A cembranoid diterpene and preparation method and application thereof

Abstract

The invention discloses a kind of abietane type diterpene and its preparation method and application, belong to the field of traditional Chinese medicine extraction and separation, natural medicine chemistry and medical technology. The preparation method of abietane type diterpene comprises the following steps: (1) the whole herb of dried Prunella vulgaris is crushed and extracted with 95% ethanol reflux to obtain an ethanol extract; (2) the ethanol extract is concentrated under reduced pressure to remove alcohol, and the solvent is recovered to obtain a crude extract of Prunella vulgaris; (3) the crude extract is chromatographed on AB‑8 macroporous resin, eluted with 60% and 80% methanol in sequence, and then subjected to LH‑20 gel column chromatography, and then obtained after solvent elution and / or recrystallization. The abietane type diterpene extracted from Prunella vulgaris by the present invention can effectively inhibit the death of nerve cells by anti-oxidative stress, thereby effectively inhibiting neuritis and combating neurodegenerative diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine extraction and separation, natural medicine chemistry and medicine, and particularly relates to a rosinan type diterpene extracted from Prunella hispida and an extraction method and application thereof. BACKGROUND

[0002] Prunella hispida is a plant of the genus Prunella in the Labiatae family, and is mainly produced in the southwest of China. At present, the main drugs developed from the genus Prunella include Prunella vulgaris (original variety), Prunella hispida, Prunella vulgaris var. incisa, and Prunella vulgaris var. angustifolia. Prunella has the effects of clearing heat and improving eyesight, and can treat red eyes, swelling, pain, headache, and swelling and knotting.

[0003] Publication No. CN117180330A discloses the application of an ethanol aqueous extract of Prunella vulgaris var. incisa in the preparation of a blood sugar lowering composition. The ethanol aqueous extract of Prunella vulgaris var. incisa has a new function of lowering blood sugar. However, the chemical components and pharmacological activities of Prunella hispida have not been reported in the literature.

[0004] With the acceleration of population aging, neurodegenerative diseases (ND) have gradually become a worldwide health problem. Neurodegenerative diseases are a class of irreversible diseases characterized by damage to brain and spinal cord neurons or loss of their myelin sheaths, resulting in dysfunction of neurons. The main pathogenesis involves genetics, oxidative stress, mitochondrial damage, metal ion disorder, energy metabolism disorder, immune abnormalities, autophagy, apoptosis, viral infection, etc. At present, there is no specific drug for the treatment of neurodegenerative diseases, and existing drugs can only temporarily relieve clinical symptoms, but still cannot stop the progression of the disease. SUMMARY

[0005] In order to solve the above problems, the present application provides a rosinan type diterpene and a preparation method and application thereof. The compound extracted from Prunella hispida has good prevention and treatment effect on neurodegenerative diseases

[0006] In order to achieve the above purpose, the present application is realized by the following technical scheme:

[0007] A rosinan type diterpene, which is a compound extracted from Prunella hispida, and the chemical structural formula of the rosinan type diterpene includes compounds 1-7:

[0008]

[0009] The present application also claims a preparation method of a rosinan type diterpene, characterized by comprising the following steps:

[0010] (1) dry the whole herb of U. thomsonii, crush it, and extract it with 95% ethanol by reflux to obtain an ethanol extract;

[0011] (2) concentrate the ethanol extract by reducing pressure to remove ethanol, and recover the solvent to obtain a crude extract of U. thomsonii;

[0012] (3) chromatograph the crude extract with AB-8 macroporous resin, elute it with 60% and 80% methanol in sequence, and then chromatograph it with LH-20 gel column, and then elute it with a solvent and / or recrystallize it to obtain the roylean-type diterpene compound of the present application.

[0013] Further, the solvent in the solvent elution in step (3) is any one or several of acetonitrile / water mixed solvent and methanol / water mixed solvent.

[0014] Further, the LH-20 gel column chromatography in step (3) obtains five fractions of Fra, Frb, Frc, Frd and Fre; the Frc fraction is analyzed by high performance liquid chromatography and eluted with a solvent to obtain compounds 1, 2, 4, 5, 6 and 7.

[0015] Further, the compounds 2 and 6 are eluted with acetonitrile / water mixed solvent.

[0016] Further, the compounds 1, 4, 5 and 7 are eluted with methanol / water mixed solvent.

