Abietane type diterpenoid as well as preparation method and application thereof

Abstract

The invention discloses abietane diterpenes as well as a preparation method and application thereof, and belongs to the technical fields of traditional Chinese medicine extraction and separation, natural pharmaceutical chemistry and medicine. The preparation method of the abietane type diterpenoid comprises the following steps: (1) crushing dried spica prunellae, and performing reflux extraction with 95% ethanol to obtain an ethanol extracting solution; (2) concentrating the ethanol extracting solution under reduced pressure to remove alcohol, and recovering a solvent to obtain a prunella vulgaris crude extract; and (3) carrying out chromatography on the crude extract by using AB-8 macroporous resin, sequentially eluting by using 60% methanol and 80% methanol, then carrying out LH-20 gel column chromatography, and then eluting by using a solvent and / or recrystallizing to obtain the abietane type diterpenoid compound. The abietane diterpenes extracted from the spica prunellae can effectively inhibit nerve cell ferroptosis through anti-oxidative stress, so that neuritis is effectively inhibited, and neurodegenerative diseases are resisted.

Description

Technical Field

[0001] The invention relates to the fields of extraction and separation of traditional Chinese medicine, natural drug chemistry and medical technology, and in particular to a rosinane-type diterpene extracted from Prunella vulgaris and an extraction method and application thereof. Background Art

[0002] Prunella vulgaris is a plant of the genus Prunella in the family Lamiaceae, mainly produced in southwest my country. At present, the main Prunella vulgaris plants developed for medicinal value include Prunella vulgaris (original variant), Prunella vulgaris, Prunella vulgaris, and Prunella vulgaris; and its main effect is to clear away heat and improve eyesight, and can treat red and swollen eyes, headaches, and reduce swelling and resolve knots.

[0003] Publication No. CN117180330A discloses the use of an ethanol aqueous solution extract of Prunella vulgaris in the preparation of a hypoglycemic composition, in which the ethanol aqueous solution extract of Prunella vulgaris has a new function of hypoglycemic. However, there is no literature report on the chemical composition and pharmacological activity of Prunella vulgaris.

[0004] With the acceleration of population aging, neurodegenerative diseases (ND) have gradually become a global health problem. Neurodegenerative diseases are a class of irreversible diseases characterized by neuronal dysfunction caused by damage to brain and spinal cord neurons or loss of their myelin sheaths. Their main pathogenesis involves genetics, oxidative stress, mitochondrial damage, metal ion disorders, energy metabolism disorders, immune abnormalities, cell autophagy, cell apoptosis, viral infection, etc. At present, there are no targeted drugs for the treatment of neurodegenerative diseases, and existing drugs can only temporarily relieve their clinical symptoms, but still cannot prevent the progression of the disease. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a rosinane-type diterpene and its preparation method and application. The compound extracted from Prunella vulgaris has a good preventive and therapeutic effect on neurodegenerative diseases

[0006] In order to achieve the above object, the present invention is implemented by the following technical solutions:

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

[0008]

[0009] The present invention also claims a method for preparing abietane-type diterpenes, characterized in that it comprises the following steps:

[0010] (1) The dried whole herb of Prunella vulgaris is crushed and extracted with 95% ethanol under reflux to obtain an ethanol extract;

[0011] (2) concentrating the ethanol extract under reduced pressure to remove alcohol, and recovering the solvent to obtain a crude extract of Prunella vulgaris;

[0012] (3) The crude extract is chromatographed on an AB-8 macroporous resin, eluted with 60% and 80% methanol in sequence, and then chromatographed on an LH-20 gel column, and then eluted with a solvent and / or recrystallized to obtain the abietane-type diterpene compound of the present invention.

[0013] Furthermore, the solvent used in the solvent elution in step (3) is any one or more of an acetonitrile / water mixed solvent and a methanol / water mixed solvent.

