CN118319964A - Cupressaceae plant extract, and preparation method and application thereof - Google Patents

Abstract

The invention relates to a natural extract, in particular to a Cupressaceae plant extract, a preparation method and application thereof, wherein the Cupressaceae plant extract is a Cupressaceae plant extract or a Platycladus orientalis extract. The extract and the biflavanoid compound thereof have obvious PDE4 enzyme inhibition activity, can be used for preparing PDE4 enzyme inhibitors, and are used for treating and/or preventing diseases related to type 4 phosphodiesterase.

Description

Cupressaceae plant extract and its preparation method and application

Technical Field

The invention relates to a natural extract, in particular to a Cupressaceae plant extract and a preparation method and application thereof.

Background technique

Cyclic nucleotide phosphodiesterases (PDEs) are hydrolases that metabolize cGMP and cAMP in the human body, effectively controlling the concentrations of cGMP and cAMP in cells, thereby regulating the biochemical effects transmitted by second messengers in the body.

Among them, phosphodiesterase type 4 (PDE4) specifically hydrolyzes cAMP into 5'-AMP, regulates the cAMP concentration in cells to be within an appropriate range, and maintains normal physiological activities. PDE4 is mainly distributed in various inflammatory cells, immune cells and airway smooth muscle cells. Inflammatory cells can be inhibited by inhibiting the activity of PDE4. The PDE4 family can be divided into four subtypes (PDE-4ABCD), among which PDE-4B is the main anti-inflammatory target, and PDE-4D is related to adverse reactions such as gastrointestinal discomfort. At present, PDE4 inhibitors have been developed into anti-inflammatory drugs, such as Roflumilast, etc., which are mainly used clinically to treat lung inflammation, especially for the treatment of asthma and chronic obstructive pulmonary disease. However, taking such drugs can cause gastrointestinal adverse reactions such as diarrhea and nausea, which limits their use. Therefore, how to overcome the adverse reactions of these PDE4 inhibitors and study new specific inhibitors has become one of the hot spots of research.

Selaginella uncinata is the whole herb of Selaginella uncinata (Desv.) Spring, a plant of the genus Selaginella in the family Selaginaceae. It is also known as phoenix tail grass, blue cypress, resurrection grass, and dragon scale grass. It has traditional effects such as clearing heat and dampness, detoxifying and removing blood stasis. It is used by the people to treat jaundice, dysentery, edema, rheumatic pain, coughing and vomiting blood, throat carbuncle, hemorrhoid fistula, knife wounds, burns, etc. Modern pharmacological experimental studies have shown that Selaginella uncinata has anti-inflammatory, anti-asthmatic, anti-viral, anti-hypoxic, and anti-tumor effects. The main chemical components are biflavonoids, fatty acids, phenolic acids, steroidal saponins, etc. Biflavonoid compounds are characteristic components of Selaginellaceae plants, and have pharmacological effects such as anti-hyperglycemia, anti-tumor, anti-viral, and anti-hypoxia. Overall, the research on Selaginella uncinata extracts is still mostly focused on anti-tumor and anti-inflammatory research, and there is no research on it as a PDE4 inhibitor. Therefore, it is very necessary to further develop and utilize Selaginella uncinata extracts.

Platycladus orientalis (L.) Franco is a tree of the genus Platycladus in the Cupressaceae family. Platycladus orientalis leaves are also known as cypress leaves, cypress leaves, and cypress leaves. The leaves are small and scaly, attached to flat branches, alternate and opposite, dark green or yellow-green, brittle and easy to break, and yellow-white in cross section. Platycladus orientalis is widely distributed and has a long history of cultivation. Literature records that Platycladus orientalis leaves have a fragrant smell and a bitter taste. They belong to the lung, liver, and spleen meridians and have the effects of relieving cough and reducing phlegm, promoting hair growth, and stopping bleeding. Platycladus orientalis leaves contain many types of medicinal active ingredients, among which flavonoids, volatile oils, and tannins have been studied more; they have multiple pharmacological effects such as antibacterial, anti-tumor, hemostasis, hair growth promotion, and anti-inflammatory.

Therefore, natural products are one of the important sources 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

In view of the technical defects existing in the above-mentioned prior art, the object of the present invention is to provide the use of extracts of Herba Lycopodii and Platycladus orientalis and the biflavonoid compounds therein in the treatment of type 4 phosphodiesterase-related diseases.