[0017] Further, the LH-20 gel column chromatography in step (3) obtains five fractions of Fra, Frb, Frc, Frd and Fre; the Frb fraction is recrystallized to obtain compound 3.

[0018] The present application also protects the use of the roylean-type diterpene extracted from U. thomsonii in the preparation of a medicament for preventing and treating nervous diseases.

[0019] The nervous diseases of the present application mainly include inflammation of nerve cells, neuron damage, increase of Aβ protein, memory decline and slow movement, and mainly include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, cerebellar atrophy, multiple sclerosis, cerebral infarction, cerebral hemorrhage, meningitis, spinocerebellar ataxia and Pick's body disease.

[0020] Further, the medicament of the present application further comprises an auxiliary material or has an auxiliary component, and the auxiliary material or the auxiliary component mainly comprises a pharmaceutical carrier commonly used in pharmacy, and / or an excipient, and / or a flavoring agent, and / or a functional agent; and the dosage form of the medicament is selected from injection, suspension, emulsion, solution, syrup, tablet, capsule, granule, powder, pill, infusion, spray and aerosol.

[0021] The rosin a diterpene extracted from the Urtica fissa, can effectively inhibit the neural cell ferroptosis through the antioxidant stress, thereby effectively inhibiting the neuroinflammation and resisting the neurodegenerative diseases. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0023] Figure 1 is a schematic diagram of the inhibitory activity of the compound of the present application on neural cell ferroptosis;

[0024] Figure 2 is a schematic diagram of the inhibitory activity of different concentrations of the compound 4 of the present application on neural cell ferroptosis;

[0025] Figure 3 is a schematic diagram of the anti-neural cell ferroptosis activity of the compound 4 of the present application by inhibiting the production of reactive oxygen species. DETAILED DESCRIPTION

[0026] In order to make the person skilled in the art better understand the present application, the technical solutions of the present application will be described clearly and completely in combination with the embodiments. Obviously, the described embodiments are only some of the embodiments of the present application, but not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0027] Embodiment 1

[0028] A preparation method of a rosin a diterpene, comprising the following steps:

[0029] (1) Dry Urtica fissa (5.0 kg) whole grass is crushed, and 95% ethanol is used for reflux extraction for 3 times, and each extraction time is 2h, to obtain an ethanol extract;

[0030] (2) The ethanol extract is concentrated under reduced pressure until there is no alcohol smell, and the solvent is recovered to obtain a Urtica fissa crude extract extract;

[0031] (3) The crude extract (360.8 g) was chromatographed on AB-8 macroporous resin, eluted with 60% and 80% methanol successively. The eluted extract (202 g) was chromatographed on LH-20 gel column (methanol) to obtain Fra, Frb, Frc, Frd and Fre five fractions. The Frc fraction was analyzed by high performance liquid chromatography to obtain compounds. After elution with acetonitrile / water 80:20, 2.0 mL / min, compounds 2 and 6 (8.5 mg) were obtained; after elution with methanol / water 75:25, 2.0 mL / min, compounds 5 (3.3 mg) and 7 (10 mg) were obtained; after elution with methanol / water 80:20, 2.0 mL / min, compounds 1 (3.5 mg) and 4 (5 mg) were obtained; and after recrystallization of the Frb fraction, compound 3 (15 mg) was obtained.

[0032] Example 2

[0033] The compounds 1, 2, 3, 4, 5, 6 and 7 obtained in Example 1 were subjected to structural identification.

[0034] Compound 1

[0035] Red oil; (c 0.011, MeOH); UV (MeOH) λ max (log ε) 229 (3.63) nm; CD (MeOH) λ max (Δε): 214 (+10.47), 234 (-2.89), 292 (+4.97) nm; 1 H and 13 C NMR data are shown in Table 1. HRESIMS: m / z 365.1349 [M+Na] + (calcd for C 20 H 22 O5Na + , 365.1359). The structural formula thereof is:

[0036]

[0037] Compound 2

[0038] Colorless oil; (c 0.008, MeOH); UV (MeOH) λ max (log ε) 325 (3.25) nm; 1 H and 13 C NMR data are shown in Table 1. HRESIMS: m / z 337.1763 [M+Na] + (calcd. for C 20 H26 O4Na + ,337.1774).

[0039] The structure of compound 3 is identified as Agastaquinone.

