroinflammation is not effectively controlled, it will lead to pain, cognitive impairment, movement disorders and even symptoms such as atherosclerosis and stroke, which seriously threaten the safety of diabetic patients. At present, the effects of drugs for the treatment of diabetes are mainly focused on pain management and blood sugar control. There are very few therapeutic drugs for neuroinflammation caused by diabetes. Therefore, it is urgent to develop anti-diabetic neuroinflammation drugs with obvious effects and low side effects.

[0003] Cortex Lycii is the dried root bark of Lycium chinese Mill. or Lycium barbarum L., a plant of the Solanaceae family. It is cold in nature and sweet in flavor, entering the lung, liver, and kidney meridians. It cools the blood, eliminates steam, clears the lungs, and reduces internal heat. It is primarily used for symptoms such as yin deficiency-induced hot flashes, night sweats due to bone steaming, cough due to lung heat, hemoptysis, epistaxis, and internal heat-induced thirst. Modern research has shown that it has pharmacological activities such as lowering blood pressure, blood lipids, and blood sugar, as well as antibacterial and anti-inflammatory, immunomodulatory, and antipyretic and analgesic properties. Both single and combined preparations have been clinically used to treat diabetic neuropathy. Compounds identified in Cortex Lycii primarily include alkaloids, flavonoids, terpenes, anthraquinones, organic acids, glycosides, and cyclic peptides. However, the active ingredient(s) responsible for treating diabetic neuroinflammation remain to be determined.

[0004] Therefore, further analysis and development of anti-diabetic neuroinflammatory components in Radix Lycii are of great significance for the application of Radix Lycii and the development of new anti-diabetic neuroinflammatory drugs or health products. Summary of the Invention

[0005] The present invention takes the bark of Lycium bark, a plant of the genus Lycium in the Solanaceae family, as the research target, studies its chemical components, obtains novel sesquiterpenoid compounds, and evaluates their anti-neuroinflammatory activity, all of which have anti-neuroinflammatory activity.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The sesquiterpenoid compounds with the following structure were isolated from Lycii cortex, a plant of the Solanaceae family:

[0008]

[0009]

[0010] As a preferred technical solution of the present invention, the sesquiterpenoid compound has the following structure:

[0011]

[0012] Another object of the present invention is to provide a method for preparing the sesquiterpenoid compound, comprising:

[0013] Step (1), the dried Chinese wolfberry bark medicinal material is subjected to reflux extraction with 75-90% ethanol, the extract is concentrated under reduced pressure to remove the solvent to obtain an extract, the extract is adsorbed on equal amounts of diatomaceous earth, and eluted with petroleum ether, ethyl acetate and methanol in sequence, the petroleum ether eluate, ethyl acetate eluate and methanol eluate are collected respectively, and are concentrated under reduced pressure to obtain a petroleum ether fraction, an ethyl acetate fraction and a methanol fraction; wherein the mass volume ratio of the dried Chinese wolfberry bark medicinal material to 75-90% ethanol is 1:10-1:20 kg / L or g / mL;

[0014] Step (2), the ethyl acetate fraction was subjected to silica gel column chromatography using a petroleum ether-ethyl acetate system and a dichloromethane-methanol system as eluents, and the same fractions were combined to obtain nine components: Fr.A to Fr.I;

[0015] Step (3): Component Fr.E was subjected to silica gel column chromatography using a dichloromethane-methanol system as an eluent to obtain ten fractions: Fr.E1 to Fr.E10; Fr.E6 was subjected to preparative HPLC chromatography (preparative high performance liquid chromatography) to obtain compound 11; Fr.E8 was subjected to preparative HPLC chromatography to obtain compound 10; Fr.E9 was subjected to preparative HPLC chromatography to obtain compounds 3, compound 4, and compound 5;

[0016] Step (4), component Fr.G was subjected to silica gel column chromatography with a dichloromethane-methanol system as an eluent to obtain twelve fractions: Fr.G1 to Fr.G12; Fr.G5 was subjected to Sephadex LH-20 gel column chromatography with a methanol-water system as an eluent to obtain six fractions: Fr.G5A to Fr.G5F; Fr.G5A was subjected to preparative HPLC chromatography to obtain compound 1 and compound 8; Fr.G10 was subjected to silica gel column chromatography with a dichloromethane-methanol system as an eluent to obtain eight fractions: Fr.G10A to G10H; Fr.G10B was subjected to preparative HPLC chromatography to obtain compound 6; Fr.G10G was subjected to preparative HPLC chromatography to obtain compound 2, compound 7, and compound 9.

[0017] In step (1), preferably, the dried Radix Lycii Bark medicinal material is subjected to reflux extraction with 75% ethanol.

[0018] Preferably, the mass volume ratio of the dried Radix Rehmanniae Root Bark and 75-90% ethanol is 1:20 kg / L.

[0019] The extraction was performed twice, and the time for each extraction was 2 hours.

[0020] In step (2), the petroleum ether-ethyl acetate system is petroleum ether:ethyl acetate = 10:1 → 0:1 V / V, and the dichloromethane-methanol system is dichloromethane-methanol = 15:1 → 5:1 V / V.

[0021] In step (3), the dichloromethane-methanol system is dichloromethane:methanol=150:1→10:1V / V.

[0022] When isolating compound 11, the preparative HPLC chromatographic conditions were: YMC-Pack ODS-A column (model AA12S05-2510WT), mobile phase of acetonitrile:water = 50:50 V / V, and a flow rate of 7-10 mL / min.

[0023] When isolating compound 10, the preparative HPLC chromatographic conditions were: YMC-Pack ODS-A column (model AA12S05-2510WT), mobile phase of acetonitrile:water = 30:70 v / v, and a flow rate of 7-10 mL / min.

[0024] When separating compound 3, compound 4, and compound 5, the preparative HPLC chromatographic conditions were: YMC-Pack ODS-A column (model AA12S05-2510WT), mobile phase acetonitrile:water = 30:70 v / v, and a flow rate of 7-10 mL / min.

[0025] In step (4), component Fr.G is subjected to silica gel column chromatography, and the dichloromethane-methanol system is dichloromethane:methanol = 100:1→20:1 V / V.

