A sesquiterpene compound extracted and separated from radix comphoricus and a preparation method and application thereof

Abstract

The present application relates to the field of extraction and separation of traditional Chinese medicine, in particular to a sesquiterpene compound extracted and separated from radix comphoricus laxifolii, a preparation method and application thereof; the compound has a molecular formula of C 15 H 18 O2, and a chemical name of 8-hydroxyl-1,5,8-trimethyl-6,7,8,9-tetrahydrobenzo[e][1]benzofuran; the present application provides an extraction, separation and purification method for the compound, and the compound is successfully obtained by adopting alcohol extraction, silica gel column chromatography, macroporous adsorption resin and other methods for separation and purification; the compound has a significant protective effect on a PC12 cell hypoxia model induced by CoCl2 at a concentration of 20 muM, and is dose-dependent, indicating that the compound has good anti-hypoxia activity, and is expected to become a drug for treating acute high altitude disease and ischemia-hypoxia related diseases.

Description

Technical Field

[0001] This invention relates to the field of extraction and separation of traditional Chinese medicine, and particularly to new compounds extracted, separated and identified from the medicinal herb Codonopsis pilosula and their preparation methods, specifically a sesquiterpene compound extracted and separated from Codonopsis pilosula, its preparation method and application. Background Technology

[0002] Codonopsis pilosula (Franch.) Nannf., Codonopsis pilosula Nannf. var. modesta (Nannf.) L. Shen, or Codonopsis tangshen Oliv. are all plants belonging to the Campanulaceae family. Codonopsis pilosula is sweet and neutral in nature, and enters the spleen and lung meridians. It has the effects of strengthening the spleen and lungs, nourishing blood and promoting body fluid production. Codonopsis pilosula is widely distributed in Shanxi, Northeast China, Gansu, and other regions of my country. Among them, the authentic medicinal material from Shanxi Province—Luzhou Codonopsis pilosula—is widely used due to its abundant resources, low price, and significant efficacy.

[0003] Codonopsis pilosula possesses various pharmacological activities, such as immunomodulation, gastrointestinal protection, and neuroprotection, effectively preventing and treating a variety of chronic diseases, and exhibiting significant medicinal and health-promoting value. The rich pharmacological activities of Codonopsis pilosula are inseparable from its diverse chemical components. Phytochemical studies have shown that Codonopsis pilosula contains polysaccharides, triterpenoids, alkynes, phenylpropanoids, alkaloids, and other chemical components. However, due to the complexity of the chemical composition of Codonopsis pilosula, with a large number of components having similar molecular weights and polarities, the isolation of new compounds is quite difficult. Furthermore, research on the pharmacological activities of Codonopsis pilosula has largely focused on extracts, with limited research on the pharmacological activities of individual chemical components. Therefore, in-depth exploration of the chemical components of Codonopsis pilosula to find the material basis corresponding to its traditional efficacy is of great significance.

[0004] Acute mountain sickness is an idiopathic disease occurring in hypoxic environments at high altitudes (above 2500m). It has a rapid onset (symptoms appear within 6-12 hours) and a high incidence (30%-90%). Studies have reported that compound preparations such as Shengnaokang Pills (composed of Codonopsis pilosula, Salvia miltiorrhiza, Panax notoginseng, etc.) and Compound Codonopsis pilosula Tablets (composed of Codonopsis pilosula, Salvia miltiorrhiza, Angelica sinensis, etc.) have anti-hypoxic effects and can be used to prevent high-altitude hypoxia reactions and treat acute mountain sickness. Other reports indicate that Codonopsis pilosula decoction, fermentation broth, and polysaccharides can all prolong the survival time of animals in hypoxic environments. Therefore, Codonopsis pilosula has medicinal potential in combating hypoxia and is expected to become a drug for treating acute mountain sickness and ischemia-hypoxia related diseases caused by hypoxia.

