Cupressaceae plant extract, method for preparing the same, and use thereof

Abstract

The present application relates to natural extracts, in particular to a cypress family plant extract and a preparation method and application thereof, and the cypress family plant extract is a Selaginella uncinata extract or a Platycladus orientalis extract. The extract and biflavonoids in the extract have significant PDE4 enzyme inhibitory activity, and can be used for preparing a PDE4 enzyme inhibitor, and are used for treating and / or preventing diseases related to type 4 phosphodiesterase.

Description

Technical Field

[0001] This invention relates to natural extracts, specifically to an extract from a cypress family plant, its preparation method, and its application. Background Technology

[0002] Cyclic nucleotide phosphodiesterases (PDEs) are hydrolytic enzymes that metabolize cGMP and cAMP in the human body. They effectively control the intracellular concentrations of cGMP and cAMP, thereby regulating the biochemical processes mediated by second messengers in the body.

[0003] Phosphodiesterase type 4 (PDE4) specifically hydrolyzes cAMP to 5'-AMP, regulating cellular cAMP concentration within an appropriate range to maintain normal physiological activity. PDE4 is mainly distributed in various inflammatory cells, immune cells, and airway smooth muscle cells. Inhibiting PDE4 activity can suppress inflammatory cells. The PDE4 family can be further divided into four subtypes (PDE-4ABCD), with PDE-4B being the main anti-inflammatory target, while PDE-4D is associated with adverse reactions such as gastrointestinal discomfort. Currently, PDE4 inhibitors have been developed into anti-inflammatory drugs, such as roflumilast, which are mainly used clinically to treat lung inflammation, especially for the treatment of asthma and chronic obstructive pulmonary disease. However, taking these drugs can cause gastrointestinal adverse reactions such as diarrhea and nausea, limiting their use. Therefore, overcoming these adverse reactions of PDE4 inhibitors and researching novel specific inhibitors has become a research hotspot.

[0004] Selaginella uncinata (Desv.) Spring, a plant belonging to the Selaginellaceae family and the Selaginella genus, is also known as Phoenix Tail Grass, Blue Ground Cypress, Resurrection Grass, and Dragon Scale Grass. It has traditional medicinal properties such as clearing heat and dampness, detoxifying, and resolving blood stasis. In folk medicine, it is used to treat jaundice, dysentery, edema, rheumatic pain, cough with hemoptysis, sore throat, hemorrhoids, cuts, and burns. Modern pharmacological studies have demonstrated that Selaginella uncinata has anti-inflammatory, anti-asthmatic, antiviral, anti-hypoxic, and anti-tumor effects. Its main chemical components include biflavonoids, fatty acids, phenolic acids, and steroidal saponins. Biflavonoids are characteristic components of Selaginellaceae plants and possess pharmacological effects such as anti-hyperglycemia, anti-tumor, antiviral, and anti-hypoxic properties. Overall, research on Selaginella uncinata extract is still largely focused on its anti-tumor and anti-inflammatory effects; its use as a PDE4 inhibitor is currently lacking. Therefore, further development and utilization of Selaginella uncinata extract is essential.

[0005] The Chinese arborvitae (Platycladus orientalis (L.) Franco), a tree belonging to the genus Platycladus in the family Cupressaceae, is known for its leaves, also called cypress leaves, clump cypress leaves, or flat cypress leaves. These small, scale-like leaves lie flat on the branches, arranged oppositely, and are dark green or yellowish-green. They are brittle and easily broken, with a yellowish-white cross-section. The Chinese arborvitae is widely distributed and has a long history of cultivation. Literature records that the leaves have a fragrant aroma and a bitter taste, and are associated with the lung, liver, and spleen meridians, possessing properties such as relieving cough and phlegm, promoting hair growth, and stopping bleeding. The leaves contain various medicinal active ingredients, among which flavonoids, volatile oils, and tannins have been extensively studied; they exhibit antibacterial, antitumor, hemostatic, hair growth-promoting, and anti-inflammatory pharmacological effects.

[0006] Therefore, natural products are an important source for finding new PDE4 inhibitors, and are of great significance for developing a new generation of PDE4 inhibitors with strong efficacy and few side effects. Summary of the Invention

[0007] In view of the technical defects existing in the prior art, the purpose of the present invention is to provide the application of extracts of Selaginella uncinata and Platycladus orientalis, as well as the biflavonoids therein, in the treatment of phosphodiesterase type 4 related diseases.

[0008] The above-mentioned objective of this invention is achieved through the following technical solution:

[0009] A first aspect of the present invention is to provide the use of cypress extracts in the treatment and / or prevention of phosphodiesterase type 4 related diseases, said cypress extracts being Selaginella uncinata extract or Platycladus orientalis extract.

[0010] Furthermore, the Selaginella uncinata extract is obtained by alcohol extraction of Selaginella uncinata. The alcohol extraction method is as follows: weigh dried Selaginella uncinata leaves, crush them into coarse powder, add 8-12 times the volume of alcohol solution and soak them three times, with the first soaking lasting 8-15 days, and the second and third soaking lasting 4-7 days each. Filter, combine the filtrates, recover the effective components of alcohol under reduced pressure, and concentrate the remaining liquid to a thick paste to obtain Selaginella uncinata extract.