[0026] The methanol-water system is methanol:water=80:20V / V.

[0027] For the separation of compounds 1 and 8, the preparative HPLC conditions were as follows: a YMC-Pack ODS-A column (model AA12S05-2510WT), a mobile phase of acetonitrile:water = 30:70 v / v, and a flow rate of 7-10 mL / min.

[0028] When Fr.G10 was subjected to silica gel column chromatography, the dichloromethane-methanol system was dichloromethane:methanol = 100:0→10:1 V / V;

[0029] When isolating compound 6, the preparative HPLC chromatographic conditions were: YMC-Pack ODS-A column (model AA12S05-2510WT), mobile phase of acetonitrile:water = 30:70 V / V, and a flow rate of 7-10 mL / min.

[0030] When preparing compound 2, compound 7, and compound 9, the preparative HPLC chromatographic conditions were: YMC-Pack ODS-A column (model AA12S05-2510WT), a mobile phase of acetonitrile:water = 30:70 v / v, and a flow rate of 7-10 mL / min.

[0031] Another object of the present invention is to provide the use of the sesquiterpenoid compound in the preparation of a drug for treating neuroinflammation.

[0032] Another object of the present invention is to provide the use of the sesquiterpenoid compound in the preparation of a drug for treating neuroinflammation induced by diabetes.

[0033] The present invention also provides a pharmaceutical composition with anti-neuroinflammation effect, wherein the pharmaceutical composition contains the sesquiterpenoid compound as an effective ingredient.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] This study isolated novel sesquiterpenoid compounds from the bark of Lycium barbarum, a plant of the Solanaceae family. In vitro cell experiments demonstrated that these compounds exhibit anti-neuroinflammatory activity and hold promise for the development of anti-neuroinflammatory drugs. They can also serve as lead compounds, providing a strong basis for the discovery of potential new anti-neuroinflammatory drugs.

[0036] The separation method of the sesquiterpenoid compounds of the present invention is simple, reliable, efficient and highly operable. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The effects of compound 1-compound 11 on BV2 cell viability.

[0038] Figure 2 The effects of compound 1 to compound 11 (dosage concentration 10 μM) on the release of NO from BV2 cells stimulated by LPS; compared with the blank group, #### P<0.0001; compared with the model group, ** P < 0.01, **** P<0.0001.

[0039] Figure 3The effects of compound 5 at different concentrations (2.5 μM, 5 μM, 10 μM, 20 μM) on the release of NO from BV2 cells stimulated by LPS are shown in Figure 2. Compared with the blank group, ## P<0.01; compared with the model group, ** P<0.01.

[0040] Figure 4 The effect of compound 5 on the levels of TNF-α, IL-1β and IL-6 in BV2 cells induced by LPS; compared with the model group, ** P<0.01.

[0041] Figure 5 The effect of compound 5 on the expression levels of COX2 and iNOS genes in BV2 cells induced by LPS; compared with the blank group, #### P<0.0001; compared with the model group, * P < 0.01, **** P<0.0001.

[0042] Figure 6 The effect of compound 5 on the expression levels of COX2 and iNOS proteins in BV2 cells induced by LPS; compared with the blank group, #### P<0.0001; compared with the model group, * P < 0.01, ** P < 0.01, *** P < 0.001, **** P<0.0001. DETAILED DESCRIPTION

[0043] Example 1

[0044] Preparation of sesquiterpenoids

[0045] Sesquiterpenoids are isolated and purified from the bark of Lycium bark, including:

[0046] Step (1), the dried Chinese wolfberry bark medicinal material (50kg) is reflux extracted twice with 1000L 75% ethanol, each extraction is 2h, the extract is concentrated under reduced pressure to remove the solvent, and an extract (7.4kg) is obtained. The extract is adsorbed on an equal amount of diatomaceous earth, and after complete drying, it is eluted with petroleum ether, ethyl acetate and methanol in sequence, and the petroleum ether eluate, ethyl acetate eluate and methanol eluate are respectively collected, and they are respectively concentrated under reduced pressure to obtain a petroleum ether fraction, an ethyl acetate fraction and a methanol fraction;

[0047] The ethyl acetate fraction (775.7 g) from step (2) was subjected to silica gel column chromatography using a gradient elution ratio of petroleum ether:ethyl acetate (10:1, 4:1, 7:3, 1:1, 0:1, V / V) and dichloromethane:methanol (15:1, 10:1, 8:1, 5:1, V / V). Identical fractions were combined to obtain nine components, designated Fr.A, Fr.B, Fr.C, Fr.D, Fr.E, Fr.F, Fr.G, Fr.H, and Fr.I.

[0048] Step (3), Fr.E (20.4 g) was subjected to silica gel column chromatography, and gradient elution with dichloromethane-methanol (150:1, 100:1, 80:1, 50:1, 30:1, 20:1, 10:1, V / V) was used to obtain 10 fractions, which were respectively recorded as Fr.E1, Fr.E2, Fr.E3, Fr.E4, Fr.E5, Fr.E6, Fr.E7, Fr.E8, Fr.E9, and Fr.E10; Fr.E6 (3.5 g) was subjected to preparative HPLC chromatography (YMC-Pack ODS-A column, model AA12S05-2510WT, mobile phase: acetonitrile: water = 50:50 V / V, flow rate: 7 mL / min) to prepare compound 11 (7.4 mg); Fr.E8 (1.5 g) was subjected to preparative HPLC chromatography (YMC-Pack ODS-A column, model AA12S05-2510WT, mobile phase: acetonitrile: water = 30:70 V / V, flow rate: 7 mL / min) to prepare compound 10 (7.3 mg); Fr.E9 (6.6 g) was subjected to preparative HPLC chromatography (YMC-Pack ODS-A column, model AA12S05-2510WT, mobile phase: acetonitrile: water = 30:70 V / V, flow rate: 7 mL / min) to prepare compound 3 (5.7 mg), compound 5 (1.9 mg), and compound 4 (4.6 mg).