The above-mentioned purpose of the present invention is achieved through the following technical solutions:

The first aspect of the present invention provides the use of a Cupressaceae plant extract in treating and/or preventing type 4 phosphodiesterase-related diseases, wherein the Cupressaceae plant extract is a Herba Corydalis extract or a Platycladus orientalis extract.

Furthermore, the Herba Eupatorii extract is obtained by alcohol extraction of the Herba Eupatorii, and the alcohol extraction method is as follows: weigh dry leaves of the Herba Eupatorii, crush them into coarse powder, add 8-12 times the volume of alcohol solution and soak them three times respectively, wherein the first time is 8-15 days, and the second and third times are 4-7 days respectively, filter, combine the filtrate, recover the effective ingredients of the alcohol under reduced pressure, and concentrate the remaining liquid to a thick paste to obtain the Herba Eupatorii extract.

Furthermore, the Platycladus orientalis extract is obtained by extracting the Platycladus orientalis alcohol, and the alcohol extraction method is as follows: weigh the Platycladus orientalis dry leaves, crush them into coarse powder, add 8-12 times the volume of alcohol solution and soak them three times respectively, wherein the first time is 2-5 days, and the second and third times are 1-3 days respectively, filter, combine the filtrate, recover the effective components of the alcohol under reduced pressure, and concentrate the remaining liquid to a thick paste to obtain the Platycladus orientalis extract containing the effective parts.

Furthermore, the effective part of the Platycladus orientalis extract is separated by macroporous resin, and the separation method is as follows: Step 1: Take macroporous resin AB-8, add 2-5 times the volume of anhydrous ethanol to soak, activate for 12-48 hours, load the activated macroporous resin into a glass column with a sand core, and rinse the macroporous resin with a large amount of distilled water until there is no ethanol smell in the rinse liquid; Step 2: Weigh the Platycladus orientalis extract, dissolve it with anhydrous ethanol, slowly drip it on the surface of the macroporous resin, adsorb for one hour, and then elute with 100% distilled water, 30% ethanol/water, 70% ethanol/water, and 100% ethanol in turn, collect it in batches, and evaporate it under reduced pressure to obtain the refined extract of the effective part of Platycladus orientalis.

The second aspect of the present invention provides the use of the biflavonoid compounds in the Cupressaceae plant extract in the treatment and/or prevention of phosphodiesterase type 4 related diseases.

Furthermore, the structure of the biflavonoid compound is selected from one of the compounds represented by formula (I)-(V):

in:

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

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

The third aspect of the present invention provides an application in the preparation of a health product or food for treating and/or preventing phosphodiesterase type 4 related diseases, wherein the health product or food comprises the above-mentioned Platycladus orientalis extract.

The fourth aspect of the present invention provides a drug for treating and/or preventing type 4 phosphodiesterase-related diseases, wherein the drug comprises one or more of the above-mentioned Cupressaceae plant extracts or biflavonoid compounds having structures shown in any one of formulas (I) to (V), and also includes pharmaceutically acceptable excipients and carriers.

Furthermore, the dosage form of the drug is capsule, tablet, pill, granule, granule or spray.

The present invention has the following beneficial effects:

The present invention conducts in-depth research on extracts of Herba Lycopodii and Platycladus orientalis, including in vivo and in vitro activity evaluation of the extracts, and uses a variety of column chromatography separation techniques and semi-preparative high performance liquid chromatography separation methods to extract and separate a series of biflavonoid compounds. 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 associated with type 4 phosphodiesterase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is an inhibition curve of compound 1 of the present invention on PDE4D;

FIG2 is a co-crystal structure of a representative compound 1 of the present invention and PDE4D;

FIG3 shows the anti-psoriasis efficacy of the Platycladus orientalis ethyl acetate extract of the present invention on an IMQ-induced psoriasis mouse model;

FIG. 4 shows the efficacy of the effective fraction separated by the Platycladus orientalis macroporous resin of the present invention and compound 1 on a mouse psoriasis model.

Detailed ways

The present invention is further described below with reference to the examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

In the following examples, NMR spectra were recorded using a Bruker 400 MHz NMR spectrometer with TMS as internal standard. Mitsubishi Chemical MCI filler (CHP20P, 75-150 μM), YMC ODS filler (12 nm, S-50 μM); other solvents and reagents: analytical grade (AR).