[0011] Furthermore, the arborvitae extract is obtained by arborvitae alcohol extraction. The alcohol extraction method is as follows: weigh the dried arborvitae leaves, crush them into coarse powder, add 8-12 times the volume of alcohol solution and soak them three times, with the first soaking lasting 2-5 days, and the second and third soaking lasting 1-3 days each. Filter, combine the filtrates, recover the effective components of alcohol under reduced pressure, and concentrate the remaining liquid to a thick paste to obtain the arborvitae extract containing the effective components.

[0012] Furthermore, the effective fraction of the Platycladus orientalis extract is obtained by separation using macroporous resin. The separation method is as follows: Step 1: Take macroporous resin AB-8, add 2-5 times its volume of anhydrous ethanol to soak, and activate for 12-48 hours. Pack the activated macroporous resin into a glass column with a frit core, and rinse the macroporous resin with a large amount of distilled water until there is no ethanol odor in the rinsing solution. Step 2: Weigh the Platycladus orientalis extract, dissolve it in anhydrous ethanol, and slowly drop it onto the surface of the macroporous resin. Adsorb for one hour, and then elute sequentially with 100% distilled water, 30% ethanol / water, 70% ethanol / water, and 100% ethanol. Collect in batches, evaporate under reduced pressure, and the refined extract of the effective fraction of Platycladus orientalis can be obtained.

[0013] A second aspect of the present invention is to provide the use of biflavonoid compounds in the extracts of the cypress family plants in the treatment and / or prevention of phosphodiesterase type 4 related diseases.

[0014] Furthermore, the structure of the biflavonoid compound is selected from one of the compounds shown in formulas (I)-(V):

[0015]

[0016] in:

[0017] R1, R2, R3, R4, R5 and R6 are selected from one of halogen, aldehyde, carboxyl, sulfonic acid, nitro, nitroso or hydrogen, respectively;

[0018] Alternatively, R1, R2, R3, R4, R5, and R6 are each independently a hydroxyl or methoxy group. Further, the structure of the compound represented by formula (I) is selected from any of the following structures:

[0019]

[0020]

[0021] A third aspect of the invention is to provide an application in the preparation of a health product or food for the treatment and / or prevention of phosphodiesterase type 4 related diseases, said health product or food comprising the above-described arborvitae extract.

[0022] A fourth aspect of the present invention is to provide a medicament for treating and / or preventing phosphodiesterase type 4-related diseases, said medicament comprising one or more of the above-described cypress extracts or biflavonoid compounds with structures shown in any of formulas (I)-(V), and further comprising pharmaceutically acceptable excipients and carriers.

[0023] Furthermore, the dosage form of the drug is capsules, tablets, pills, granules, powders, or sprays.

[0024] The present invention has the following beneficial effects:

[0025] This invention conducts an in-depth study on extracts of Selaginella uncinata and Platycladus orientalis, including in vitro and in vivo activity evaluation of the extracts, and the extraction and separation of a series of biflavonoid compounds using various column chromatography separation techniques and semi-preparative high performance liquid chromatography methods. These compounds have significant PDE4 enzyme inhibitory activity and can be used to prepare PDE4 enzyme inhibitors for the treatment and / or prevention of diseases related to phosphodiesterase type 4. Attached Figure Description

[0026] Figure 1 The inhibition curve of compound 1 of the present invention against PDE4D is shown.

[0027] Figure 2 This is the eutectic structure of the representative compound 1 of this invention and PDE4D;

[0028] Figure 3 The present invention demonstrates the anti-psoriasis efficacy of the ethyl acetate extract of Platycladus orientalis in an IMQ-induced mouse model of psoriasis.

[0029] Figure 4 This invention describes the effective fraction of macroporous resin from Platycladus orientalis and the efficacy of compound 1 in a mouse psoriasis model. Detailed Implementation

[0030] The present invention will be further illustrated below with reference to embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0031] In the following examples, NMR spectra were recorded using a Bruker 400 MHz nuclear magnetic resonance spectrometer with TMS internal standard. Mitsubishi Chemical MCI packing material (CHP20P, 75–150 μM) and YMC ODS packing material (12 nm, S–50 μM) were used; other solvents and reagents were analytical grade (AR).

[0032] Example 1: Extraction and separation process of Selaginella uncinata and its inhibitory activity against PDE4

[0033] I. Preparation of Selaginella uncinata extract and its inhibitory activity against PDE4

[0034] Weigh out dried leaves of Selaginella uncinata, crush them into coarse powder, add 10 times the volume of 95% ethanol and soak them 3 times, the first time for 10 days, the second and third times for 5 days each, filter, combine the filtrates, recover the ethanol under reduced pressure, and concentrate the remaining liquid to a thick paste to obtain the total ethanol extract of Selaginella uncinata.

[0035] The ethanol extract of Selaginella uncinata was suspended in 4 times its weight of water and extracted sequentially with petroleum ether, ethyl acetate, and n-butanol in equal volumes to water (each solvent was extracted 3 times consecutively) to obtain petroleum ether, ethyl acetate, n-butanol extracts and aqueous solutions. The extracts were evaporated to dryness under reduced pressure to obtain petroleum ether extract, ethyl acetate extract, n-butanol extract and aqueous concentrate, and their PDE4D inhibitory activity was tested.