[0049] Step (4), Fr.G (17.3 g) was subjected to silica gel column chromatography and gradient eluted with dichloromethane-methanol (100:1, 80:1, 50:1, 30:1, 20:1, V / V) to obtain Fr.G1, Fr.G2, Fr.G3, Fr.G4, Fr.G5, Fr.G6, Fr.G7, Fr.G8, Fr.G9, Fr.G10, Fr.G11, and Fr.G12; Fr.G5 (1.5 g) was subjected to Sephadex chromatography. LH-20 gel column chromatography, with methanol-water (80:20 V / V) as eluent, separated and obtained 6 fractions, which were respectively named Fr.G5A, Fr.G5B, Fr.G5C, Fr.G5D, Fr.G5E, and Fr.G5F; Fr.G5A (178.2 mg) was subjected to preparative HPLC chromatography (YMC-Pack ODS-A column, model AA12S05-2510WT, mobile phase: acetonitrile: water = 30:70 V / V, flow rate: 7 mL / min), to prepare compound 1 (3.1 mg) and compound 8 (2.7 mg); Fr.G10 (2.5 g) was subjected to silica gel column chromatography, dichloromethane-methanol (100:0, 80:1, 50:1, 25:1, 10:1, V / V) gradient elution, separation to obtain 10 fractions, respectively recorded as Fr.G10A, Fr.G10B, Fr.G10C, Fr.G10D, Fr.G10E, Fr.G10F, Fr.G10G, Fr.G10H, Fr.G10B (117.8 mg) was subjected to preparative HPLC chromatography (YMC-Pack ODS-A column, model AA12S05-2510WT, mobile phase: acetonitrile: water = 30:70 V / V, flow rate: 7 mL / min) was used to prepare compound 6 (2.1 mg). Fr.G10G (846.5 mg) was subjected to preparative HPLC chromatography (YMC-Pack ODS-A column, model AA12S05-2510WT, mobile phase: acetonitrile: water = 30:70 V / V, flow rate: 7 mL / min) to prepare compound 7 (67.9 mg), compound 9 (13.7 mg) and compound 2 (2.4 mg).

[0050] Structural analysis of compounds

[0051] Compound 1

[0052]

[0053] Compound 1 is a brown powder. The UV methanol spectrum shows a maximum absorption wavelength of 214 nm. The IR spectrum indicates the presence of hydroxyl groups (3443 cm -1 ), carbonyl (1735cm -1 ) group, HRESI(+)MS showed 267.1593[M+H]+ (The calculated value is C 15 H 23 O4, 267.1591), combined 13 C NMR (Table 3) showed that the molecular formula is C 15 H 22 O4, molecular weight is 266, and unsaturation is 5. 1 H NMR showed four methyl proton signals δ H 1.25 (3H, s, H-12), δ H 1.23 (3H, s, H-13), δ H 1.91 (3H, s, H-14), δ H 1.64 (3H, s, H-15), three pairs of methylene proton signals δ H 2.06 (1H, dd, J=17.7, 6.2Hz, H-1α), δ H 2.25 (1H, m, H-1β), δ H 1.76 (1H, dd, J=13.0, 6.2Hz, H-2α), δ H 1.46 (1H, m, H-2β), δ H 2.18(1H, d, J=16.8Hz, H-4α), δ H 1.94 (1H, d, J=16.8Hz, H-4β); 13 C NMR has 5 carbon atoms in the low field signal, namely C-5 (125.8), C-6 (131.1), C-8 (159.3), C-9 (119.7), and C-10 (172.2), indicating that there are two double bonds and one carbonyl group in compound 1, and it shows that there are 15 carbon atoms. 1 H- 1 The presence of H-8 / H-9 in the H COSY spectrum indicates the presence of an unsaturated △8,9 double bond. In HMBC, δ H 2.06 (1H, dd, J = 17.7, 6.2 Hz, H-1α) and C-3 (74.1), C-5 (125.8) have long-range correlation; δ H 1.76 (1H, dd, J = 13.0, 6.2 Hz, H-2α) and C-4 (34.6), C-6 (131.1) have long-range correlation; δ H 2.18 (1H, d, J = 16.8 Hz, H-4α) and C-2 (26.7), C-6 (131.1) have long-range correlations, indicating the existence of a six-membered ring with a △5,6 double bond, δ H 1.25 (3H, s, H-12), δ H1.23 (3H, s, H-13) and C-3 (74.1), C-11 (74.9) have long-range correlations, indicating that an isopropyl group is connected to the C-3 position. H The presence of long-range correlations between 1.91 (3H, s, H-14) and C-1 (30.3) and C-5 (125.8) indicates that the methyl group at position 14 is connected to C-6, δ H 7.61 (1H, d, J = 5.6 Hz, H-8) and C-10 (172.2); δ H 6.02 (1H, d, J = 5.6 Hz, H-9) and C-7 (90.8) have long-range correlations, indicating the presence of α, β-pentaunsaturated lactone, δ H The presence of long-range correlations at 1.64 (3H, s, H-15) and C-7 (90.8), C-5 (125.8), and C-8 (159.3) indicates that the methyl group at position 15 is directly connected to C-7, the α, β-five-membered unsaturated lactone is connected to the 5-position of the six-membered ring, and the last two hydroxyl groups are adjacent to C-3 and 11. Combined with HSQC, the planar structure of compound 1 was determined. By calculating the carbon spectrum and processing the data, two correlation lines were obtained, and the DP4 of compound 1 was 0.04. + The value is 100%, DP4 of (3R,7S)-10 + was 0, and combined with the calculation of ECD, the absolute configuration of compound 1 was finally determined to be 3S, 7S, and it was named Lyciiterpenoid E.