Example 1 Extraction and separation process of Herba Corydalis and its inhibitory activity on PDE4

1. Preparation of Herba Corydalis Extract and Its Inhibitory Activity against PDE4

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

The ethanol extract of Herba Eupatorii was suspended in 4 times the mass of water, and extracted with petroleum ether, ethyl acetate, and n-butanol in equal volumes to water (each solvent was extracted 3 times in a row) to obtain petroleum ether, ethyl acetate, n-butanol extracts and aqueous solutions, which were evaporated under reduced pressure to obtain petroleum ether extracts, ethyl acetate extracts, n-butanol extracts and aqueous concentrates, which were tested for PDE4D inhibitory activity.

PDE4 enzyme activity was determined by liquid scintillation counting. As a cAMP-preferred isozyme, 3H-cAMP was selected as the substrate for the PDE4 inhibitory activity test. The test method is summarized as follows: prepare Assay buffer (50mMTris-HCl, 10mM MgCl ₂ /MnCl ₂ , 1.0mM DTT, pH 7.5); dilute the PDE4D protein (recombinantly expressed by the research group, for specific methods, see the published literature: Biochemical Pharmacology 130 (2017) 51–59) with Assay buffer to an appropriate multiple and place on ice for use; dilute the substrate ^3H -cAMP with Assay buffer to an appropriate concentration (the reading of the control group is about 20,000 cpm); dissolve and dilute the test compound with DMSO to the set concentration; take 58μL of the diluted substrate to 1.5mL In the EP tube, add 2 μL of the prepared test compound of the set concentration, mix evenly, then add 40 μL of the diluted enzyme solution, mix gently, and then stand at room temperature for 15 minutes; add 200 μL of 0.2M ZnSO ₄ (to terminate the reaction) and 0.2N Ba(OH) ₂ solution (to precipitate the ring-opened ³ H-cAMP after the reaction) to the reaction system, centrifuge at high speed (14800rpm) for 5 minutes, take 430 μL of the supernatant solution containing unreacted ³ H-cAMP and add it to 2mL of scintillation fluid, shake and mix thoroughly, and measure the radioactivity reading with a liquid scintillation counter. Each group of experiments is set up with a substrate group (C ₀ ), a target protein group ( _CE ), a positive control group (rolipram, _CP ) and an inhibitor group ( _CI ), and the inhibition rate of the inhibitor is calculated by the formula Inhibition Rate = [1-(C _{0 -CI} )/(C _{0 -CE} )]*100%. When determining the specific IC ₅₀ value of the inhibitor (the IC ₅₀ value is the inhibitor concentration when the inhibition rate reaches 50%), a concentration gradient of 8-10 half-fold dilutions was set according to the results of the initial inhibition rate, and after the inhibition rates were determined, the specific IC ₅₀ value was obtained by nonlinear fitting using GraphPadPrism software, and the mean and standard deviation were obtained by independent repetition three times. The results of the inhibitory activity of the extract of Herba Eupatorii on PDE4D are shown in Table 1 below.

Table 1 Inhibitory activity of Herba Eupatorii extract on PDE4D

Herba Eupatoriae Extract 100μg/ml 10μg/ml 1μg/ml 0.1μg/ml 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%

2. Isolation and identification of biflavonoids from Herba Eupatorii and their inhibitory activity against PDE4

(I) Preparation of biflavonoid compounds 1-13

The ethyl acetate extract with the best PDE4 inhibitory activity was selected, and was first preliminarily segmented using an MCI column and eluted with methanol: water = 1:9→10:0. After TLC detection, it was merged into 4 fractions (Fr.A-Fr.D), and then repeatedly purified using an ODS column. Finally, it was further purified by high-performance liquid chromatography under (acetonitrile: water) and (methanol: water) conditions to obtain 13 biflavonoid monomer compounds.