[0014] Furthermore, in step (3), LH-20 gel column chromatography was used to obtain five fractions, namely Fra, Frb, Frc, Frd and Fre; wherein the Frc fraction was subjected to high performance liquid chromatography analysis and solvent elution to obtain compound 1, compound 2, compound 4, compound 5, compound 6 and compound 7.

[0015] Furthermore, the compound 2 and compound 6 are obtained by eluting with an acetonitrile / water mixed solvent.

[0016] Furthermore, the compound 1, compound 4, compound 5 and compound 7 were obtained by eluting with a methanol / water mixed solvent.

[0017] Furthermore, in step (3), LH-20 gel column chromatography was used to obtain five fractions, namely Fra, Frb, Frc, Frd, and Fre; wherein the Frb fraction was recrystallized to obtain compound 3.

[0018] The present invention also protects the use of abietane-type diterpenes extracted from Prunella vulgaris in the preparation of drugs for preventing, treating and curing neurological diseases.

[0019] The neurological diseases described in the present invention mainly include nerve cell inflammation, neuron damage, increased Aβ protein, memory loss, and bradykinesia, including 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 body disease.

[0020] Furthermore, the medicine of the present invention also includes auxiliary materials or auxiliary ingredients, and the auxiliary materials or auxiliary ingredients mainly include pharmaceutical carriers and / or excipients and / or flavoring agents and / or functional agents commonly used in pharmacy; and the dosage form of the medicine is selected from injections, suspensions, emulsions, solutions, syrups, tablets, capsules, granules, powders, pills, granules, sprays, and aerosols.

[0021] The abietane-type diterpenes and the preparation method and application thereof of the present invention have the beneficial effects that the abietane-type diterpenes extracted from Prunella vulgaris can effectively inhibit ferroptosis of nerve cells by resisting oxidative stress, thereby effectively inhibiting neuritis and combating neurodegenerative diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0023] Figure 1 Schematic diagram of the inhibitory activity of the compounds of the present invention on ferroptosis of neural cells;

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

[0025] Figure 3 This is a schematic diagram showing that compound 4 of the present invention exerts anti-ferroptosis activity of neuronal cells by inhibiting the production of reactive oxygen species. DETAILED DESCRIPTION

[0026] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] Example 1

[0028] A method for preparing abietane-type diterpenes comprises the following steps:

[0029] (1) The whole dried Prunella vulgaris herb (5.0 kg) was crushed and extracted with 95% ethanol under reflux for 3 times, each time for 2 h, to obtain an ethanol extract;

[0030] (2) concentrating the ethanol extract under reduced pressure until there is no alcohol taste, and recovering the solvent to obtain a crude extract of Prunella vulgaris;

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

[0032] Example 2

[0033] The structures of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7 obtained in Example 1 were identified.

[0034] Compound 1

[0035] Red oily substance; (c 0.011,MeOH); UV(MeOH)λ max (logε)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 O 5 Na + ,365.1359). Its structural formula is:

[0036]

[0037] Compound 2

[0038] Colorless oil; (c 0.008,MeOH); UV(MeOH)λ max (logε)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 C20 H 26 O 4 Na + ,337.1774).

[0039] The structural formula is:

[0040]

[0041] Table 1 NMR 600 (MHz for Compounds 1 and 2 1 H and 150MHz for 13 C)

[0042]

[0043]

[0044] Compound 3

[0045] Dark red solid. 1 H NMR (600 MHz, CDCl 3 )δ13.12(s,7-OH),8.79(1H,d,J=10.5Hz,H-1),7.30(1H,s,H-6),6.29(1H,d,J=10.5Hz,H-2),4.08(3H,s,OCH3),3.40(1 H,hept,J=7.1Hz,H-15),1.49(3H,s,H-18),1.49(3H,s,H-19),1.28(3H,d,J=7.0Hz,H-16),1.28(3H,d,J=7.0Hz,H-17). 13 C NMR (151 MHz, CDCl 3 )δ201.9(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). Therefore, the compound 3 was identified as Agastaquinone. Its structural formula is:

[0046]

[0047] Compound 4

[0048] Red flocculent solid. 1H NMR (600MHz, CDCl3) δ13.13 (s, 14-OH), 5.76 (s, 11-OH), 3.35 (1H, dt, J = 14.1 Hz, H-1β), 3.31 (1H, m, H-15), 2.73 (1H, dd, J = 16.5, 14.7 Hz, H-6β), 2.62 (2H, dd, J = 8.5, 6.6 Hz, H-2), 2.54 (1H, dd, J = 16.5, 2 .9Hz,H-6α),2.40(1H,dd,J=14.7,2.8Hz,H-5α),1.98(1H,dt,J=14.0,8.4Hz,H-1α),1.43(3H,s,H- 20), 1.40 (3H, d, J = 7.4Hz, H-16), 1.39 (3H, d, J = 7.4Hz, H-17), 1.17 (3H, s, H-18), 1.16 (3H, s, H-19). 13C NMR (151MHz,CDCl3)δ216.1(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). Therefore, compound 4 was identified as 12-O-methylcandelabrone. The structural formula is:

[0049]

[0050] Compound 5

[0051] Colorless oil. 1H NMR (600MHz, CDCl3) δ7.16 (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 .9Hz,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=12.0,6.0Hz,H-6 ),1.28(3H,s,H-19),1.26(3H,s,H-20),1.22(3H,d,J=6.9Hz,H-16),1.22(3H,d,J=6.9Hz,H-17). 13C NMR (151MHz,CDCl3)δ178.1(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 basically consistent with those reported in the literature, so the compound 5 is identified as 4-epi-dehydroabietic acid. The structural formula is:

[0052]

[0053] Compound 6

[0054] Colorless oil. 1H NMR (600MHz, CDCl3) δ7.25 (1H, dd, J = 8.4, 1.8Hz, H-12), 7.22 (1H, d, J = 8.3Hz, H-11), 7.14 (1H, d, J = 2.1Hz, H-14), 5.33 (1H.s, H-16a), 5.03 (1H, t, J = 1.4Hz, H-16b), 2.92 (1H, dd, J = 16.8, 4.8Hz, 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 (151MHz,CDCl3)δ184.2(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). Therefore, the compound 6 was identified as Angustanoic acid E. Its structural formula was obtained as:

[0055]

[0056] Compound 7

[0057] Transparent oily liquid. 1H NMR (600MHz, CDCl3) 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 (600MHz, CDCl3) 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, compound 7 was identified as Dehy The structural formula of droabietinol is:

[0058]

[0059] Example 3

[0060] Application of abietane-type diterpenes extracted from Prunella vulgaris in preparing drugs for preventing, treating and curing neurological diseases.

[0061] Monomer compounds were selected for biological activity screening, and the results showed that compound 4 had significant inhibitory activity on RSL3-induced ferroptosis in HT-22 neurons, with an EC50 of 0.77±0.08μM. Further studies found that the compound could significantly inhibit the release of ROS in HT22 neurons, indicating that compound 4 may exert ferroptosis inhibitory activity by resisting oxidative stress.

[0062] Verification of the inhibitory activity of the compounds of the present invention on ferroptosis of HT-22 neurons induced by RSL3

[0063] HT22 cells were cultured in high-glucose DMEM medium. The cells were placed in a cell culture incubator set at 37°C and CO 2 The concentration is 5%, which is used for subculture. Cells in the logarithmic growth phase were taken for the experiment. HT22 cells in the logarithmic growth phase were inoculated into a 96-well plate at a density of 5000 cells / well, and 90 μL of cell suspension was added to each well. After inoculation, the cells continued to be cultured in the incubator, and subsequent experimental operations were performed. Set up a blank control and a negative control, with three parallels in each group, and culture for one day. 10 μL RSL3 inducer and 10 μL sample (compound 1-7) were added to each well at the same time and incubated for 24 hours. Add 10 μL of MTT stain, and after 4 hours, aspirate the supernatant, add 100 μL DMSO, and measure the OD value. The activity inhibition graph is shown in the figure Figure 1 As shown. Figure 1 It can be seen that the compounds of the present invention, especially compound 1 and compound 4, show effective inhibitory activity on ferroptosis of HT-22 neurons induced by ferroptosis ( Figure 1 ).