[0054] Compound 2

[0055]

[0056] Compound 2 is a white powder. The UV methanol spectrum shows that the maximum absorption wavelengths are 216 and 283 nm. The IR spectrum indicates the presence of hydroxyl groups (3244 cm -1 ) and fragrance (1588, 1460cm -1 ) group, HRESI(+)MS showed 235.1689[M+H] + (The calculated value is C 15 H 23 O2, 235.1693), combined 13 C NMR (Table 3) showed that the molecular formula is C 15 H 22 O3, molecular weight is 234, unsaturation degree is 5. NMR data show that it is similar to Lycifuranone A, but 13 C NMR shows one less carbonyl carbon than Lycifuranone A. 1There are two more methylene hydrogens connected to O in H NMR. H 3.21 (1H, m, H-1β), δ H 3.45 (1H, m, H-1α), H-1β / H-2β in COSY spectrum, δ in HMBC H This was confirmed by the presence of long-range correlations at 1.69 (1H, m, H-2β) and C-1 (63.0). The NOESY spectra of H-3β / H-5β, H-3β / H-1β, and H-1β / H-2β confirmed the relative configuration of compound 2. The presence of a positive Cotton effect at 210 nm in the circular dichroism spectrum indicated that the calculated and experimental values ​​for 3S-2 were consistent, confirming the absolute configuration of compound 2 and naming it Lyciiterpenoid F.

[0057] Compound 3

[0058]

[0059] Compound 3 is a colorless crystal (methanol). The UV methanol spectrum shows that the maximum absorption wavelengths are 209 and 215 nm. The IR spectrum indicates the presence of hydroxyl groups (3373, 3301, 3280 cm -1 ) and fragrance (1602, 1581cm -1 ) group, HRESI(-)MS showed 249.1503[MH] - (The calculated value is C 15 H 21 O3, 249.1496), combined 13 C NMR (Table 3) showed that the molecular formula is C 15 H 22 O3, molecular weight is 250, unsaturation degree is 5. By analyzing NMR data, it is found to be similar to the compound Lyciiterpenoid C. 1 The characteristic signals provided by the HNMR (600MHz, CDCl3) spectrum revealed that the compound Lyciiterpenoid G had an additional O-linked methine signal δ compared to Lyciiterpenoid C. H 5.09 (1H, m, H-1) but lacks a pair of methylene proton signals. In the 1H-1H COSY spectrum, H-1 and H-2 are correlated, and H-7 and H-8 are correlated. At the same time, in the HMBC spectrum, δ HLong-range correlations at 5.09 (1H, m, H-1) and C-2 (33.9) and C-3 (75.7) confirmed that Lyciiterpenoid G possesses an additional hydroxyl group at the C-1 position compared to Lyciiterpenoid C. The NOESY spectrum, showing H-2β / H-1 and H-2β / H3-13, confirmed the relative configuration of the compound. X-ray single crystal diffraction analysis confirmed its absolute configuration as 1R, 3R, and the compound was named Lyciiterpenoid G.

[0060] Compound 4

[0061]

[0062] Compound 4 is a white powder. The UV methanol spectrum shows that the maximum absorption wavelengths are 205, 270, and 277 nm. The IR spectrum indicates the presence of hydroxyl groups (3349 cm -1 ) and fragrance (1622, 1579cm -1 ) group, HRESI(-)MS showed 263.1638[MH] - (The calculated value is C 16 H 23 O3, 263.1653), combined 13 C NMR (Table 3) showed that the molecular formula is C 16 H 24 O3, molecular weight is 264, unsaturation degree is 5. The NMR data are similar to those of compound 3, except that the substituent at position 1 is methoxy. 1 The characteristic signals provided by the H NMR (600MHz, CDCl3) spectrum show that there is a methoxyl signal δ in the high field region. H 3.53 (3H, s, H-16), 13 C NMR (150MHz, CDCl3) showed that the compound had 16 carbon signals. At the same time, in the HMBC spectrum, δ H The presence of long-range correlations at 3.53 (3H, s, H-16) and C-1 (75.7) suggested a planar structure for compound 4. The NOESY spectrum revealed H3-16 / 3-OH, H3-12 / H-2β, and H-2β / H-4β, confirming the relative configuration of the compound. The presence of a negative Cotton effect at 222 nm in the circular dichroism spectrum indicated agreement between the calculated and experimental values ​​for (1R,3R)-4, confirming the absolute configuration of compound 4, which was named Lyciiterpenoid H.

[0063] Compound 5

[0064]

[0065] Compound 5 is a yellow powder. The UV methanol spectrum shows that the maximum absorption wavelengths are 208, 269, and 277 nm. The IR spectrum indicates the presence of hydroxyl groups (3404 cm -1 ) and aroma (1565, 1510cm- 1 ) group, HRESI(+)MS showed 265.1784[M+H] + (The calculated value is C 16 H 25 O3, 265.1798), combined 13 C NMR (Table 3) showed that the molecular formula is C 16 H 24 O3, with a molecular weight of 264 and a degree of unsaturation of 5. Comparison with the NMR spectrum of compound 4 revealed substantial agreement, but the NOESY spectrum revealed H3-16 / H-2β, H3-13, and H-4β, confirming the relative configuration of the compound. The circular dichroism spectrum showed a positive Cotton effect at 208 nm, indicating agreement between the calculated and experimental values ​​for (1S,3R)-5. This confirmed the absolute configuration of compound 5, which is a diastereomer with compound 4 and was named Lyciiterpenoid I.

[0066] Compound 6

[0067]

[0068] Compound 6 is a brown powder. The UV methanol spectrum shows that the maximum absorption wavelengths are 217 and 266 nm. The IR spectrum indicates the presence of hydroxyl groups (3415 cm -1 ) and fragrance (1592, 1541cm -1 ) group, HRESI(-)MS showed 263.1663[MH] - (The calculated value is C 16 H 23 O3, 263.1653), combined 13 C NMR (Table 3) showed that the molecular formula is C 16 H 24 O3, molecular weight is 264, unsaturation degree is 5. 1 H NMR and 13 C NMR comparison showed that the results were basically the same, except that there was no methyl substitution at C-9 and a methyl substitution at C-7. 1 H- 1 There is a correlation between H-8 and H-9 in the H COSY spectrum; at the same time, in the HMBC spectrum, δ H 7.29 (1H, d, J = 7.9 Hz, H-9) and C-1 (76.5) have long-range correlation; δ H2.27 (3H, s, H-14) and C-6 (132.2) have long-range correlation. The planar structure of compound 6 was determined by HSQC, but due to 1 There is no 3-OH signal in the H NMR spectrum, so the relative configuration of compound 6 cannot be determined using the NOESY spectrum. By calculating the carbon spectrum and processing the data, two related straight lines are obtained: DP4 when 1-OCH3 and 3-OH are on opposite sides. + The value is 100%, DP4 on the same side + The absolute configuration of compound 6 was finally determined to be 1S, 3R by combining the calculation of ECD, and it was named Lyciiterpenoid J.