Specifically, the preparation method of compound 1-13 comprises the following steps:

S1: The fraction Fr.C was separated by ODS column to obtain two fractions Fr.C1 and Fr.C2. The Fr.C1 fraction was prepared and purified by HPLC, using methanol-water (containing 0.1% acetic acid, 50:50, 6 mL/min) as the mobile phase, and the detection wavelength was 254 nm to obtain compound 1 (200 mg, t _R = 13 min). The Fr.C2 fraction was separated by ODS silica gel column, using methanol: water = 3:7 → 10:0 gradient elution, and combined into 15 fractions (Fr.C2a-Fr.C2o) after TLC detection. The Fr.C2m fraction was subjected to silica gel column chromatography, using dichloromethane: methanol = 100: 1 → 10: 1 gradient elution, and after TLC detection and 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 a detection wavelength of 254 nm to obtain 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 a detection wavelength of 254 nm to obtain compound 11 (4.5 mg, t _R =18 min) and compound 12 (3.6 mg, t _R =27 min).

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

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

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

¹ H NMR (DMSO-d ₆ , 400 MHz): δ _H 13.10 (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＝2.0 Hz, H-2′), 8.00 (1H, d, J＝8.5 Hz, 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 = 8.5 Hz, H-3″′, 5″′), 6.47 (1H, d, J = 2.0 Hz, H-8), 6.20 (1H, d, J = 2.0 Hz, H -6)

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

¹ H NMR (DMSO-d ₆ , 400 MHz): 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＝2.0 Hz, H-8), 6.73 (2H, d, J＝8.5, H -3"',5'")), 6.42 (1H, s, H-6")), 6.32 (1H, s, J = 2.0 Hz, H-6), 3.80 (3H, s, OCH ₃ ).

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

¹ H NMR (DMSO-d ₆ , 400 MHz): δ _H 13.09 (1H, s, OH-5″), 12.92 (1H, s, OH-5), 8.19 (1H, dd, J＝8.5, 2.0 Hz, H-6′), 8.06 (1H, d, J = 2.0 Hz, 2′), 7.50 (2H, d, J = 8.5 Hz, 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 = 2.0 Hz, H-8), 6.42 (1H, s, H-6'), 6.20 (1H, d, J = 2.0 Hz, H-6″), 3.78 (3H, s, 4′-OCH _3, )

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

¹ H NMR (DMSO-d ₆ , 400 MHz): δ _H 3.76 (3H, s, OCH ₃ ), 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″′)

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

¹ H NMR (DMSO-d ₆ , 400 MHz): δ _H 13.08 (1H, s, OH-5″), 12.98 (1H, s, OH-5), 8.02 (1H, d, J＝2.0 Hz, H- 2′), 7.99(1H, dd, J＝8.5, 2.0 Hz, H-6′), 7.69(2H, d, J＝8.5 Hz, 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 = 2.0 Hz, H-8), 6.36 (1H, s, H-6"), 6.18 (1H, d, J = 2.0 Hz, H-6), 3.73 (3H, s, 4") ′-OCH ₃ )

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

¹ H NMR (DMSO-d ₆ , 400 MHz): δ _H 13.08 (1H, s, OH-5"), 12.91 (1H, s, OH-5), 8.22 (1H, dd, J = 8.5, 2.0 Hz, H-6'), 8.09 (1H, d, J = 2.0 Hz, H-2'), 7.49 (2H, d, J = 8.5 Hz, H-2"', H-6"'), 7.36 (1H, d, J = 8.5 Hz, 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 = 8.5 Hz, H-3"', H-5"'), 6.39 (1H, s, 6"), 6.36 (1H, s), 3.83 (3H, s, 7-OCH ₃ ),3.79(3H,s,4′-OCH ₃ )

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

¹ H NMR (DMSO-d ₆ , 400 MHz): δ _H 13.06 (1H, s, OH-5"), 12.96 (1H, s, OH-5), 8.04 (1H, s, H-2'), 8.02 (1H, d, J = 8.5, 2.0 Hz, H-6'), 7.68 (2H, d, J = 8.5 Hz, H-2"', H-6"'), 7.15 (1H, d, J = 8.5 Hz, H-5'), 6.91 (2H, d, J = 8.5 Hz, 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 = 2.0 Hz, H-6), 3.81 (3H, s, OCH ₃ -7),3.74(3H,s,OCH ₃ -4′″)