[0036] PDE4 enzyme activity was determined using liquid scintillation counting. As a cAMP-preferred isoenzyme, 3H-cAMP was selected as the substrate for PDE4 inhibitory activity testing. The assay method is summarized as follows: Prepare Assaybuffer (50 mM Tris-HCl, 10 mM MgCl2 / MnCl2, 1.0 mM DTT, pH 7.5); dilute PDE4D protein (recombinantly expressed by our research group; specific methods can be found in the published literature: Biochemical Pharmacology 130(2017)51–59) with Assaybuffer to an appropriate fold and place on ice; dilute the substrate with Assaybuffer. 3 H-cAMP was diluted to a suitable concentration (the control group reading was approximately 20,000 cpm); the test compound was dissolved and diluted with DMSO to the set concentration; 58 μL of the diluted substrate was transferred to a 1.5 mL EP tube, 2 μL of the prepared test compound at the set concentration was added, and the mixture was thoroughly mixed. Then, 40 μL of the diluted enzyme solution was added, and the mixture was gently mixed. The mixture was then allowed to stand at room temperature for 15 min; 200 μL of 0.2 M ZnSO4 (to terminate the reaction) and 0.2 N Ba(OH)2 solution (to precipitate the ring-opening reaction product) were added to the reaction system sequentially. 3 H-cAMP was centrifuged at high speed (14800 rpm) for 5 min, and 430 μL containing unreacted cAMP was collected. 3 The supernatant of H-cAMP was added to 2 mL of scintillation fluid, thoroughly vortexed, and the radioactivity reading was measured using a liquid scintillation counter. Each experiment included a substrate group (C0) and a target protein group (C1). E ), positive control group (coripram, C P ) and inhibitor group (C I The inhibition rate of the inhibitor is given by the formula Inhibition Rate = [1 - (C0 - C)] / [C0 - C] I ) / (C0-C E The specific IC50 of the inhibitor was calculated as 100%. 50 Value (IC) 50When the inhibitor concentration (value at which the inhibition rate reaches 50%) is used, 8-10 half-dilution concentration gradients are set based on the initial inhibition rate results. After measuring the inhibition rate at each gradient, the specific IC50 is obtained by nonlinear fitting using GraphPadPrism software. 50 The values ​​were calculated and the mean and standard deviation were obtained by independently repeating the experiment three times. The results of the inhibitory activity of Selaginella uncinata extract against PDE4D are shown in Table 1 below.

[0037] Table 1. Inhibitory activity of Selaginella uncinata extract against PDE4D

[0038] Total Ethanol Extraction 126.38％ 113.35％ 99.60％ 28.98％ water 105.47％ 99.81％ 25.72％ n-Butanol 106.91％ 117.25％ 100.84％ 66.68％ Ethyl acetate 112.52％ 110.48％ 92.82％ 28.02％ petroleum ether 35.61％ 9.78％

[0039] II. Isolation and Identification of Biflavonoids from Selaginella uncinata and Their Inhibitory Activity Against PDE4

[0040] (I) Preparation of biflavonoid compounds 1-13

[0041] The ethyl acetate extract with the best PDE4 inhibitory activity was selected and initially fractionated using an MCI column. Elution was performed with methanol:water = 1:9 → 10:0. The fractions were merged into four fractions (Fr.A-Fr.D) by TLC. The fractions were then repeatedly purified using an ODS column and finally further purified by high performance liquid chromatography under (acetonitrile:water) and (methanol:water) conditions to obtain 13 biflavonoid monomers.

[0042] Specifically, the preparation method of compounds 1-13 includes the following steps:

[0043] S1: The fraction Fr.C was separated by an ODS column to obtain two fractions, Fr.C1 and Fr.C2. Fr.C1 was purified by HPLC using methanol-water (containing 0.1% acetic acid, 50:50, 6 mL / min) as the mobile phase and 254 nm as the detection wavelength, yielding compound 1 (200 mg, t). R =13 min). The Fr.C2 fraction was separated by ODS silica gel column chromatography with a methanol:water gradient of 3:7 to 10:0. After TLC analysis, the fractions were combined into 15 fractions (Fr.C2a-Fr.C2o). The Fr.C2m fraction was subjected to silica gel column chromatography with a dichloromethane:methanol gradient of 100:1 to 10:1. After TLC analysis, compound 9 (1.2 mg) was obtained. The Fr.C2h fraction was purified by HPLC using methanol-water (containing 0.1% acetic acid, 60:40, 2 mL / min) as the mobile phase and 254 nm as the detection wavelength, yielding compound 10 (5.2 mg, t). R=26 min). The Fr.C2g fraction was purified by HPLC using acetonitrile-water (containing 0.1% acetic acid, 40:60, 2 mL / min) as the mobile phase and 254 nm as the detection wavelength, yielding compound 11 (4.5 mg, t). R =18min) and compound 12 (3.6mg, t R =27min).

[0044] S2: Fraction Fr.D was separated by ODS silica gel column chromatography using a methanol:water gradient of 5:5 → 10:0. The fractions were analyzed by TLC and combined into 14 fractions (Fr.D1–Fr.D14). Fraction Fr.D4 was further purified by HPLC using acetonitrile-water (containing 0.1% acetic acid, 45:55, 2 mL / min) as the mobile phase and 254 nm as the detection wavelength, yielding compound 2 (1.8 mg, t). R =16min), compound 3 (5.2mg, t R =21min) and compound 4 (1.7mg, t R =25 min). The Fr.D5 fraction was purified by HPLC using acetonitrile-water (containing 0.1% acetic acid, 45:55, 2 mL / min) as the mobile phase and 254 nm as the detection wavelength, yielding compound 5 (5.5 mg, t). R =29min) and compound 13 (3.9mg, t R =34 min). The Fr.D6 fraction was further separated and purified by HPLC. The mobile phase was acetonitrile-water (containing 0.1% acetic acid, 50:50, 2 mL / min), and the detection wavelength was 254 nm, yielding compound 6 (3.8 mg, t). R =30min) and compound 8 (8.6mg, t R =35 min). The Fr.D7 fraction was further separated and purified by HPLC. The mobile phase was acetonitrile-water (containing 0.1% acetic acid, 50:50, 2 mL / min), and the detection wavelength was 254 nm, yielding compound 7 (3.1 mg, t). R =45min).