[0069] Compound 7

[0070]

[0071] Compound 7 is a colorless oil. The UV methanol spectrum shows that the maximum absorption wavelengths are 229 and 264 nm. The IR spectrum indicates the presence of hydroxyl groups (3384, 3369 cm -1 ) and fragrance (1663, 1596cm -1 ) group, HRESI(-)MS showed 249.1489[MH] - (The calculated value is C 15 H 21 O3, 249.1496), combined 13 C NMR (Table 3) showed that the molecular formula is C 15 H 22 O3, molecular weight is 250, unsaturation degree is 5. 1 H NMR and 13 C NMR comparison showed that they were basically consistent, except that there was no methoxy substitution at the C-1 position and the methyl substitution at the C-9 position was changed to hydroxymethyl. 1 The H NMR (600 MHz, CDCl3) spectrum showed a methylene signal connected to O, δ H 4.67 (2H, s, H-15), three pairs of methylene signals δ H 1.69 (1H, m, H-1), δ H 2.05 (1H, m, H-1), δ H 2.91 (2H, m, H-2), δ H 2.72 (1H, m, H-1), three methyl signals δ H 1.31 (3H, s, H-12), δ H 1.34 (3H, s, H-13), δ H2.22 (3H, s, H-14); δ in COSY spectrum H 1.69 (1H, m, H-1), δ H 2.05 (1H, m, H-1) and δ H 2.91 (2H, m, H-2) is correlated; this is also confirmed by the HMBC spectrum, δ H 4.62 (2H, s, H-15) and C-8 (125.3), C-10 (134.0) have long-range correlations, δ H 2.07 (1H, m, H-1) and C-10 (134.0) showed long-range correlation. Combined with HSQC, the planar structure of compound 7 was determined, but due to 1 There was no 3-OH signal in the H NMR spectrum, so the NOESY spectrum could not determine the relative configuration of compound 7. The absolute configuration of compound 7 was finally determined to be 3R by ECD calculation, and it was named Lyciiterpenoids K.

[0072] Compound 8

[0073]

[0074] Compound 8 is a colorless oil. The UV methanol spectrum shows that the maximum absorption wavelengths are 205 and 244 nm. The IR spectrum indicates the presence of hydroxyl groups (3412 cm -1 ) and carbonyl (1661cm -1 ) group, HRESI(-)MS showed 233.1549[MH] - (The calculated value is C 15 H 21 O2, 233.1547), combined 13 C NMR (Table 3) showed that the molecular formula is C 15 H 22 O2, molecular weight 234, unsaturation 5. NMR data are similar to the spectrum of Dehydrocarissone, except that there is no methyl substitution at C-4 position, but there is a methyl substitution at C-1 position. The δ H 2.39 (1H, m, H-6) and δ H 1.50 (1H, m, H-7) is correlated. H 2.01 (3H, brs) and C-10 (43.0) have long-range correlation; δ HThis was confirmed by the presence of long-range correlations at 6.11 (1H, brs, H-4) and C-6 (33.9). The NOESY spectrum showed H-9β / H3-15, H3-13, H3-13 / H-8β, and H-7α / H-6α. The circular dichroism spectrum showed a negative Cotton effect at 260 nm, indicating agreement between the calculated and experimental values ​​for (10S,7R)-8. The absolute configuration of compound 8 was confirmed and named Lyciiterpenoid L.

[0075] Compound 9

[0076]

[0077] Compound 9 is a white powder. The UV methanol spectrum shows a maximum absorption wavelength of 234 nm. The IR spectrum indicates the presence of hydroxyl groups (3449, 3371 cm -1 ) and carbonyl (1647cm -1 ) group, HRESI(-)MS showed 251.1665[MH] - (The calculated value is C 15 H 23 O3, 251.1653), combined 13 C NMR (Table 3) showed that the molecular formula is C 15 H 22 O3, molecular weight 252, unsaturation 4. Comparison of NMR revealed that compound 9 and carissone are basically identical, with the difference being that the △4,5 double bond in carissone is changed to △1,2 in compound 9; the methyl substitution at the C-4 position in carissone is changed to a methyl substitution at the C-1 position in compound 9; the methyl substitution at the C-10 position in carissone is changed to a methyl substitution at the 1-OH position in compound 9; and compound 9 has an additional methyl substitution at the C-15 position. H 1.94 (1H, m, H-9) and δ H 1.40 (1H, m, H-8) is correlated; δ H 5.82 (1H, s, H-2) and C-10 (73.6) have long-range correlation; δ H There is a long-range correlation between 1.98 (3H, brs, H-14) and C-10 (73.6); δ H 1.94 (1H, m, H-9) and C-1 (161.9) have long-range correlation; δ H1.02 (3H, s, H-15) and C-4 (48.1), C-5 (39.5), C-6 (33.4) have long-range correlations that confirm these differences. In the NOESY spectrum, H-9α / H3-15, H3-13, the relative configurations of C-5 and C-7 can be determined. Since the hydroxyl group at the C-10 position has no signal in NMR, two correlation lines are obtained by calculating the carbon spectrum and processing the data. When C-10 is on the same side as C-5 and 7, DP4 + 0%, while C-10 and C-5, 7 are on the opposite side DP4 + The relative configuration of compound 9 was determined to be 100%. The absolute configuration of compound 9 was determined to be 5S, 7S, 10S by ECD calculation and named Lyciterpenoid M.