[0040]

[0041] Table 1 NMR 600 (MHz for 1 H and 150 MHz for 13 C)

[0042]

[0043]

[0044] Compound 3

[0045] Dark red solid. 1 H NMR (600 MHz, CDC13) δ 13.12 (s, 7-OH), 8.79 (1H, d, J = 10.5 Hz, H-1), 7.30 (1H, s, H-6), 6.29 (1H, d, J = 10.5 Hz, H-2), 4.08 (3H, s, OCH3), 3.40 (1H, hept, J = 7.1 Hz, H-15), 1.49 (3H, s, H-18), 1.49 (3H, s, H-19), 1.28 (3H, d, J = 7.0 Hz, H-16), 1.28 (3H, d, J = 7.0 Hz, H-17). 13 C NMR (151 MHz, CDC13) δ 201.9 (C-3), 191.0 (C-14), 183.6 (C-11), 162.7 (C-7), 160.0 (C-12), 157.3 (C-5), 139.8 (C-1), 137.6 (C-13), 128.7 (C-10), 126.5 (C-2), 122.5 (C-9), 122.2 (C-6), 114.0 (C-8), 61.0 (-OCH3), 48.4 (C-4), 27.9 (C-18), 27.9 (C-19), 24.4 (C-15), 20.4 (C-16). 20.4 (C-17). The structure of compound 3 is identified as Agastaquinone.

[0046]

[0047] Compound 4

[0048] Red flocculent solid. 1H NMR (600 MHz, CDC13) δ 13.13 (s, 14-OH), 5.76 (s, 11-OH), 3.35 (1H, dt, J = 14.1 Hz, H-1P), 3.31 (1H, m, H-15), 2.73 (1H, dd, J = 16.5, 14.7 Hz, H-6P), 2.62 (2H, dd, J = 8.5, 6.6 Hz, H-2), 2.54 (1H, dd, J = 16.5, 2.9 Hz, H-6a), 2.40 (1H, dd, J = 14.7, 2.8 Hz, H-5a), 1.98 (1H, dt, J = 14.0, 8.4 Hz, H-la), 1.43 (3H, s, H-20), 1.40 (3H, d, J = 7.4 Hz, H-16), 1.39 (3H, d, J = 7.4 Hz, H-17), 1.17 (3H, s, H-18), 1.16 (3H, s, H-19).13C NMR (151 MHz, CDC13) δ 216.1 (C-3), 204.5 (C-7), 158.2 (C-14), 152.5 (C-12), 139.0 (C-9), 133.4 (C-11), 126.9 (C-13), 112.1 (C-8), 62.3 (-OCH3), 48.9 (C-5), 47.1 (C-4), 39.0 (C-10), 36.4 (C-1), 35.6 (C-6), 34.6 (C-2), 27.1 (C-18), 26.2 (C-15), 21.0 (C-19), 20.4 (C-16), 20.4 (C-17), 17.6 (C-20). The compound 4 was identified as 12-O-methylcandelabrone. Its structural formula is:

[0049]

[0050] Compound 5

[0051] Colorless oil. 1H NMR (600 MHz, CDC13) δ 7.16 (1H, d, J = 8.1 Hz, H-11), 6.99 (1H, dd, J = 8.1, 2.1 Hz, H-12), 6.89 (1H, d, J = 2.1 Hz, H-14), 2.85 (1H, m, H-7), 2.80 (1H, m, H-15), 2.29 (1H, d, J = 12.9 Hz, H-1), 2.22 (1H, d, J = 12.9 Hz, H-5), 1.85 (1H, m, H-6), 1.76 (1H, m, H-3), 1.74 (1H, m, H-2), 1.53 (1H, dd, J = 12.0, 6.0 Hz, H-6), 1.28 (3H, s, H-19), 1.26 (3H, s, H-20), 1.22 (3H, d, J = 6.9 Hz, H-16), 1.22 (3H, d, J = 6.9 Hz, H-17).13C NMR (151 MHz, CDC13) δ 178.1 (COOH-19), 145.8 (C-9), 145.7 (C-13), 135.2 (C-8), 127.0 (C-14), 125.6 (C-11), 124.1 (C-12), 53.0 (C-5), 44.1 (C-4), 39.5 (C-1), 38.5 (C-10), 37.7 (C-3), 33.6 (C-15), 32.2 (C-7), 29.0 (C-18), 24.1, (C-16), 24.0 (C-17), 23.5 (C-20), 21.2 (C-6), 20.1 (C-2). The above data are in good agreement with the literature reported, thus the compound 5 was identified as 4-epi-dehydroabietic acid. The structural formula is obtained as:

[0052]

[0053] Compound 6

[0054] Colorless oil. 1H NMR (600 MHz, CDC13) δ 7.25 (1H, dd, J = 8.4, 1.8 Hz, H-12), 7.22 (1H, d, J = 8.3 Hz, H-11), 7.14 (1H, d, J = 2.1 Hz, H-14), 5.33 (1H. s, H-16a), 5.03 (1H, t, J = 1.4 Hz, H-16b), 2.92 (1H, dd, J = 16.8, 4.8 Hz, H-7β), 2.81 (1H, m, H-7α), 2.28 (1H, m, H-1β), 2.26 (1H, m, H-3β), 2.20 (1H, m, H-6α), 2.12 (3H, s, H-17), 2.06 (1H, m, H-6β), 2.01 (1H, m, H-2β), 1.62 (1H, m, H-2α), 1.57 (1H, m, H-5), 1.38 (1H, m, H-1α), 1.34 (3H, s, H-18), 1.13 (3H, s, H-20), 1.08 (1H, m, H-3α).13C NMR (151 MHz, CDC13) δ 184.2 (C-19), 147.5 (C-9), 143.1 (C-15), 138.3 (C-13), 135.2 (C-8), 126.2 (C-14), 125.6 (C-11), 123.2 (C-12), 111.8 (C-16), 53.0 (C-5), 44.1 (C-4), 39.4 (C-1), 38.6 (C-10), 37.5 (C-3), 32.2 (C-7), 28.9 (C-18), 23.2 (C-20), 21.9 (C-17), 21.0 (C-6), 20.0 (C-2). The compound 6 was identified as Angustanoic acid E. Its structural formula is:

[0055]

[0056] Compound 7

[0057] Transparent oily liquid. 1H NMR (600 MHz, CDC13) δ: 7.19 (1H, d, J = 8.4 Hz, H-12), 7.09 (1H, dd, J = 8.4, 2.4 Hz, H-11), 6.89 (1H, d, J = 1.8 Hz, H-14), 3.88 (1H, dd, J = 11.4, 2.4 Hz, H-18a), 3.56 (1H, dd, J = 11.4, 1.2 Hz, H-18b), 2.93 (1H, m, H-6a), 2.85 (1H, m, H-6b), 2.33 (1H, m, H-15), 1.99 (2H, m, H-7), 1.90 (2H, m, H-2), 1.72 (1H, m, H-5), 1.52 (2H, m, H-3), 1.24 (9H, d, J =, H-16, 17), 1.07 (3H, s, H-20). 13C NMR (600 MHz, CDC13) δ: 147.07 (C-14), 145.50 (C-13), 134.47 (C-8), 126.72 (C-14), 124.38 (C-12), 123.85 (C-11), 65.16 (C-18), 51.18 (C-5), 38.82 (C-1), 38.60 (C-4), 37.37 (C-10), 35.11 (C-3), 33.34 (C-15), 30.95 (C-7), 26.73 (C-20), 25.70 (C-16), 23.91 (C-17), 23.89 (C-6), 19.13 (C-2), 18.92 (C-19) Therefore, the compound 7 is identified as Dehydroabietinol. Its structural formula is:

[0058]

[0059] Example 3

[0060] Use of rosin diterpenes of the abietane type extracted from U. setaceus in the preparation of a medicament for the prevention and treatment of neurological diseases.

[0061] The monomer compound was selected for biological activity screening, and the results showed that compound 4 had significant inhibitory activity on RSL3-induced HT-22 neural cell ferroptosis, and the EC50 was 0.77±0.08 μM. Further research found that the compound can significantly inhibit the release of ROS of HT22 neural cells, indicating that compound 4 may play a ferroptosis inhibitory activity by antioxidant stress.