[0005] Therefore, this invention studies the chemical composition and anti-hypoxia activity of Codonopsis pilosula, a traditional Chinese medicine from Shanxi, in order to find compounds with better activity. It is hoped that this study can provide a reference for researchers studying this genus of plants, so as to develop new drugs that can be applied in clinical practice. Summary of the Invention

[0006] The present invention overcomes the shortcomings of the prior art, and the technical problem to be solved is to provide a sesquiterpene compound extracted and isolated from Codonopsis pilosula.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a sesquiterpene compound extracted and isolated from Codonopsis pilosula, with the molecular formula: C 15 H 18 O2, chemical name: 8-hydroxyl-1,5,8-trimethyl-6,7,8,9-tetrahydrobenzo[e][1]benzofuran, chemical structural formula:

[0008] This invention also provides a method for preparing sesquiterpene compounds extracted and isolated from Codonopsis pilosula, comprising the following steps:

[0009] (1) Take dried Codonopsis pilosula root, heat and reflux with ethanol to extract, combine the extracts and concentrate under reduced pressure to obtain concentrated extract;

[0010] (2) Disperse the extract in water, extract with an extractant, concentrate under reduced pressure to obtain the extractant-extracted fraction;

[0011] (3) The extractant fractions were separated by vacuum silica gel column chromatography. The solvent system initially used petroleum ether / ethyl acetate with a volume ratio of 15 / 1→10 / 1→5 / 1→3 / 1→1 / 1; then dichloromethane / methanol with a volume ratio of 50 / 1→30 / 1→20 / 1→10 / 1→5 / 1→3 / 1→1 / 1→0 / 1 for gradient elution. The eluent was concentrated and analyzed by silica gel TLC. Components with similar spots were combined to obtain five fractions, namely: Fr. A, Fr.B, Fr.C, Fr.D, Fr.E; where Fr.A is the sample obtained by elution, concentration and evaporation with a petroleum ether / ethyl acetate volume ratio of 15 / 1→10 / 1→5 / 1→3 / 1, Fr.B is the sample obtained by elution, concentration and evaporation with a petroleum ether / ethyl acetate volume ratio of 1 / 1 and a dichloromethane / methanol volume ratio of 50 / 1→30 / 1, and Fr.C is the sample obtained by elution, concentration and evaporation with a dichloromethane / methanol volume ratio of 20 / 1.

[0012] (4) Fr.A, Fr.B, and Fr.C were separated by D-101 macroporous resin column chromatography. The solvent system used ethanol / water gradient elution. After the eluent was concentrated, it was analyzed by high performance liquid chromatography (HPLC). The components with similar elution time periods were combined to obtain three fractions: Fr.a, Fr.b, and Fr.c. The elution time of Fr.a was 5–30 min, the elution time of Fr.b was 25–45 min, and the elution time of Fr.c was 40–60 min.

[0013] (5) The three fractions Fr.a, Fr.b, and Fr.c were separated by ODS reverse column chromatography. The solvent system was methanol / water. The elution was carried out by setting a gradient concentration according to the HPLC analysis results. The eluted components were detected by HPLC. The components with similar elution time periods were combined to obtain six fractions: Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, and Fr.6. Among them, the elution time of Fr.5 was 45-60 min.

[0014] (6) Fr.5 was separated by silica gel column chromatography. The solvent system first used petroleum ether / ethyl acetate with a volume ratio of 30 / 1→25 / 1→20 / 1→15 / 1→10 / 1→8 / 1→5 / 1→3 / 1→2 / 1→1 / 1; then dichloromethane / methanol was used for gradient elution with a volume ratio of 30 / 1→25 / 1→20 / 1→15 / 1→0 / 1. The eluent was concentrated and then detected by silica gel TLC. Components with similar spots were combined to obtain ten fractions, namely: Fr.5.1, Fr.5.2, Fr.5.3, Fr.5.4, Fr.5.5, Fr.5.6, Fr.5.7, Fr.5.8, Fr.5.9, and Fr.5.10. Among them, Fr.5.5 was the sample obtained by elution, concentration, and evaporation to dryness with a petroleum ether / ethyl acetate volume ratio of 8 / 1→5 / 1.