[0078] Compound 10

[0079]

[0080] Compound 10 is a yellow oil. The UV methanol spectrum shows that the maximum absorption wavelengths are 216, 271, and 279 nm. The IR spectrum indicates the presence of hydroxyl groups (3380 cm -1 ), carbonyl (1705cm -1 ) and fragrance (1600, 1575cm -1 ) group, HRESI(-)MS showed 249.1503[MH] - (The calculated value is C 15 H 21 O3, 249.1496), combined 13 C NMR (Table 3) showed that the molecular formula is C 15 H 22 O3, with a molecular weight of 250 and a degree of unsaturation of 5. NMR data showed a similar structure to Lyciterpenoid D, with the difference being that compound 10 has a hydroxymethyl substitution at C-1, no methyl substitution at C-5, a methyl substitution at C-3, and a carbonyl carbon at C-9. 13 In C NMR, the low field shows that C-9 (213.7) is the carbonyl carbon. Combined with the HMBC δ H There is a long-range correlation between 1.34 (6H, s, H-11, 12) and C-9 (213.7); δ H There is a long-range correlation between 4.77 (2H, s, H-13) and C-2 (135.5); δ H The presence of long-range correlations at 2.40 (3H, s, H-15) and C-2 (135.5) and C-4 (128.3) confirmed the structure of compound 10, which was named Lyciiterpenoid N.

[0081] Compound 11

[0082]

[0083] Compound 11 was a brown solid, as determined by HRESI(-)MS (291.1599 [MH] - ) and NMR data confirmed that its molecular formula is C 17 H 24 O4. Compound 11 1 H NMR (Table 2) and 13 The C NMR spectrum (Table 3) was similar to that of compound 10, with the only difference being the presence of an acetyl group at C-1 in compound 11, rather than the hydroxymethyl group at C-1 in compound 10. This was further confirmed by HMBC correlations of H3-14 with C-1, H2-13 with C-2 and C-16, and H3-17 with C-16. Compound 11 was named Lyciterpenoid O.

[0084] Table 1. Proton spectrum (CDCl3 600MHz) data of compounds 1-5

[0085]

[0086] Table 2. Proton spectrum (CDCl3600 MHz) data of compounds 6-11

[0087]

[0088] Table 3. Carbon spectrum (CDCl3 150 MHz) data of compounds 1-11

[0089]

[0090] Example 2

[0091] Graph Pad Prism 7 software was used for statistical analysis and graphing of data, with mean ± standard deviation (SEM) presented. One-way ANOVA was used to compare differences between multiple groups, and Tukey's variance test was used for univariate analysis of differences between two groups. P < 0.05 indicated statistically significant differences between groups.

[0092] Study on the anti-neuritis effect and mechanism of the compound in vitro

[0093] 1. Cell culture

[0094] BV2 cells (mouse microglial cells) were inoculated in DMEM medium containing 10% fetal bovine serum and cultured in a 5% CO2 cell culture incubator at 37°C. The medium was changed in time according to the cell adhesion.

[0095] 2. CCK-8 assay to detect the effects of compounds 1-11 on BV2 cell viability

[0096] BV2 cells were seeded in 96-well plates, with approximately 2 × 10 BV2 cells in each well. 4 The cells were divided into a blank group (Control group) and a drug-treated group. The culture medium of the blank group contained 1‰ DMSO, and the culture medium of the drug-treated group contained a culture medium containing the sesquiterpene compound to be tested (the sesquiterpene compound was dissolved in DMSO to prepare a stock solution with a concentration of 30 mM, which was then diluted to 30 μM with culture medium). The cells were cultured in an incubator with 5% CO2 and 37°C for 24 h. The supernatant in the 96-well plate was removed, and 100 μL of culture medium containing 10 μL of CCK-8 was added to each well. The cells were incubated at 5% CO2 and 37°C for 2 h. The absorbance of each well was measured at 490 nm using a microplate reader, and the cell viability of the compound was calculated based on the absorbance.

[0097] The results are as follows Figure 1 As shown, at a dosage concentration of 30 μM, compounds 1 to 11 had no obvious toxicity to BV2 cells.

[0098] 3. Griess method for measuring the effects of compounds 1-11 on the NO content in LPS-induced BV2 cells

[0099] A blank group (Control group), a model group (LPS 1 μg / mL), an LPS (1 μg / mL) + compound group, and an LPS (1 μg / mL) + control group were set up. The sesquiterpenoid compounds and curcumin to be tested were prepared in DMSO to a 30 mM stock solution and then diluted to 10 μM in culture medium.

[0100] BV2 cells were grown at 3 × 10 4Cells were seeded at a density of 100 μM in 96-well plates and cultured overnight. The following procedures were performed for each group: the LPS+compound group was cultured with medium containing the test sesquiterpenoid compound (10 μM) for 2 h, then replaced with medium containing LPS (1 μg / mL) for stimulation and continued to be cultured for 24 h; the LPS+control group was cultured with medium containing curcumin (10 μM) for 2 h, then replaced with medium containing LPS (1 μg / mL) for 24 h; the model group was cultured with fresh medium for 2 h, then replaced with medium containing LPS (1 μg / mL) for 24 h; and the control group was cultured with fresh medium for a total of 26 h. Cell supernatants were then collected, and NO levels were determined using the Griess method. OD values ​​were measured at 540 nm using a microplate reader.

[0101] like Figure 2 As shown, compounds 1-11 were able to inhibit the abnormal release of NO induced by LPS at a dosage concentration of 10 μM, among which compound 5 showed the best activity.

[0102] According to the above experiment, the effects of different concentrations (2.5 μM, 5 μM, 10 μM, 20 μM) of compound 5 on the release of NO from LPS-stimulated BV2 cells were investigated. Figure 3 Compound 5 inhibited the abnormal release of NO in BV2 cells in a dose-dependent manner, and its effect at 10 μM was comparable to that of curcumin (dosage concentration 20 μM).

[0103] 4. ELISA kit to measure anti-inflammatory factors

[0104] A blank group (Control group), a model group (1 μg / mL LPS), an LPS+Compound 5 group (1 μg / mL LPS, 2.5, 5, and 10 μM Compound 5), and an LPS+Control group (1 μg / mL LPS, 20 μM Curcumin) were set up. The sesquiterpenoid compounds and curcumin to be tested were prepared into a 10 mM stock solution with DMSO and then diluted to the corresponding concentration with culture medium.