[0062] Verification of the ferroptosis inhibitory activity of the compound of the present application on RSL3-induced HT-22 neural cells

[0063] HT22 cells were cultured using high glucose DMEM medium. The cells were placed in a cell incubator, set at 37°C and 5% CO2 concentration, for subculture. Cells in the logarithmic growth phase were used for experiments. Logarithmic growth phase HT22 cells were seeded into a 96-well plate at a density of 5000 cells / well, with 90 μL of cell suspension added to each well. After seeding, the cells were incubated in the incubator and subsequent experimental operations were performed. Blank controls, negative controls, three replicates per group, were set up and incubated for one day, with 10 μL of RSL3 inducer and 10 μL of sample (compounds 1-7) added to each well simultaneously, and incubated for 24 h. 10 μL of MTT staining agent was added, and after 4 h, the supernatant was aspirated, 100 μL of DMSO was added, and the OD value was measured. The activity inhibition graph is shown in Figure 1 As can be seen from Figure 1 , the compounds of the present application, especially compound 1 and compound 4, show effective inhibitory activity on induced HT-22 neural cell ferroptosis Figure 1 .

[0064] HT22 cells were seeded in a 96-well plate at a density of 5000 cells / well for 24 h. Then, ferroptosis inducer RSL3 (1 μM) and compound 4 at the indicated concentrations (20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.3125 μM) were added in triplicate and incubated for 24 h, after which MTT solution (10 μL, 10 mg / mL) was added to each well and incubated for 4 h. The supernatant was aspirated, 100 μL of DMSO was added, and the absorbance was measured at 490 nm using a microplate spectrophotometer after shaking for about 30 min at room temperature on a shaker, as shown in Figure 2 The results show that compound 4 exhibits significant ferroptosis inhibition at different concentration gradients, with an EC50 value of 0.77 ± 0.08 μM (Fer-1: 0.06 ± 0.002 μM) Figure 2 C and Figure 2 D).

[0065] Verification of the inhibitory effect of the compound on the release of ROS in HT22 neural cells

[0066] HT22 cells were seeded in 6-well plates at a density of 300,000 cells / well for 24 hours. Then, Fer-1 (1 mM), RSL3 (1 mM) and different concentrations of compound 4 (0.3125 mM, 0.625 mM, 1.25 mM) were added to the 6-well plates. After 4 hours of reaction, the supernatant medium was aspirated, H2PCFDA (1 mL, 10 mM) was added, incubated in an incubator for 40 minutes (37°C), then washed twice with serum-free medium, and photographed with a fluorescence microscope under dark conditions. Further studies found that compound 4 had obvious activity in inhibiting the generation of reactive oxygen species, indicating that compound 4 may exert an iron death effect on nerve cells HT22 through the oxidative stress pathway Figure 3

[0067] Reference herein to "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment can be included in at least one embodiment of the application. The appearances of the phrase that the phrase in various places in the specification are not necessarily all referring to the same embodiment, or are necessarily referring to some particular embodiment, or are mutually exclusive in reference to some other embodiments.

[0068] It should be noted finally that: the embodiments disclosed in the application are only the preferred embodiments of the application, and are only used to explain the technical solutions of the application, but not to limit the application; although the application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement to part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the application.​

Claims

1. A method for preparing abietane-type diterpenes, characterized in that: The following steps are involved: (1) The dried whole herb of Prunella vulgaris was crushed and reflux-extracted with 95% ethanol to obtain an ethanol extract; (2) The ethanol extract is concentrated under reduced pressure to remove alcohol, and the solvent is recovered to obtain a crude extract of Prunella vulgaris; (3) The crude extract was chromatographed on AB-8 macroporous resin, eluted with 60% and 80% methanol in sequence, and then chromatographed on LH-20 gel column to obtain five fractions, namely Fra, Frb, Frc, Frd, and Fre. The Frc fraction was subjected to high performance liquid chromatography to obtain compound 2, which was then eluted with acetonitrile / water to obtain abietane-type diterpene compound 2 and compound 6. The compound was eluted with methanol / water to obtain abietane-type diterpene compound 1, compound 4, compound 5, and compound 7. The Frb fraction is recrystallized to obtain abietane-type diterpene compound 3; The structural formulas of the abietane-type diterpene compounds 1, 2, 3, 4, 5, 6 and 7 are: .

2. Use of abietane-type diterpenes in the preparation of drugs for preventing, treating and treating neurological diseases, characterized in that: The abietane-type diterpene is the compound 4 described in claim 1, and its structural formula is: .

3. The use according to claim 2, characterized in that: The neurological disease is selected from the group consisting of nerve cell inflammation, neuron damage, increased Aβ protein, memory loss, and bradykinesia.

4. The use according to claim 2, characterized in that: The drug also includes pharmaceutically acceptable excipients.

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