[0015] (7) Fr.5.5 was eluted isocratically through a semi-preparative column, and the eluent was concentrated and evaporated to dryness to obtain the compound. The retention time of the compound was in the range of 26.7-27.7 min.

[0016] Furthermore, in step (1), the ethanol concentration is 70%, the solid-liquid ratio is 1:3, and the reflux extraction is performed three times, each time for 1.5 hours. The three reflux extractions ensure more thorough extraction.

[0017] Furthermore, in step (2), the extractant is ethyl acetate, the material-to-liquid ratio is 1:1, and the extraction is performed 4 times.

[0018] Furthermore, in step (3), the silica gel for mixing is 100-200 mesh, and the amount of silica gel used for mixing is 1.5 times that of the extract; the silica gel for chromatography in silica gel column chromatography is 200-300 mesh, and the amount of chromatography silica gel used is 10 times that of the silica gel used for mixing; in gradient elution, each gradient is eluted for 3 column volumes, and each gradient is applied once; in TLC detection, the thin-layer plate is GF254, the developing solvent is petroleum ether / ethyl acetate volume ratio 3 / 1, and concentrated sulfuric acid ethanol solution is sprayed for color development.

[0019] Furthermore, in step (4), the amount of D-101 macroporous resin used is 15 times that of the sample; the eluent is 30%, 60%, and 95% ethanol in sequence; in gradient elution, each gradient is eluted for 3 column volumes, approximately 500 mL each time; the HPLC analysis conditions are: 10-100% methanol, 60 min, detection wavelength 210 nm.

[0020] Furthermore, in step (5), the amount of ODS used for mixing the sample is 1.5 times the amount of sample; the amount of ODS used in the chromatography column is 4 times the amount of ODS used for mixing the sample; the gradient concentration elution conditions are: Fr.a is 10%, 35%, 60%, 100%, Fr.b is 45%, 65%, 85%, 100%, Fr.c is 70%, 80%, 90%, 100%; in the gradient elution, each gradient elution has 3 retention volumes, approximately 500 mL each time.

[0021] Furthermore, in step (6), during gradient elution, each gradient elutes 3 column volumes, and each gradient is applied once.

[0022] Furthermore, in step (7), the semi-preparative column conditions are as follows: the mobile phase is 50% acetonitrile-water solution, the flow rate is 3.5 ml / min, the detection wavelength is 210 nm and 254 nm, the column type is YMC-Pack ODS-A, and the specifications are 250 × 10 mm and 5 μm.

[0023] In addition, this invention also provides the application of the sesquiterpene compounds extracted and isolated from Codonopsis pilosula in the preparation of drugs for the prevention and treatment of acute mountain sickness.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. This invention provides a novel sesquiterpene compound derived from Codonopsis pilosula.

[0026] 2. This invention provides a method for the extraction, separation and purification of the above-mentioned compounds. The method uses alcohol extraction, silica gel column chromatography, macroporous adsorption resin and other methods for separation and purification, and successfully obtains new compounds. The operation method is simple and fast, and the compounds separated by this method have high purity.

[0027] 3. This invention evaluates the anti-hypoxia activity of the above-mentioned compound at the cellular level. The compound, at a concentration of 20 μM, exhibits significant protective effects against CoCl2-induced PC12 cell hypoxia in a dose-dependent manner, indicating that it possesses good anti-hypoxia activity and holds promise as a drug for treating acute mountain sickness and ischemia-hypoxia-related diseases caused by hypoxia. Attached Figure Description

[0028] Figure 1 This is a flowchart illustrating the extraction, separation, and purification process of the compounds in this invention.

[0029] Figure 2 This is the HR-ESIMS spectrum of the compound of this invention.

[0030] Figure 3 This is the UV spectrum of the compound of the present invention.