[0045] (II) Structure and identification of biflavonoid compounds 1-13

[0046] (1) The structure and NMR data of compound 1 are as follows:

[0047]

[0048] 1 H NMR (DMSO-d6, 400MHz): δ H13.10(1H,s,OH-5″),12.97(1H,s,OH-5),10.79(1H,s,OH-7),10.26(1H,s,4′″-OH ),8.01(1H,d,J=2.0Hz,H-2′),8.00(1H,d,J=8.5Hz,H-6′),7.57(2H,d,J=8.5Hz,H -2″′,6″′),7.16(1H,d,J=8.5Hz,H-5′),6.82(1H,s,H-3″),6.77(1H,s,H-3),6.73 (2H,d,J=8.5Hz,H-3″′,5″′),6.47(1H,d,J=2.0Hz,H-8),6.20(1H,d,J=2.0Hz,H-6)

[0049] (2) The structure and NMR data of compound 2 are as follows:

[0050]

[0051] 1 H NMR (DMSO-d6, 400MHz): δ H 13.08(1H,s,OH-5″),12.98(1H,s,OH-5),8.03(1H,dd,J=8.5,2.0,H-6′),8.02( 1H,d,J=2.0,H-2′),7.55(2H,d,J=8.5,H-2″′,6′″),7.17(1H,d,J=8.5,H-5′),6 .87(1H,s,H-3),6.77(1H,s,H-3″),6.75(1H,d,J=2.0Hz,H-8),6.73(2H,d,J=8. 5,H-3″′,5′″),6.42(1H,s,H-6″),6.32(1H,s,J=2.0Hz,H-6),3.80(3H,s,OCH3).

[0052] (3) The structure and NMR data of compound 3 are as follows:

[0053]

[0054] 1 H NMR (DMSO-d6, 400MHz): δ H13.09(1H,s,OH-5″),12.92(1H,s,OH-5),8.19(1H,dd,J=8.5,2.0Hz,H-6′),8.06( 1H,d,J=2.0Hz,2′),7.50(2H,d,J=8.5Hz,H-2″′,6″′),7.35(1H,d,J=8.5Hz,H-5′), 6.92(1H,s,H-3″),6.80(1H,s,H-3),6.72(2H,d,J=8.5Hz,H-3″′,5″′),6.50(1H,d ,J=2.0Hz,H-8),6.42(1H,s,H-6'),6.20(1H,d,J=2.0Hz,H-6″),3.78(3H,s,4′-OCH 3, )

[0055] (4) The structure and NMR data of compound 4 are as follows:

[0056]

[0057] 1 H NMR (DMSO-d6, 400MHz): δ H 3.76(3H,s,OCH3),6.19(1H,d,J=2.0Hz,H-6),6.45(1H,d,J=2.0Hz,H-8 ),6.83(1H,s,H-3),7.16(1H,d,J=8.5Hz,H-5′),8.02(1H,d,J=2.0Hz,H- 2′),8.02(1H,dd,J=8.5,2.0Hz,H-6′),6.41(1H,s,H-6″),6.89(1H,s,H- 3″),6.93(2H,d,J=8.5Hz,H-3′″,5′″),7.69(2H,d,J=8.5Hz,H-2″′,6″′)

[0058] (5) The structure and NMR data of compound 5 are as follows:

[0059]

[0060] 1 H NMR (DMSO-d6, 400MHz): δ H13.08(1H,s,OH-5″),12.98(1H,s,OH-5),8.02(1H,d,J=2.0Hz,H-2′),7.99(1H,dd,J =8.5,2.0Hz,H-6′),7.69(2H,d,J=8.5Hz,H-2″′,H-6″′),7.12(1H,d,J=8.5Hz,H-5′), 6.89(2H,d,J=8.5Hz,H-3″′,H-5″′),6.87(1H,s,H-3″),6.81(1H,s,H-3),6.42(1H,d ,J=2.0Hz,H-8),6.36(1H,s,H-6″),6.18(1H,d,J=2.0Hz,H-6),3.73(3H,s,4″′-OCH3)

[0061] (6) The structure and NMR data of compound 6 are as follows:

[0062]

[0063] 1 H NMR (DMSO-d6, 400MHz): δ H 13.08(1H,s,OH-5″),12.91(1H,s,OH-5),8.22(1H,dd,J=8.5,2.0Hz,H-6′),8.09(1 H,d,J=2.0Hz,H-2′),7.49(2H,d,J=8.5Hz,H-2″′,H-6″′),7.36(1H,d,J=8.5Hz,H-5 ′),7.00(1H,s,H-3″),6.80(1H,s,H-8),6.79(1H,s,H-3),6.72(2H,d,J=8.5Hz,H-3 ″′,H-5″′),6.39(1H,s,6″),6.36(1H,s),3.83(3H,s,7-OCH3),3.79(3H,s,4′-OCH3)