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

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

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

¹ H NMR (DMSO-d ₆ , 400 MHz): δ _H 13.08 (1H, s, OH-5"), 12.94 (1H, s, OH-5), 8.18 (1H, dd, J = 8.5.2.0 Hz, H-6'), 8.04 (1H, dd, J = 2.0 Hz, H-2'), 7.62 (2H, dd, J = 8.5, H-2", 6"), 7.38 (1H, d, J = 8.5 Hz, 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 = 2.0 Hz, H-6), 3.91 (3H, s, OCH ₃ ),3.85(3H,s,OCH ₃ ),3.80(3H,s,OCH ₃ )

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

¹ H NMR (Methanol-d ₄ , 400 MHz): δ _H 7.59 (2H, d, J＝8.5 Hz, H-2″′, H-6″′), 7.46 (1H, s, H-2′), 7.44 (1H, d, J = 2.0 Hz, H-6'), 7.07 (1H, d, J = 8.5 Hz, H-5'), 6.81 (2H, d, J = 8.5 Hz, 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＝12.6 Hz, H-2), 3.18 (1H, dd, J＝17.0, 12.6 Hz, H-3), 2.77 (1H, d, J＝17.0 Hz, H-3)

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

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

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

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

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

¹ H NMR (DMSO-d ₆ , 400 MHz): δ _H 13.18 (1H, s, OH-5″), 12.88 (1H, s, OH-5), 7.99 (2H, d, J＝8.5 Hz, H-2′, 6′), 7.94 (2H, d, J＝8.5 Hz, H-2′″, 6′″), 7.02 (2H, d, J＝8.5 Hz, H-3′, 5′), 6.93 (2H, d, J＝8.5 Hz, 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)

After obtaining the above 13 biflavonoid compounds, the same method (PDE4 enzyme activity was determined by liquid scintillation counting method) was used to test their inhibitory activity against PDE4. As shown in Table 2 below, the 13 biflavonoid compounds all had good inhibitory activity against PDE4. Among them, compound 1 had an inhibitory IC ₅₀ of 12nM against PDE4D as shown in Figure 1, and the further analyzed cocrystal structure also confirmed that compound 1 was tightly bound to the catalytic pocket of the target PDE4D, as shown in Figure 2.

Table 2 Inhibitory activity of biflavonoid monomer compounds isolated from Herba Eupatorii on PDE4D

Example 2 Extraction and separation process of Platycladus orientalis and its inhibitory activity on PDE4

1. Preparation of Platycladus orientalis extract and its inhibitory activity against PDE4

Weigh the dried leaves of Platycladus orientalis, grind them into coarse powder, add 10 times the volume of methanol and soak them for 3 times, 2 days for the first time, 1 day for the second and third times respectively, filter, combine the filtrate, recover ethanol under reduced pressure, and concentrate the remaining liquid to a thick paste to obtain the total methanol extract of Platycladus orientalis.

The methanol extract of Platycladus orientalis was suspended in 4 times the mass of water, and extracted with petroleum ether, ethyl acetate, and n-butanol in equal volumes to water (each solvent was extracted 3 times in succession) to obtain petroleum ether, ethyl acetate, and n-butanol extracts and aqueous phase solutions, which were evaporated to dryness under reduced pressure to obtain petroleum ether extracts, ethyl acetate extracts, n-butanol extracts, and aqueous phase concentrates, which were tested for PDE4D inhibitory activity, respectively, and the determination method was consistent with the above method (PDE4 enzyme activity was determined by liquid scintillation counting method). The results are shown in Table 3 below.

Table 3 Inhibitory activity of Platycladus orientalis extract on PDE4D

2. Isolation and identification of biflavonoids from Platycladus orientalis and their inhibitory activity against PDE4

(I) Preparation of biflavonoid compounds A and B

The ethyl acetate extract with the best PDE4 inhibitory activity was selected, and the components of the ethyl acetate extract were preliminarily confirmed by HPLC (mobile phase method 0-30min, water:acetonitrile=92:8→10:90; 30-40min, water:acetonitrile=10:90→10:90). The spectral results confirmed that the ethyl acetate extract was composed of 5 main components (Fr.A－Fr.E). It was then purified by C18 reverse phase silica gel column, and finally further purified by preparative liquid chromatography under (acetonitrile:water) conditions to obtain 5 flavonoid monomer compounds.