[0031] Figure 4 The compounds of this invention 1 H-NMR spectrum (600MHz, CDCl3).

[0032] Figure 5 The compounds of this invention 13 C-NMR spectrum (150MHz, CDCl3).

[0033] Figure 6 The HSQC spectrum (600MHz, CDCl3) of the compound of this invention is shown.

[0034] Figure 7 The HMBC spectrum (600MHz, CDCl3) of the compound of this invention is shown.

[0035] Figure 8 The effect of different concentrations of compounds on the viability of PC12 cells.

[0036] Figure 9 To investigate the effect of different concentrations of CoCl2 on PC12 cell viability, a model group was compared with a normal group. * P<0.05, *** P<0.001.

[0037] Figure 10 To investigate the effects of different concentrations of compounds on the viability of CoCl2-induced hypoxic PC12 cells, a comparison was made between the model group and the normal group. *** P<0.001; Drug-treated group vs. model group, ## P<0.01 ### P<0.001.

[0038] Figure 11To investigate the effects of different concentrations of compounds on ROS levels in CoCl2-induced hypoxic PC12 cells, a comparison was made between the model group and the normal group. *** P<0.001; Drug-treated group vs. model group, ### P<0.001. Detailed Implementation

[0039] The present invention will be further described below with reference to specific embodiments.

[0040] Example 1

[0041] A method for preparing sesquiterpenoid compounds extracted and isolated from Codonopsis pilosula ( Figure 1 This includes the following steps:

[0042] (1) Take 90 kg of dried Codonopsis pilosula root, cut it into sections, add 3 times the volume of 70% ethanol, and heat under reflux to extract three times, 1.5 h each time. Combine the extracts and concentrate under reduced pressure at 50°C to obtain 10 kg of ethanol extract.

[0043] (2) The ethanol extract in paste form was dispersed in 50L of purified water and extracted four times with an equal volume of ethyl acetate to obtain an ethyl acetate extract. The ethyl acetate extract was concentrated under reduced pressure at 50°C to a paste form to obtain 504g of the ethyl acetate extract fraction.

[0044] (3) The ethyl acetate fraction was separated by vacuum silica gel column chromatography. The silica gel used for mixing was 100-200 mesh, and the amount of silica gel used was 1.5 times that of the extract. The silica gel used for chromatography was 200-300 mesh, and the amount of chromatography silica gel used was 10 times that of the silica gel used for mixing. The solvent system first used petroleum ether / ethyl acetate with a volume ratio of 15 / 1→10 / 1→5 / 1→3 / 1→1 / 1; then dichloromethane / methanol was used with a volume ratio of 50 / 1→30 / 1→20 / 1→10 / 1→5 / 1→3 / 1→1 / 1→0 / 1 for gradient elution. Each gradient consisted of 3 column volumes, and each gradient was repeated once. The eluent was concentrated and evaporated to dryness under reduced pressure at 50°C. An appropriate amount of sample was dissolved in methanol and detected by silica gel TLC. The detection conditions were: GF254 thin-layer plate, petroleum ether / ethyl acetate (3 / 1) as the developing solvent, and concentrated sulfuric acid ethanol solution as the developing solvent. Combining components with similar spots yielded five fractions: Fr. A 240g (sample obtained by elution and concentration at a petroleum ether / ethyl acetate volume ratio of 15 / 1→10 / 1→5 / 1→3 / 1, and then evaporated to dryness), Fr. B 40g (sample obtained by elution and concentration at a petroleum ether / ethyl acetate volume ratio of 1 / 1, followed by dichloromethane / methanol volume ratio of 50 / 1→30 / 1, and then evaporated to dryness), Fr. C 79g (sample obtained by elution and concentration at a dichloromethane / methanol volume ratio of 20 / 1, and then evaporated to dryness), Fr. D 64g (sample obtained by elution and concentration at a dichloromethane / methanol volume ratio of 10 / 1→5 / 1→3 / 1, and then evaporated to dryness), and Fr. E 56g (sample obtained by elution and concentration at a dichloromethane / methanol volume ratio of 1 / 1→0 / 1, and then evaporated to dryness).