[0064] (7) The structure and NMR data of compound 7 are as follows:

[0065]

[0066] 1 H NMR (DMSO-d6, 400MHz): δ H13.06(1H,s,OH-5″),12.96(1H,s,OH-5),8.04(1H,s,H-2′),8.02(1H,d,J=8.5,2.0H z,H-6′),7.68(2H,d,J=8.5Hz,H-2″′,H-6″′),7.15(1H,d,J=8.5Hz,H-5′),6.91(2H,d ,J=8.5Hz,H-3″′,H-5″′),6.89(1H,s,H-3″),6.87(1H,s,H-3),6.73(1H,s,H-8),6.40 (1H,s,H-6″),6.34(1H,d,J=2.0Hz,H-6),3.81(3H,s,OCH3-7),3.74(3H,s,OCH3-4′″)

[0067] (8) The structure and NMR data of compound 8 are as follows:

[0068]

[0069] 1 H NMR (DMSO-d6, 400MHz): δ H 13.05(1H,s,OH-5″),12.92(1H,s,OH-5),8.18(1H,dd,J=8.5,2.0Hz,H-6′),8.05(1H,d,J=2.0H z,H-2′),7.61(2H,d,J=8.5Hz,H-2″′,H-6″′),7.35(1H,d,J=8.5Hz,H-5′),6.93(2H,d,J=8.5Hz ,H-3″′,H-5″′),6.91(1H,s,J=2.0Hz,H-3″),6.88(1H,d,J=2.0Hz,H-3),6.48(1H,d,J=2.0Hz,H -8),6.42(1H,s,H-6″),6.20(1H,d,J=2.0Hz,H-6),3.79(3H,s,OCH3-4′),3.75(3H,s,OCH3-4′″)

[0070] (9) The structure and NMR data of compound 9 are as follows:

[0071]

[0072] 1 H NMR (DMSO-d6, 400MHz): δ H13.08(1H,s,OH-5″),12.94(1H,s,OH-5),8.18(1H,dd,J=8.5.2.0Hz,H-6′),8.04(1H,dd, J=2.0Hz,H-2′),7.62(2H,dd,J=8.5,H-2",6"),7.38(1H,d,J=8.5Hz,H-5),7.02(1H,s,H- 3″),7.00(2H,d,J=8.5,H-3'"5"),6.90(1H,s,H-3),6.70(1H,d,J=2.0,H-8),6.48(1H,s, H-6"),6.21(1H,d,J=2.0Hz,H-6),3.91(3H,s,OCH3),3.85(3H,s,OCH3),3.80(3H,s,OCH3)

[0073] (10) The structure and NMR data of compound 10 are as follows:

[0074]

[0075] 1 H NMR (Methanol-d4, 400MHz): δ H 7.59(2H,d,J=8.5Hz,H-2″′,H-6″′),7.46(1H,s,H-2′),7.44(1H,d,J=2.0Hz,H -6′),7.07(1H,d,J=8.5Hz,H-5′),6.81(2H,d,J=8.5Hz,H-3″′,H-5″′),6.61(1 H,s,H-3″),6.37(1H,s,H-6″),5.91(1H,s,H-8),5.88(1H,s,H-6),5.44(1H,d, J=12.6Hz,H-2),3.18(1H,dd,J=17.0,12.6Hz,H-3),2.77(1H,d,J=17.0Hz,H-3)

[0076] (11) The structure and NMR data of compound 11 are as follows:

[0077]

[0078] 1 H NMR (DMSO-d6, 400MHz): δ H 13.14(1H,s,5-OH),7.45(2H,d,J=8.5Hz,H-2′,6-′),6.75(1H,s,H-8),6.72(2H,d,J=8.5Hz,H-3′,H-5′),6.41(1H,s,H-6)

[0079] (12) The structure and NMR data of compound 12 are as follows:

[0080]

[0081] 1 H NMR (DMSO-d6, 400MHz): δ H 13.28(1H,s,OH-5″),13.03(1H,s,OH-5),7.99(2H,d,J=8.5Hz,H-2′,6′),7.55(2H,d,J=8.5Hz,H-2″′,6″′),6.97(2H,d,J=8.5Hz ,H-3′,5′),6.84(1H,s,H-3),6.76(1H,d,J=2Hz,H-8),6.76(2H,d,J=8.5Hz,H-3″′,H-5″′),6.72(1H,s,H-3″),6.37(1H,s,H-6″)

[0082] (13) The structure and NMR data of compound 13 are as follows:

[0083]

[0084] 1 H NMR (DMSO-d6, 400MHz): δ H 13.18(1H,s,OH-5″),12.88(1H,s,OH-5),7.99(2H,d,J=8.5Hz,H-2′,6′),7.94(2H,d,J=8.5Hz,H-2′″,6′″),7.02(2H,d,J=8.5Hz,H- 3′,5′),6.93(2H,d,J=8.5Hz,H-3′″,5′″),6.83(1H,s,H-3),6.79(1H,s,H-3″),6.62(1H,s,H-8″),6.47(1H,s,H-8),6.19(1H,s,H-6)

[0085] After obtaining the above 13 biflavonoid compounds, their inhibitory activity against PDE4 was tested using the same method (PDE4 enzyme activity was determined by liquid scintillation counting). As shown in Table 2 below, all 13 biflavonoid compounds exhibited good inhibitory activity against PDE4. Among them, compound 1 showed the best inhibitory activity against PDE4 (IC50). 50 Reaching 12nM Figure 1 As shown, and further analysis of the eutectic structure confirmed that compound 1 was tightly bound within the catalytic pocket of the target PDE4D, as... Figure 2 As shown.