[0105] BV2 cells were grown at 5 × 10 4The cells were seeded in a 96-well plate at a density of 100 cells / mL. After overnight culture, the LPS+compound 5 group was replaced with a culture medium containing the sesquiterpene compound to be tested. After 2 h of culture, it was replaced with a culture medium containing LPS (1 μg / mL) for stimulation and continued to be cultured for 24 h. The LPS+control group was replaced with a culture medium containing curcumin (20 μM). After 2 h of culture, it was replaced with a culture medium containing LPS (1 μg / mL) and continued to be cultured for 24 h. The model group was replaced with fresh culture medium. After 2 h of culture, it was replaced with a culture medium containing LPS (1 μg / mL) and continued to be cultured for 24 h. The control group was replaced with fresh culture medium for a total of 26 h. The cell supernatant was collected, divided into 120 μL / tube, and stored in a -20°C refrigerator. Inflammatory factors were determined using an ELISA kit (Hunan Aifang Biotechnology Co., Ltd.).

[0106] Add the standard and sample to the bottom of the ELISA plate wells according to the grouping. Seal the plate with a sealing film and incubate at 37°C for 30 minutes. Remove the sealing film, discard the liquid, fill each well with wash solution, let it stand for 30 seconds, then discard it, repeat five times, and let it stand to dry. Except for the blank group, add 50μL of ELISA reagent to each well, incubate at 37°C for 30 minutes, discard the liquid, fill each well with wash solution, let it stand for 30 seconds, then discard it, repeat five times, and let it stand to dry. First, add 50μL of color developer A to each well, then add 50μL of color developer B, mix gently, and develop at 37°C in the dark for 10 minutes (at which point the blue color immediately turns yellow). Then add stop solution to terminate the reaction. Use the blank group culture medium well to zero the reaction. Within 15 minutes after adding the stop solution, measure the absorbance (OD value) of each well at 450nm. Use the concentration and OD value of the standard to calculate the standard curve, substitute the OD value of the sample into the standard curve, calculate the sample concentration, and then multiply it by the dilution factor to get the actual concentration of the sample.

[0107] The results are as follows Figure 4 As shown in the results, compared with the Control group, LPS significantly stimulated the release of TNF-, IL-1β and IL-6 in BV2 cells, while compound 5 (2.5, 5, 10 μM) could inhibit the release of TNF-, IL-1β and IL-6 in a dose-dependent manner.

[0108] 5. Real-time fluorescence quantitative PCR experiment

[0109] Real-time fluorescence quantitative PCR was used to analyze the effect of compound 5 on the expression of inflammatory factor genes in LPS-induced BV2 cells.

[0110] BV2 cells were grown at 3 × 10 5Cells were seeded at a density of 100 cells / well in a 24-well plate and grouped as in the "ELISA kit for anti-inflammatory cytokine assay," differing only in that the cells were cultured for an additional 6 hours after the addition of LPS. Total RNA was first extracted, and a sample premix was prepared according to Table 4. Reverse transcription was then performed using a thermocycler with the program set to "25°C, 5 min → 42°C, 30 min → 85°C, 5 min." The resulting reverse transcription sample was stored at -20°C or -80°C. Real-time fluorescence quantitative PCR was performed using primer designs shown in Table 5 and the PCR reaction system shown in Table 6.

[0111] The total RNA extraction process is as follows:

[0112] (1) After the cells were administered and cultured for 6 hours, the culture medium was discarded, and the 24-well plate was rinsed with pre-cooled PBS. 1 mL of Trizol was added, and the plate was lysed on ice for 5 minutes. The plate was inverted and allowed to stand for 5 minutes. 200 μL of chloroform was added, the plate was tightly covered and vortexed for 15 seconds. The plate was allowed to stand at room temperature for 2-3 minutes, and then allowed to stand on ice for 15 minutes. The plate was centrifuged at 12,000 rpm for 20 minutes at 4°C, and 350 μL of the supernatant was transferred to a new enzyme-free EP tube.

[0113] (2) Add an equal volume of isopropanol, gently invert the tube 10 times, and place on ice for 10 minutes. Centrifuge at 12,000 rpm at 4°C for 20 minutes, discard the supernatant, and the precipitate at the bottom of the tube is RNA.

[0114] (3) Slowly add 500 μL of 75% ethanol (prepared with DEPC water and stored at 4 degrees) along the wall, gently tap the EP tube, centrifuge at 12,000 rpm at 4°C for 5 minutes, repeat once, slowly discard the ethanol, dry at room temperature for 10 minutes until translucent, add 20 μL of DEPC water to dissolve, and use Nanodrop to detect RNA purity.

[0115] Table 4. Reverse transcription reaction system

[0116]

[0117] Table 5. QRT-PCR primer sequences

[0118]

[0119] Table 6. PCR amplification reaction system

[0120]

[0121] Depend on Figure 5As shown, compared to the control group, 6 hours after LPS treatment of BV2 cells, COX2 and iNOS gene expression levels were significantly increased. However, when compound 5 (2.5, 5, and 10 μM) was administered 2 hours beforehand, the expression levels of these genes decreased in a concentration-dependent manner compared to the model group. Furthermore, compared to curcumin, compound 5 exhibited a stronger inhibitory effect on COX2 and iNOS gene expression at a lower concentration.

[0122] 6. Western blot experiment

[0123] BV2 cells were cultured at 1 × 10 6 The cells were seeded in 6-well plates at a density of 100 cells / mL and grouped as in the "ELISA kit for anti-inflammatory factor assay". After 24 h of treatment according to the groups, the culture medium was discarded, the 6-well plates were rinsed with pre-cooled PBS, 120 μL RIPA was added on ice and lysed on ice for 30 min, and then the cells of each group were collected by cell scraping. The cells were centrifuged at 12,000 rpm at 4°C for 20 min, and part of the supernatant was taken to determine the protein concentration by the BCA method. An appropriate volume of PBS and protein loading buffer was added to the remaining supernatant to adjust the protein concentration of each group of samples to be consistent. The samples were heated in a 100°C metal bath for 10 min to denature the proteins, and the proteins were stored at -20°C.