[0045] (4) Fr.A, Fr.B, and Fr.C were separated by D-101 macroporous resin column chromatography, with the amount of D-101 macroporous resin being 15 times the sample volume. The solvent system used was ethanol / water, and elution was performed sequentially with 30%, 60%, and 95% ethanol, with each gradient elution consisting of 3 column volumes, approximately 500 mL per elution. The eluent was concentrated and evaporated to dryness under reduced pressure at 50°C. An appropriate amount of sample was dissolved in methanol and analyzed by HPLC. The analytical method was: 10-100% methanol, 60 min, detection wavelength 210 nm. Components with similar peak times were combined to obtain three fractions: Fr.a 11 g (peak time 5-30 min), Fr.b 30 g (peak time 25-45 min), and Fr.c 106 g (peak time 40-60 min).

[0046] (5) The three fractions Fr.a, Fr.b, and Fr.c were separated by reverse-phase column chromatography using ODS. The amount of ODS used for mixing the sample was 1.5 times that of the sample, and the amount of ODS in the chromatography column was 4 times that of the ODS used for mixing the sample. The solvent system used was methanol / water. Based on the above HPLC analysis results, gradient concentrations were set for elution. The specific conditions were as follows: Fr.a (10%, 35%, 60%, 100%), Fr.b (45%, 65%, 85%, 100%), and Fr.c (70%, 80%, 90%, 100%). Three retention volumes were used for each gradient elution, approximately 500 mL per run. The eluent was concentrated and evaporated to dryness under reduced pressure at 50°C. An appropriate amount of sample was dissolved in methanol and analyzed by HPLC. Components with similar elution times were combined to obtain six fractions: Fr.1 24g (elution time 3–22 min), Fr.2 26g (elution time 15–35 min), Fr.3 29g (elution time 25–45 min), Fr.4 18g (elution time 38–52 min), Fr.5 20g (elution time 45–60 min), and Fr.6 7g (elution time 55–60 min).

[0047] (6) Fr.5 was separated by silica gel column chromatography. The solvent system initially used petroleum ether / ethyl acetate with a volume ratio of 30 / 1→25 / 1→20 / 1→15 / 1→10 / 1→8 / 1→5 / 1→3 / 1→2 / 1→1 / 1; then, dichloromethane / methanol was used for gradient elution with a volume ratio of 30 / 1→25 / 1→20 / 1→15 / 1→0 / 1. Each gradient consisted of 3 column volumes, and each gradient was repeated once. The eluent was concentrated to dryness under reduced pressure at 50°C, and an appropriate amount of sample was dissolved in methanol and analyzed by silica gel TLC. Combining components with similar spots yielded ten fractions: Fr. 5.1 0.12 g (sample obtained by elution and concentration to dryness with a petroleum ether / ethyl acetate volume ratio of 30 / 1), Fr. 5.2 0.074 g (sample obtained by elution and concentration to dryness with a petroleum ether / ethyl acetate volume ratio of 25 / 1→20 / 1), Fr. 5.3 1.3257 g (sample obtained by elution and concentration to dryness with a petroleum ether / ethyl acetate volume ratio of 15 / 1), Fr. 5.4 1.6625 g (sample obtained by elution and concentration to dryness with a petroleum ether / ethyl acetate volume ratio of 10 / 1), Fr. 5.5 1.6008 g (sample obtained by elution and concentration to dryness with a petroleum ether / ethyl acetate volume ratio of 8 / 1→5 / 1), Fr. 5.6 2.1995g (sample obtained by elution, concentration and evaporation with a petroleum ether / ethyl acetate volume ratio of 3 / 1), Fr.5.7 1.9632g (sample obtained by elution, concentration and evaporation with a petroleum ether / ethyl acetate volume ratio of 2 / 1), Fr.5.8 4.4394g (sample obtained by elution, concentration and evaporation with a petroleum ether / ethyl acetate volume ratio of 1 / 1 and a dichloromethane / methanol volume ratio of 30 / 1), Fr.5.9 0.9319g (sample obtained by elution, concentration and evaporation with a dichloromethane / methanol volume ratio of 25 / 1→20 / 1), Fr.5.10 1.2536g (sample obtained by elution, concentration and evaporation with a dichloromethane / methanol volume ratio of 15 / 1→0 / 1).