Specifically, the preparation and identification method of compounds Fr.A-Fr.E includes the following steps:

S1: Dissolve 1g of ethyl acetate extract of Platycladus orientalis in 100mL of methanol, evaporate under reduced pressure to remove the small polar solvent in the sample, and repeat this step. Dissolve the obtained sample in 100mL of methanol, add three times the mass of C18 silica gel powder, mix well, evaporate under reduced pressure to obtain dry silica gel powder. Use water-acetonitrile as the mobile phase (elution with water: acetonitrile = 9:1→10:0, 15mL/min, 500mL), and the detection wavelength is 254nm to preliminarily purify the five compounds. When water/acetonitrile = 20%, fraction Fr.A is obtained. The solvent gradient is maintained. After the peak is completely eluted, the solvent gradient is increased. When water/acetonitrile = 34%, fraction Fr.B is obtained. The solvent gradient is maintained. After the peak is completely eluted, the solvent gradient is increased. When water/acetonitrile = 45%, fraction Fr.C is obtained. The solvent gradient is maintained. After the peak is completely eluted, the solvent gradient is increased. When water/acetonitrile = 68%, fraction Fr.D is obtained. The solvent gradient is maintained. After the peak is completely eluted, the solvent gradient is increased. When water/acetonitrile = 78%, fraction Fr.E is obtained. The solvent gradient is maintained. The solvent is collected and evaporated under reduced pressure to obtain a dry powder.

A small amount of the obtained fractions Fr.A-Fr.E were dissolved in chromatographic grade methanol, and the purity of each component was confirmed by HPLC, and compared with the HPLC spectrum of the ethyl acetate extract to confirm whether the peak time was consistent. Each fraction was further purified by preparative liquid phase (the mobile phase method was kept consistent), and compound A, compound B, compound C, compound D and compound E were obtained through repeated purification. Among them, the biflavonoid compound A and compound B isolated from Platycladus orientalis were consistent with the structure and NMR data of compound 1 and compound 13 isolated from Herba Euphorbiae, and they had common active ingredients.

(II) Structure and identification of biflavonoid compounds A and B

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

¹ H NMR (DMSO-d ₆ , 400 MHz): δ _H 13.10 (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＝2.0 Hz, H-2′), 8.00 (1H, d, J＝8.5 Hz, 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 = 8.5 Hz, H-3″′, 5″′), 6.47 (1H, d, J = 2.0 Hz, H-8), 6.20 (1H, d, J = 2.0 Hz, H -6)

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

¹ H NMR (DMSO-d ₆ , 400 MHz): δ _H 13.18 (1H, s, OH-5″), 12.88 (1H, s, OH-5), 7.99 (2H, d, J＝8.5 Hz, H-2′, 6′), 7.94 (2H, d, J＝8.5 Hz, H-2′″, 6′″), 7.02 (2H, d, J＝8.5 Hz, H-3′, 5′), 6.93 (2H, d, J＝8.5 Hz, 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)

The inhibitory activity of the above two biflavonoid compounds on PDE4 was tested using the same method. The test results were the same as the inhibition of PDE4 by compound 1 and compound 13 isolated from ophiopogon aviculare. Their IC50 (nM) values are not repeated here, indicating that compound A and compound B isolated from Platycladus orientalis have the same pharmacological effects as compound 1 and compound 13 isolated from ophiopogon aviculare, and both can inhibit PDE4.

3. Isolation method of effective parts from Platycladus orientalis and PDE4 inhibitory activity

Select macroporous resin AB-8, add 2 times the volume of anhydrous ethanol to soak, activate the macroporous resin for 24 hours, load the activated macroporous resin into a glass column with a sand mold, and rinse the macroporous resin with a large amount of distilled water until there is no ethanol smell in the rinse liquid. Weigh the methanol extract of Platycladus orientalis, dissolve it with anhydrous ethanol, slowly drip it on the surface of the macroporous resin, and adsorb it for one hour. Then elute it with 100% distilled water, 30% ethanol/water, 70% ethanol/water, and 100% ethanol in turn, collect it in batches, evaporate it under reduced pressure, and perform HPLC quantitative analysis on the obtained components, obtain the elution component with a higher content of selected compound A as 100% ethanol, and perform PDE4 inhibitory activity test, and use the same test method (PDE4 enzyme activity is determined by liquid scintillation counting method) for testing, and the results are shown in Table 4 below.