[0124] After 90 minutes of electrophoresis at room temperature, transfer the proteins to a PVDF membrane by electrophoresis in an ice-water bath for 60 minutes. Block the membrane for 90 minutes at room temperature and wash three times with TBST. Incubate with the primary antibody overnight at 4°C and wash three times with TBST. Incubate with the secondary antibody for 1 hour at room temperature and wash three times with TBST. Finally, cover the entire surface of the PVDF membrane with ECL working solution and react at room temperature for 1 minute. The membrane was then placed in an automated chemiluminescence imaging system to obtain final band data.

[0125] The results are as follows Figure 6 As shown, under LPS stimulation, the expression levels of COX2 and iNOS proteins in BV2 cells were significantly increased compared with the blank group, while compound 5, when treated 2 h in advance, downregulated the expression levels of COX2 and iNOS proteins in a dose-dependent manner.

[0126] In summary, the sesquiterpenoid compounds of the present invention have good application prospects in the preparation of drugs for treating neuroinflammation, and provide a reference for clinical discovery of potential anti-neuroinflammation lead compounds from Lycium bark.

Claims

1. Sesquiterpenoid compounds with the following structure:

2. Sesquiterpenoid compounds with the following structure:

3. The method for preparing the sesquiterpenoid compound according to claim 1, wherein: include: Step (1), the dried Chinese wolfberry bark is subjected to reflux extraction with 75-90% ethanol, the extract is concentrated under reduced pressure to remove the solvent, and an extract is obtained. The extract is adsorbed on equal amounts of diatomaceous earth, and eluted with petroleum ether, ethyl acetate and methanol in sequence, and the petroleum ether eluate, ethyl acetate eluate and methanol eluate are respectively collected and concentrated under reduced pressure to obtain a petroleum ether fraction, an ethyl acetate fraction and a methanol fraction; Step (2), the ethyl acetate fraction was subjected to silica gel column chromatography using a petroleum ether-ethyl acetate system and a dichloromethane-methanol system as eluents, and the same fractions were combined to obtain nine components: Fr.A to Fr.I; Step (3): Component Fr.E was subjected to silica gel column chromatography using a dichloromethane-methanol system as an eluent to obtain ten fractions: Fr.E1 to Fr.E10; Fr.E6 was subjected to preparative HPLC chromatography to obtain compound 11; Fr.E8 was subjected to preparative HPLC chromatography to obtain compound 10; Fr.E9 was subjected to preparative HPLC chromatography to obtain compounds 3, 4, and 5; Step (4), component Fr.G was subjected to silica gel column chromatography with a dichloromethane-methanol system as an eluent to obtain twelve fractions: Fr.G1 to Fr.G12; Fr.G5 was subjected to Sephadex LH-20 gel column chromatography with a methanol-water system as an eluent to obtain six fractions: Fr.G5A to Fr.G5F; Fr.G5A was subjected to preparative HPLC chromatography to obtain compound 1 and compound 8; Fr.G10 was subjected to silica gel column chromatography with a dichloromethane-methanol system as an eluent to obtain eight fractions: Fr.G10A to G10H; Fr.G10B was subjected to preparative HPLC chromatography to obtain compound 6; Fr.G10G was subjected to preparative HPLC chromatography to obtain compound 2, compound 7, and compound 9.

4. The method for preparing a sesquiterpenoid compound according to claim 3, wherein: In step (1), the mass volume ratio of the dried Radix Lycii and 75-90% ethanol is 1:10-1:20 kg / L or g / mL.

5. The method for preparing a sesquiterpenoid compound according to claim 3, wherein: In step (2), the petroleum ether-ethyl acetate system is petroleum ether:ethyl acetate = 10:1 → 0:1 V / V, and the dichloromethane-methanol system is dichloromethane-methanol = 15:1 → 5:1 V / V.

6. The method for preparing a sesquiterpenoid compound according to claim 3, wherein: In step (3), the dichloromethane-methanol system is dichloromethane:methanol=150:1→10:1V / V; When isolating compound 11, the preparative HPLC chromatography conditions were as follows: chromatographic column, mobile phase acetonitrile:water = 50:50 V / V, flow rate 7-10 mL / min; When isolating compound 10, the preparative HPLC chromatography conditions were as follows: chromatographic column, mobile phase acetonitrile:water = 30:70 V / V, flow rate 7-10 mL / min; When separating compound 3, compound 4, and compound 5, the preparative HPLC chromatographic conditions were as follows: chromatographic column, mobile phase of acetonitrile:water = 30:70 V / V, and a flow rate of 7-10 mL / min.

7. The method for preparing a sesquiterpenoid compound according to claim 3, wherein: In step (4), component Fr.G is subjected to silica gel column chromatography, and the dichloromethane-methanol system is dichloromethane:methanol = 100:1→20:1 V / V; The methanol-water system is methanol:water=80:20V / V; When separating compounds 1 and 8, the preparative HPLC chromatographic conditions were as follows: chromatographic column, mobile phase acetonitrile:water = 30:70 V / V, flow rate 7-10 mL / min; When Fr.G10 was subjected to silica gel column chromatography, the dichloromethane-methanol system was dichloromethane:methanol = 100:0 → 10:1 V / V; When isolating compound 6, the preparative HPLC chromatography conditions were as follows: chromatographic column, mobile phase acetonitrile:water = 30:70 V / V, flow rate 7-10 mL / min; When preparing compound 2, compound 7, and compound 9, the preparative HPLC chromatography conditions were as follows: a chromatographic column, a mobile phase of acetonitrile:water = 30:70 V / V, and a flow rate of 7-10 mL / min.

8. Use of the sesquiterpenoid compound according to claim 1 or 2 in the preparation of a medicament for treating neuroinflammation.

9. Use of the sesquiterpenoid compound according to claim 1 or 2 in the preparation of a medicament for treating neuroinflammation induced by diabetes.

10. A pharmaceutical composition having anti-neuroinflammatory properties, characterized in that: The pharmaceutical composition comprises the sesquiterpenoid compound according to claim 1 or 2 and a pharmaceutically acceptable carrier or excipient.

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