[0048] (7) After isocratic elution with a semi-preparative column, Fr.5.5 was concentrated and evaporated to dryness to obtain 1.6 mg of the new compound. The retention time of the compound was in the range of 26.7-27.7 min. The semi-preparative column conditions were as follows: mobile phase was 50% acetonitrile-water solution, flow rate was 3.5 ml / min, detection wavelength was 210 nm and 254 nm, and the column type was YMC-Pack ODS-A with specifications of 250 × 10 mm and 5 μm.

[0049] Example 2

[0050] Structural identification of compounds

[0051] The structural identification of a sesquiterpene compound extracted and isolated from Codonopsis pilosula includes the following steps:

[0052] The compound prepared in Example 1 of this invention is a brownish-yellow oil; it turns blue-purple in 10% concentrated vanillin sulfate and is readily soluble in solvents such as ethyl acetate. HR-ESIMS Figure 2 The quasi-molecular ion peak is given, [M+Na]. + The peak value is m / z 253.1206 (calcd for C). 15 H 18 O2Na, 253.12O4), the molecular weight of the compound was determined to be 230, and the deduced molecular formula is C. 15 H 18 O2, calculated degree of unsaturation is 7. [α] 20D -25.0 (c 0.008, MeOH), maximum UV absorption wavelength is 280 nm ( Figure 3 ).

[0053] 1 H-NMR (600MHz, CDCl3, Figure 4 In the spectrum, δ H The values ​​of 7.25 (1H, d, J = 1.4 Hz) and 7.13 (1H, s) are presumed to be signals from two double-bonded protons; δ H The values ​​3.27 (1H, d, J = 16.7 Hz), 3.23 (1H, d, J = 16.7 Hz), 2.84 (1H, dd, J = 17.2, 8.2 Hz), and 2.74 (1H, dt, J = 17.2, 6.0 Hz) are presumed to be proton signals from two sets of magnetically inequivalent methylene groups; δ H 2.37 (3H, d, J = 1.4 Hz), 2.32 (3H, s), and 1.41 (3H, s) are presumed to be three groups of methyl signals.

[0054] 13 C-NMR (150MHz, CDCl3, Figure 5 The spectrum shows 15 carbon signals, combined with HSQC ( Figure 6 ) Spectral findings, δ C The values ​​154.4, 141.0, 133.4, 128.2, 127.5, 125.0, 116.1, and 110.7 are presumed to be carbon signals from double bonds, δ C 68.7 is presumably a carbon ionomer signal, δ C 41.4, 35.7, and 24.5 are presumed to be three methylene carbon signals, δ C Signals 28.8, 20.6, and 11.4 are presumed to be three methyl carbon signals. All C-H direct correlation signals were assigned using HSQC data (Table 1).

[0055] Table 1 Compounds 1 H and 13C data (600 / 150MHz, CDCl3)

[0056]

[0057]