[0086] Table 2. Inhibitory activity of biflavonoid monomers isolated from Selaginella uncinata against PDE4D.

[0087]

[0088]

[0089] Example 2: Extraction and separation process of Platycladus orientalis and its inhibitory activity against PDE4

[0090] I. Preparation of Platycladus orientalis extract and its inhibitory activity against PDE4

[0091] Weigh out dried arborvitae leaves, crush them into coarse powder, add 10 times their volume of methanol and soak them 3 times, the first time for 2 days, the second and third times for 1 day each, filter, combine the filtrates, recover ethanol under reduced pressure, concentrate the remaining liquid to a thick paste, and obtain the total methanol extract of arborvitae.

[0092] The methanol extract of Platycladus orientalis was suspended in 4 times its weight of water and extracted sequentially with an equal volume of petroleum ether, ethyl acetate, and n-butanol (each solvent was extracted 3 times consecutively) to obtain petroleum ether, ethyl acetate, n-butanol extracts, and an aqueous phase solution. These were evaporated to dryness under reduced pressure to obtain petroleum ether extract, ethyl acetate extract, n-butanol extract, and aqueous concentrate. PDE4D inhibitory activity was then tested using the same method as described above (PDE4 enzyme activity was determined using liquid scintillation counting). The results are shown in Table 3 below.

[0093] Table 3. Inhibitory activity of Platycladus orientalis extract against PDE4D

[0094]

[0095]

[0096] II. Isolation and Identification of Biflavonoids from Platycladus orientalis and Their Inhibitory Activity Against PDE4

[0097] (I) Preparation of biflavonoid compounds A and B

[0098] The ethyl acetate extract with the best PDE4 inhibitory activity was selected. The components of the ethyl acetate extract were first preliminarily identified by HPLC (mobile phase method 0-30 min, water:acetonitrile = 92:8 → 10:90; 30-40 min, water:acetonitrile = 10:90 → 10:90). The chromatographic results confirmed that the ethyl acetate extract contained 5 major components (Fr.A-Fr.E). The extract was then purified using a C18 reversed-phase silica gel column and finally further purified by preparative liquid chromatography under (acetonitrile:water) conditions to obtain 5 flavonoid monomers.

[0099] Specifically, the preparation and identification methods of compounds Fr.A-Fr.E include the following steps:

[0100] S1: Dissolve 1 g of ethyl acetate extract of Platycladus orientalis in 100 mL of methanol, and evaporate under reduced pressure to remove the small polar solvents from the sample. Repeat this step. Dissolve the obtained sample in 100 mL of methanol, add three times the mass of C18 silica gel powder, mix well, and evaporate under reduced pressure to obtain dry silica gel powder. Use water-acetonitrile as the mobile phase (elution ratio of water:acetonitrile = 9:1 → 10:0, 15 mL / min, 500 mL), and a detection wavelength of 254 nm to perform preliminary purification of five compounds. When the water / acetonitrile ratio is 20%, fraction Fr.A is obtained. Maintaining the solvent gradient, after complete peak elution, the solvent gradient is increased. When the water / acetonitrile ratio is 34%, fraction Fr.B is obtained. Maintaining the solvent gradient, after complete peak elution, the solvent gradient is increased. When the water / acetonitrile ratio is 45%, fraction Fr.C is obtained. Maintaining the solvent gradient, after complete peak elution, the solvent gradient is increased. When the water / acetonitrile ratio is 68%, fraction Fr.D is obtained. Maintaining the solvent gradient, after complete peak elution, the solvent gradient is increased. When the water / acetonitrile ratio is 78%, fraction Fr.E is obtained. Maintaining the solvent gradient, the solvent is collected, and the product is evaporated under reduced pressure to obtain a dry powder.

[0101] Small amounts of the obtained fractions Fr.A-Fr.E were dissolved in chromatographic grade methanol, and the purity of each component was confirmed by HPLC. The chromatograms were compared with those of the ethyl acetate extract to confirm the consistency of peak times. Each fraction was further purified by preparative liquid chromatography (using a consistent mobile phase and method). Repeated purification yielded compounds A, B, C, D, and E. Among them, the biflavonoid compounds A and B isolated from Platycladus orientalis were structurally and NMR consistent with compounds 1 and 13 isolated from Selaginella uncinata, indicating the presence of common pharmacologically active components.