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

[0065] Verification of the compounds' ability to inhibit 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 μM), RSL3 (1 μM) and different concentrations of compound 4 (0.3125 μM, 0.625 μM, 1.25 μM) were added to the 6-well plates. After 4 hours of reaction, the supernatant medium was aspirated and H 2PCFDA (1 mL, 10 μM) was incubated in an incubator for 40 minutes (37°C), then washed twice with serum-free medium, and photographed under a fluorescence microscope under dark conditions. Further studies found that compound 4 had significant activity in inhibiting the production of reactive oxygen species, indicating that compound 4 may play an anti-ferroptosis role in HT22 neurons through the oxidative stress pathway ( Figure 3 ).

[0067] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present invention. The appearance of the phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0068] Finally, it should be noted that the embodiments disclosed in the present invention are only preferred embodiments of the present invention, which are only used to illustrate the technical solutions of the present invention rather than to limit them. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A rosinane-type diterpene, characterized in that: The abietane-type diterpenes are compounds extracted from Prunella vulgaris. The chemical structural formulas of the abietane-type diterpenes include compounds 1-7:

2. A method for preparing abietane-type diterpene according to claim 1, characterized in that: The following steps are involved: (1) The dried whole herb of Prunella vulgaris is crushed and extracted with 95% ethanol under reflux to obtain an ethanol extract; (2) concentrating the ethanol extract under reduced pressure to remove alcohol, and recovering the solvent to obtain a crude extract of Prunella vulgaris; (3) The crude extract is chromatographed on an AB-8 macroporous resin, eluted with 60% and 80% methanol in sequence, and then chromatographed on an LH-20 gel column, and then eluted with a solvent and / or recrystallized to obtain the abietane-type diterpene compound of the present invention.

3. The method for preparing abietane-type diterpenes according to claim 2, characterized in that: The solvent used in the solvent elution in step (3) is any one or more of an acetonitrile / water mixed solvent and a methanol / water mixed solvent.

4. The method for preparing abietane-type diterpenes according to claim 2, characterized in that: In step (3), LH-20 gel column chromatography is used to obtain five fractions, namely Fra, Frb, Frc, Frd and Fre; wherein the Frc fraction is subjected to high performance liquid chromatography analysis and solvent elution to obtain compound 1, compound 2, compound 4, compound 5, compound 6 and compound 7.

5. The method for preparing abietane-type diterpenes according to claim 4, characterized in that: The compound 2 and compound 6 were obtained by eluting with an acetonitrile / water mixed solvent.

6. The method for preparing abietane-type diterpenes according to claim 4, characterized in that: The compound 1, compound 4, compound 5 and compound 7 are obtained by eluting with a methanol / water mixed solvent.

7. The method for preparing abietane-type diterpenes according to claim 2, characterized in that: In step (3), LH-20 gel column chromatography was used to obtain five fractions, namely Fra, Frb, Frc, Frd and Fre; wherein the Frb fraction was recrystallized to obtain compound 3.

8. Use of the abietane-type diterpene according to claim 1 or 2 in the preparation of drugs for preventing, treating and treating neurological diseases.

9. The use according to claim 8, characterized in that: The neurological diseases include nerve cell inflammation, neuron damage, increased Aβ protein, memory loss, and bradykinesia.

10. The use according to claim 8, characterized in that: The medicament also includes pharmaceutically acceptable excipients or auxiliary ingredients.

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