[0058] HMBC ( Figure 7 In the spectrum, H-1(δ) H 2.84, 2.74) and C-2(δ C 35.7) / C-3(δ C 68.7) / C-4(δ C 41.4) / C-10(δ C 127.5) is related, H-2(δ) H 1.97, 1.85) and C-3(δ C 68.7) / C-4(δ C 41.4) / C-10(δ C 127.5) is related, H-4(δ) H 3.27, 3.23) and C-5 (δ C 128.2) / C-10(δ C 127.5) is related, H-15 (δ) H 1.41) and C-2(δ C 35.7) / C-3(δ C 68.7) / C-4(δ C 41.4) is related. Based on the chemical shift value of C-3, it is inferred that a hydroxyl group is attached at C-3, thus yielding ring A as shown in the figure; H-8 (δ H 7.13) and C-6(δ) C 125.0) / C-7(δ C 154.4) / C-10(δ C 127.5) is related, H-14 (δ) H 2.32) and C-8(δ) C 110.7) / C-9(δ C 133.4) / C-10(δ C 127.5) is related, combined with H-4 (δ) H 3.27, 3.23) and C-6 (δ C 125.0) is related, and it is speculated that there is a ring B as shown in the figure, while ring A and ring B are coupled through C-5-C-10; H-12 (δ H 2.32) and C-6(δ) C 125.0) / C-7(δ C154.4) / C-11(δ C 116.1) is related, H-13(δ) H 2.37) and C-6(δ) C 125.0) / C-11(δ C 116.1) / C-12(δ C 141.0) is related, and it is speculated that there is a ring C as shown in the figure, while ring B and ring C are coupled through C-6-C-7.

[0059] In summary, the planar structure of the compound of the present invention is shown in the figure below:

[0060]

[0061] Example 3

[0062] Screening of compounds for anti-hypoxia activity

[0063] Screening for the anti-hypoxia activity of sesquiterpenoid compounds extracted and isolated from Codonopsis pilosula includes the following steps:

[0064] Selection and Culture of PC12 Cells: PC12 cells were derived from rat adrenal pheochromocytoma and are commonly used to study hypoxia-induced neuronal injury, hypoxia / reoxygenation neuronal injury, oxidative stress neuronal injury, and neurodegenerative diseases. PC12 cells were purchased from Wuhan Plnosai Life Science Technology Co., Ltd., and cultured in a medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (1×10⁻⁶). 5 Cells were cultured in RPMI 1640 medium (μg / L). Cells were incubated at 37°C in a 5% CO2 incubator.

[0065] CCK-8 assay for cell viability: The CCK-8 assay is a highly sensitive, non-radioactive colorimetric detection method based on WST-8, widely used for cell proliferation and toxicity assays. WST-8 is reduced by dehydrogenases in cells to a highly water-soluble yellow formazanine product by the electron carrier 1-methoxy-5-methylphenazine onium sulfate dimethyl ester. The amount of formazanine produced is directly proportional to the number of viable cells. The number of viable cells can be determined by measuring the absorbance at a wavelength of 450 nm. Cell viability = (OD200 - OD200) / (WST ... 给药组 -OD 空白组 ) / (OD 对照组 -OD 空白组 )

[0066] Evaluation of the compound's cytotoxicity: PC12 cells in logarithmic growth phase were seeded with 100 μL (approximately 1 × 10⁻⁶ cells). 4Cells were cultured in 96-well plates at 37°C in a 5% CO2 incubator for 24 hours. After cell attachment, different concentrations of the compound (0, 0.1, 10, 50, 100, 200, and 500 μM) were added and incubated for another 24 hours. After 24 hours, 10 μL of CCK-8 solution was carefully added to each well in the dark and incubated for 3 hours. The absorbance was measured at 450 nm using a microplate reader. Cell viability was measured to evaluate the cytotoxicity of different concentrations of the compound. Each experiment was repeated three times, with four replicates per experiment.

[0067] Establishment of the CoCl2 Chemical Hypoxia Model: The CoCl2 chemical hypoxia model is widely used to simulate in vitro hypoxia or hypoxia / ischemia-induced nerve damage or apoptosis. The mechanism is that cobalt ions can promote the dissociation of tumor suppressor proteins from HIF-1α, thereby preventing the ubiquitination and degradation of HIF-1α, resulting in a significant increase in intracellular HIF-1α protein levels. This activates the expression of downstream hypoxia response elements of the protein, thereby causing a hypoxic response in the cell. The specific method is as follows: PC12 cells in logarithmic growth phase are seeded with 100 μL (approximately 1 × 10⁻⁶ cells). 4 Cells were cultured in 96-well plates at 37°C in a 5% CO2 incubator for 24 hours. After cell attachment, different concentrations of CoCl2 (0, 100, 200, 300, 400, 500, and 1000 μM) were added and the cells were cultured for 12 hours. After 12 hours, cell viability was measured using the CCK8 assay to determine the optimal modeling conditions.