[0102] (II) Structure and identification of biflavonoid compounds A and B

[0103] (1) The structure and NMR data of compound A are as follows:

[0104]

[0105] 1 H NMR (DMSO-d6, 400MHz): δ H13.10(1H,s,OH-5″),12.97(1H,s,OH-5),10.79(1H,s,OH-7),10.26(1H,s,4′″-OH ),8.01(1H,d,J=2.0Hz,H-2′),8.00(1H,d,J=8.5Hz,H-6′),7.57(2H,d,J=8.5Hz,H -2″′,6″′),7.16(1H,d,J=8.5Hz,H-5′),6.82(1H,s,H-3″),6.77(1H,s,H-3),6.73 (2H,d,J=8.5Hz,H-3″′,5″′),6.47(1H,d,J=2.0Hz,H-8),6.20(1H,d,J=2.0Hz,H-6)

[0106] (2) The structure and NMR data of compound B are as follows:

[0107]

[0108] 1 H NMR (DMSO-d6, 400MHz): δ H 13.18(1H,s,OH-5″),12.88(1H,s,OH-5),7.99(2H,d,J=8.5Hz,H-2′,6′),7.94(2H,d,J=8.5Hz,H-2′″,6′″),7.02(2H,d,J=8.5Hz,H- 3′,5′),6.93(2H,d,J=8.5Hz,H-3′″,5′″),6.83(1H,s,H-3),6.79(1H,s,H-3″),6.62(1H,s,H-8″),6.47(1H,s,H-8),6.19(1H,s,H-6)

[0109] The inhibitory activity of the two biflavonoid compounds against PDE4 was tested using the same method. The test results were the same as those of compounds 1 and 13 isolated from Selaginella uncinata, which inhibited PDE4. Their IC50 (nM) values ​​are not repeated here. This indicates that compounds A and B isolated from Platycladus orientalis have the same efficacy as compounds 1 and 13 isolated from Selaginella uncinata, and can all inhibit PDE4.

[0110] III. Methods for isolating effective components from Platycladus orientalis and their PDE4 inhibitory activity

[0111] Macroporous resin AB-8 was selected and soaked in anhydrous ethanol with a volume 2 times that of the resin for 24 hours to activate the macroporous resin. The activated macroporous resin was loaded into a glass column with a sand mold, and the macroporous resin was rinsed with a large amount of distilled water until there was no ethanol smell in the rinsing solution. The methanol extract of Platycladus orientalis was weighed and dissolved in anhydrous ethanol, and then slowly dripped onto the surface of the macroporous resin and adsorbed for one hour. Subsequently, it was eluted successively with 100% distilled water, 30% ethanol / water, 70% ethanol / water, and 100% ethanol, collected in batches, evaporated under reduced pressure, and the obtained components were quantitatively analyzed by HPLC. The elution fraction with a higher content of the selected compound A was 100% ethanol, and the PDE4 inhibitory activity test was carried out. The same test method (the activity of PDE4 enzyme was determined by liquid scintillation counting method) was used for the test, and the results are shown in Table 4 below.

[0112] Table 4 Inhibitory activity of the effective part separated by macroporous resin of Platycladus orientalis on PDE4D

[0113] Effective components for separating macroporous resins from Platycladus orientalis 105.44％ 74.23％

[0114] Example 3 Therapeutic application of ethyl acetate extract of Platycladus orientalis in diseases related to phosphodiesterase type 4 I. Anti-psoriatic effect of ethyl acetate extract of Platycladus orientalis in vivo

[0115] The Platycladus orientalis extract with inhibitory effect on PDE4 obtained by extraction was selected as the test sample to evaluate its therapeutic effect in a psoriasis animal model. The specific experimental materials, methods and results are described as follows.

[0116] (I) Experimental materials

[0117] SPF-grade BALB / c mice, male, 6 - 8 weeks old, weighing 18 - 22 g, were purchased from Beijing SPF Biotechnology Co., Ltd. (License number: SCXK (Beijing) 2019 - 010). Ambient temperature: 20 - 25 °C, humidity 50 - 70%, free access to food and water. Imiquimod (Hubei Keyi Pharmaceutical Co., Ltd., 5%), Calcipotriol (LEO Laboratories Limited).

[0118] (II) Experimental methods

[0119] BALB / c mice were randomly divided into 4 groups with 6 mice in each group. Group A was the normal control group, Group B was the imiquimod-induced model group (IMQ, 62.5 mg applied topically on the back), Group C was the imiquimod + positive drug calcipotriol group (IMQ + Calcipotriol), and Group D was the imiquimod + 10% ethyl acetate extract of Platycladus orientalis group (IMQ + 10% Platycladus orientalis extract). The back of the mice was selected as the experimental observation area. One day before the experiment, the hair on the back surface of the mice was shaved off to expose a hairless area of 2 cm × 3 cm. The control group was applied with 62.5 mg of petrolatum daily, and the mice in the other groups were given 62.5 mg of 10% imiquimod ointment daily. After the drug was absorbed, the drug was administered. The skin lesions of the mice were observed daily, photographed, and scored according to the PASI criteria (graded from 0 to 4) to evaluate the therapeutic effect of the compound on psoriatic mice.

[0120] (III) Experimental results

[0121] As Figure 3 shown, after imiquimod was given to the back of the mice, psoriasis-like symptoms such as erythema and scales appeared, indicating successful model establishment. After the ethyl acetate extract of Platycladus orientalis was given, the psoriasis-like symptoms such as erythema, scales, and thickening in the mice were improved, indicating that the ethyl acetate extract of Platycladus orientalis can play a role in treating psoriasis.

[0122] II. In vivo anti-psoriasis effects of the effective fraction separated by macroporous resin from Platycladus orientalis and Compound 1 (Compound A)

[0123] The effective fraction separated by macroporous resin from Platycladus orientalis and Compound 1, the PDE4 inhibitor obtained by extraction and separation, were selected as representatives to evaluate their therapeutic effects in psoriasis cell models and animal models. The specific experimental materials, methods, and results are described as follows.