Table 4 Inhibitory activity of effective fractions isolated from Platycladus orientalis macroporous resin on PDE4D

name 2μg/ml 0.2μg/ml Effective Parts of Platycladus orientalis Macroporous Resin Separation 105.44% 74.23%

Example 3 Application of Platycladus orientalis ethyl acetate extract in the treatment of phosphodiesterase type 4 related diseases 1. In vivo anti-psoriasis effect of Platycladus orientalis ethyl acetate extract

The Platycladus orientalis extract with an inhibitory effect on PDE4 was selected as a test sample to evaluate its therapeutic effect in an animal model of psoriasis. The specific experimental materials, methods and results are described as follows.

1. Experimental Materials

SPF BALB/c mice, male, 6-8 weeks old, weighing 18-22 g, were purchased from Sbefore (Beijing) Biotechnology Co., Ltd. (License No.: 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).

(II) Experimental methods

BALB/c mice were randomly divided into 4 groups, 6 mice in each group, group A-normal control group, group B-imiquimod modeling group (IMQ, 62.5 mg applied to the back), group C-imiquimod + positive drug calcipotriol (IMQ+Calcipotriol), group D-imiquimod + 10% Thuja orientalis ethyl acetate extract (IMQ+10% Thuja orientalis extract). The back of the mouse was selected as the experimental observation area. The hair on the back surface of the mouse was cut off 1 day before the experiment to expose a 2cm×3cm hairless area. The control group was smeared with 62.5mg vaseline every day, and the mice in the other groups were given 62.5mg 10% imiquimod ointment every day. The drug was given after the drug was absorbed. The skin lesions of the mice were observed every day, photographed and recorded, and scored according to the PASI criteria (graded 0-4) to evaluate the therapeutic effect of the compound on psoriasis mice.

(III) Experimental results

As shown in Figure 3, after the mice were given imiquimod on the back, they developed psoriasis-like symptoms such as erythema and scaling, indicating that the model was successfully established. After the mice were given the ethyl acetate extract of Platycladus orientalis, the psoriasis-like symptoms such as erythema, scaling and hypertrophy were improved, indicating that the ethyl acetate extract of Platycladus orientalis can play a role in treating psoriasis.

II. In vivo anti-psoriasis effects of the effective fraction isolated from Platycladus orientalis macroporous resin and compound 1 (compound A)

The effective fraction of PDE4 inhibitor isolated by macroporous resin of Platycladus orientalis and compound 1 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.

(1) Experimental Materials

SPF BALB/c mice, male, 6-8 weeks old, weighing 18-22 g, were purchased from Sbefor (Beijing) Biotechnology Co., Ltd. (License No.: SCXK (Beijing) 2019-010). Ambient temperature: 20-25°C, humidity 50-70%, free access to food and water. Imiquimod (IMQ, Hubei Keyi Pharmaceutical Co., Ltd., 5%) Apremilast (BiDe Pharmaceutical).

(2) Experimental methods

BALB/c mice were randomly divided into 4 groups, 6 mice in each group, group A-normal control group, group B-imiquimod modeling group (IMQ, 62.5 mg applied on the back), group C-imiquimod + positive drug apremilast (IMQ+Apremilast), group D-imiquimod + Platycladus orientalis macroporous resin separation effective part (IMQ+P.L.-MacRe), group E-imiquimod + compound 1 (IMQ+1). The back of the mouse was selected as the experimental observation area. The hair on the back surface of the mouse was cut off 1 day before the experiment to expose a 2cm×3cm hairless area. The control group was smeared with 62.5mg vaseline every day, and the mice in the other groups were given 62.5mg10% imiquimod ointment every day. The drug was given after the drug was absorbed. The skin lesions of the mice were observed every day, photographed and recorded, and scored according to the PASI criteria (graded 0-4) to evaluate the therapeutic effect of the compound on psoriasis mice.