[0068] Screening for the anti-hypoxia activity of compounds: PC12 cells in logarithmic growth phase were seeded with 100 μL (approximately 1 × 10⁻⁶ cells). 4 Cells were cultured in 96-well plates at 37°C in a 5% CO2 incubator for 24 hours. After cell attachment, different concentrations of the compound (20, 40, and 80 μM) were added for pretreatment. After 12 hours of pretreatment, the medium was replaced with 400 μM CoCl2, and the cells were incubated for another 12 hours. After 12 hours, cell viability was measured using the CCK8 assay to determine the anti-hypoxia activity of the compound.

[0069] Effects of compounds on ROS levels in CoCl2-induced hypoxic PC12 cells: ROS is a general term for oxygen-containing free radicals or peroxides that easily form free radicals in organisms, such as superoxide anion (O3). 2- ), hydroxyl radicals (OH) - Hydrogen peroxide (H2O2) is a common source of oxygen deficiency. When the body is hypoxic, ROS production increases, disrupting the body's oxidative balance and leading to functional impairment. Therefore, eliminating free radicals and reducing oxidative stress can mitigate the damage caused by hypoxia. The specific method is as follows: Take PC12 cells in the logarithmic growth phase and seed them with 1 mL (approximately 1 × 10⁻⁶ cells).5 Cells were cultured in 12-well plates at 37°C in a 5% CO2 incubator for 24 hours. After cell attachment, different concentrations of the compound (20, 40, and 80 μM) were added for pretreatment. After 12 hours of pretreatment, the medium was replaced with 400 μM CoCl2, and the cells were cultured for another 12 hours. After 12 hours, the cell culture medium was aspirated, and ROS expression in each group of cells was detected according to the ROS kit instructions to clarify the effect of the compound on ROS content in CoCl2-induced hypoxic PC12 cells.

[0070] Figure 8 The results showed that, compared with the normal group, there was no significant difference in cell viability when PC12 cells were cultured with different concentrations of the compound for 24 hours, indicating that the compound had no significant cytotoxicity within the set concentration range.

[0071] Figure 9 The results showed that PC12 cells treated with different concentrations of CoCl2 for 12 hours all exhibited some degree of damage compared to the normal group. When the CoCl2 concentration exceeded 300 μM, cell viability significantly decreased; under 400 μM CoCl2 treatment, cell viability was approximately 50% of that in the normal group. Therefore, the optimal chemical hypoxia modeling condition was determined to be: PC12 cells treated with 400 μM CoCl2 for 12 hours.

[0072] Figure 10 The results showed that, compared with the normal group, the cell viability of the model group was significantly decreased; compared with the model group, the cell viability of the drug-treated group was significantly increased in a dose-dependent manner. These results indicate that the compound has a protective effect against CoCl2-induced hypoxic damage to cells, exhibiting good anti-hypoxia activity.

[0073] Figure 11 The results showed that, compared with the normal group, the intracellular ROS content in the model group was significantly increased; compared with the model group, the intracellular ROS content in all drug-treated groups was reduced, and the ability to inhibit ROS increased with increasing drug concentration. These results indicate that the protective effect of this compound on hypoxic nerve cells may be related to the inhibition of ROS release.

Claims

1. The application of a sesquiterpene compound extracted and isolated from Codonopsis pilosula in the preparation of drugs for the prevention and treatment of acute mountain sickness, characterized in that, The chemical structural formula of the sesquiterpene compound is as follows: .

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