[0124] (1) Experimental materials

[0125] SPF-grade BALB / c mice, male, 6 - 8 weeks old, weighing 18 - 22 g, were purchased from Beijing SPF Biotechnology Co., Ltd. (License number: SCXK(Beijing)2019 - 010). Ambient temperature: 20 - 25°C, humidity 50 - 70%, with free access to food and water. Imiquimod (IMQ, Hubei Keyi Pharmaceutical Co., Ltd., 5%), Apremilast (Bidepharm).

[0126] (2) Experimental methods

[0127] BALB / c mice were randomly divided into four groups of six mice each: Group A - normal control group; Group B - imiquimod modeling group (IMQ, 62.5 mg applied to the back); Group C - imiquimod + positive control drug apremilast (IMQ + Apremilast); Group D - imiquimod + arborvitae macroporous resin for effective fraction separation (IMQ + PL-MacRe); and Group E - imiquimod + compound 1 (IMQ + 1). The back of the mice was selected as the experimental observation area. One day before the experiment, the hair on the back of the mice was shaved to expose a 2cm × 3cm hairless area. The control group received 62.5 mg of petrolatum daily, while the other groups received 62.5 mg of 10% imiquimod ointment daily, administered after absorption. The skin lesions of the mice were observed and photographed daily, and scored according to the PASI criteria (0-4) to evaluate the therapeutic effect of the compounds on psoriatic mice.

[0128] (3) Experimental Results

[0129] a) The effective components and compound 1 isolated from the macroporous resin of Platycladus orientalis significantly improved psoriasis-like symptoms in imiquimod-induced mice.

[0130] like Figure 4 As shown in (A)-(E), mice developed psoriasis-like symptoms such as erythema and scaling after being given imiquimod on their backs, indicating that the model was successfully established. When mice were given apremilast, the effective fraction isolated from arborvitae macroporous resin and compound 1, the symptoms of erythema, scaling and hypertrophy in the mice were improved, indicating that arborvitae extract can play a role in the treatment of psoriasis.

[0131] b) HE staining showed that the effective components and compound 1 isolated from the macroporous resin of Platycladus orientalis significantly improved the pathological condition of the dorsal skin in imiquimod-induced model mice.

[0132] like Figure 4 (F) HE staining showed that on the seventh day of the experiment, the epidermis of the normal group consisted of only 1 to 3 layers of cells, and the sebaceous glands / hair follicles were elongated and hollow. The epidermis of the model group and the drug-treated group was 2 to 3 times thicker than that of the normal group. Parakeratosis was observed in the stratum corneum, and inflammatory cell infiltration was observed in the dermis, accompanied by many cell fragments. The effective components separated from the macroporous resin of Platycladus orientalis and Compound 1 could reduce the thickness of the epidermis. The epidermis was close to that of the normal control group, the dermis had slightly more cell accumulation, and the sebaceous gland structure remained intact.

[0133] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The use of cypress family plant extracts in the preparation of drugs for the treatment and / or prevention of psoriasis, wherein the cypress family plant extract is Platycladus orientalis extract; wherein the Platycladus orientalis extract is ethyl acetate extract of Platycladus orientalis macroporous resin from which the effective fraction is separated; The preparation method of the ethyl acetate extract of Platycladus orientalis is as follows: weigh dried Platycladus orientalis leaves, crush them into coarse powder, and soak them three times with 8-12 times their volume of methanol solution. The first extraction takes 2-5 days, and the second and third extractions each take 1-3 days. The mixture is filtered, and the filtrates are combined. The effective components of the alcohol are recovered under reduced pressure, and the remaining liquid is concentrated to a thick paste to obtain the methanol extract of Platycladus orientalis. The methanol extract of Platycladus orientalis is suspended in 4 times its weight of water and extracted three times consecutively with petroleum ether, ethyl acetate, and n-butanol in equal volumes to water, respectively, to obtain petroleum ether, ethyl acetate, n-butanol extracts and an aqueous phase solution. The ethyl acetate extract is evaporated to dryness under reduced pressure to obtain the ethyl acetate extract. The method for separating the effective components of the macroporous resin of Platycladus orientalis is as follows: Step 1: Weigh the dried leaves of Platycladus orientalis, crush them into coarse powder, and soak them three times with 8-12 times the volume of methanol solution. The first soaking time is 2-5 days, and the second and third soaking times are 1-3 days each. Filter the solution, combine the filtrates, and recover the effective components of the alcohol under reduced pressure. Concentrate the remaining liquid to a thick paste to obtain the methanol extract of Platycladus orientalis containing the effective components. Step 2: Take macroporous resin AB-8, add 2-5 times the volume of anhydrous ethanol to soak it, and activate it for 12-48 hours. Then, pack the activated macroporous resin into a glass column with a sand core and rinse the macroporous resin with plenty of distilled water until there is no ethanol smell in the rinsing solution. Step 3: Weigh the methanol extract of Platycladus orientalis, dissolve it in anhydrous ethanol, and slowly add it dropwise to the surface of macroporous resin. Adsorb for one hour, then elute with 100% distilled water, 30% ethanol / water, 70% ethanol / water, and 100% ethanol in sequence. Collect in batches, evaporate under reduced pressure, and you can obtain the refined extract of the effective part of Platycladus orientalis.

2. A medicine for treating and / or preventing psoriasis, characterized in that, The drug comprises the arborvitae extract of claim 1, and further comprises pharmaceutically acceptable excipients and carriers.

3. The drug according to claim 2, characterized in that, The dosage form of the drug is capsule, tablet, pill, granule, powder, or spray.

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