(3) Experimental results

a) The effective fraction separated by Platycladus orientalis macroporous resin and compound 1 can significantly improve the psoriasis-like symptoms of the skin of mice induced by imiquimod

As shown in Figure 4 (A)-(E), mice developed psoriasis-like symptoms such as erythema and scaling after being given imiquimod on the back, indicating that the model was successfully established. When apremilast, the effective fraction separated by macroporous resin of Platycladus orientalis and compound 1 were given, the symptoms of erythema, scaling and hypertrophy of the mice were improved, indicating that Platycladus orientalis extract can play a role in treating psoriasis.

b) HE staining showed that the effective fraction separated by Platycladus orientalis macroporous resin and compound 1 could significantly improve the pathological condition of the back skin of mice induced by imiquimod

As shown in Figure 4 (F), HE staining showed that on the seventh day of the experiment, the epidermis of the normal group had only 1 to 3 layers of cells, and the sebaceous glands/hair follicles were long and hollow; the epidermis thickness of the model group and the drug-treated group was 2-3 times that of the normal group, the stratum corneum showed incomplete keratinization, and the dermis showed inflammatory cell infiltration accompanied by a lot of cell fragments; the effective part separated by macroporous resin of Platycladus orientalis and the compound 1 group could reduce the thickness of the epidermis, and the epidermis was close to that of the normal control group, the dermis had slightly more cell accumulation, and the sebaceous gland structure was still intact.

The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims (10)

1. Use of a Cupressaceae plant extract in the treatment and/or prevention of phosphodiesterase type 4 related diseases, wherein the Cupressaceae plant extract is a Herba Corydalis extract or a Platycladus orientalis extract. 2. The use according to claim 1 is characterized in that the Herba Eupatorii extract is obtained by alcohol extraction of the Herba Eupatorii, and the alcohol extraction method is as follows: weigh dry leaves of the Herba Eupatorii, crush them into coarse powder, add 8-12 times the volume of alcohol solution and soak them three times respectively, wherein the first time is 8-15 days, and the second and third times are 4-7 days respectively, filter, combine the filtrate, recover the effective ingredients of the alcohol under reduced pressure, and concentrate the remaining liquid to a thick paste to obtain the Herba Eupatorii extract. 3. The use according to claim 1 is characterized in that the Platycladus orientalis extract is obtained by extracting thuja alcohol, and the alcohol extraction method is as follows: weigh the dry leaves of Platycladus orientalis, crush them into coarse powder, add 8-12 times the volume of alcohol solution and soak them three times respectively, wherein the first time is 2-5 days, and the second and third times are 1-3 days respectively, filter, combine the filtrate, recover the effective ingredient of the alcohol under reduced pressure, and concentrate the remaining liquid to a thick paste to obtain the Platycladus orientalis extract containing the effective part. 4. The use according to claim 3, characterized in that the effective part of the Platycladus orientalis extract is separated by macroporous resin, and the separation method is as follows: Step 1: Take the macroporous resin AB-8, add 2-5 times the volume of anhydrous ethanol to soak, activate for 12-48 hours, put the activated macroporous resin into a glass column with a sand core, and rinse the macroporous resin with a large amount of distilled water until there is no ethanol smell in the rinse liquid; Step 2: Weigh the Platycladus orientalis extract, dissolve it with anhydrous ethanol, slowly drip it on the surface of the macroporous resin, adsorb it for one hour, then elute it with 100% distilled water, 30% ethanol/water, 70% ethanol/water, and 100% ethanol in sequence, collect it in batches, and evaporate it under reduced pressure to obtain the refined extract of the effective part of Platycladus orientalis. 5. The use according to claim 1, characterized in that the biflavonoid compounds in the Cupressaceae plant extract are used in the treatment and/or prevention of phosphodiesterase type 4 related diseases. 6. The use according to claim 5, characterized in that the structure of the biflavonoid compound is selected from one of the compounds represented by formula (I)-(V): in: R1, R2, R3, R4, R5 and R6 are each selected from one of halogen, aldehyde, carboxyl, sulfonic acid, nitro, nitroso or hydrogen; Alternatively, R1, R2, R3, R4, R5 and R6 are each independently hydroxy or methoxy. 7. The use according to claim 6, characterized in that the structure of the compound represented by formula (I) is selected from any one of the following structures: 8. An application in the preparation of a health product or food for treating and/or preventing phosphodiesterase type 4 related diseases, wherein the health product or food comprises the Platycladus orientalis extract according to claim 1. 9. A drug for treating and/or preventing phosphodiesterase type 4 related diseases, characterized in that the drug comprises one or more of the Cupressaceae plant extract according to claim 1 or the biflavonoid compounds with structures shown in any one of formulas (I) to (V) in claim 6, and also comprises pharmaceutically acceptable excipients and carriers. 10. The drug according to claim 9, characterized in that the drug is in the form of capsules, tablets, pills, granules, granules or sprays.