WO2021249237A1 - Triepoxyhexahydrochromone a, and pharmaceutical composition and use thereof - Google Patents

Abstract

Provided in the present invention are triepoxyhexahydrochromone A (PFC-34), and a pharmaceutical composition thereof and the use thereof in the preparation of a drug for preventing and treating neurodegenerative diseases and Parkinson's disease. The triepoxyhexahydrochromone A (PFC-34) has a significant protective effect on MPP +-induced PC12 damage, and can be used in the preparation of a pharmaceutical composition or a dietary supplement composition for preventing and treating Parkinson's disease.

Description

Triepoxyhexahydrochromone A and its pharmaceutical composition and its application

This application claims the priority of a Chinese patent application filed with the Chinese Patent Office on June 08, 2020, the application number is 202010512429.4, and the invention title is "Triepoxyhexahydrochromone A and its pharmaceutical composition and its application", which The entire content is incorporated into this application by reference.

Technical field

The invention belongs to the technical field of medicine, and particularly relates to a triepoxyhexahydrochromone A compound and its pharmaceutical composition and its application in the preparation of drugs for preventing and treating Parkinson's disease.

Background technique

Parkinson’s disease is a common neurological disease in middle-aged and elderly people. At present, levodopa replacement therapy is mainly used to treat Parkinson's disease, but it cannot prevent the progression of Parkinson's disease. The curative effect begins to decline after 3 to 5 years. There are not only anorexia, nausea, dizziness, mental disorders, and dyskinesias. Response, long-term use can also lead to "switching phenomenon", end-of-dose phenomenon and dystonia [Sun Jing, Xiong Hang, Yao Yuxi. Progress in the treatment of Parkinson's disease. Medical Review, 2020, 26(2): 1157- 1160,1165.]. Therefore, looking for new therapeutic drugs and methods is a hot and difficult point of research.

The PC12 cell injury model induced by 1-methyl-4-phenyl pyridinium ion (MPP ⁺ ) is currently recognized as a screening model for the treatment of Parkinson’s disease [a.Delavar MR, Baghi M,Safaeinejad Z,Kiani-Esfahani A,Ghaedi K,Nasr-Esfahani MH.Differential expression of miR-34a,miR-141,and miR-9 in MPP ⁺ -treated differentiated PC12 cells as a model of Parkinson's disease.Gene ,2018,662:54-65; b.Lin KH,Li CY,Hsu YM,Tsai CH,Tsai FJ,Tang CH,Yang JS,Wang ZH,Yin MC.Oridonin,a natural diterpenoid,protected NGF-differentiated PC12 cells against MPP ⁺ -and kainic acid-induced injury. Food and Chemical Toxicology, 2019,133:110765].

So far, there have been no reports of triepoxyhexahydrochromone A and its activity against neurodegenerative diseases and Parkinson's disease in the prior art.

Summary of the invention

When the present invention uses the MPP ⁺ induced PC12 cell damage model to screen pharmacologically active ingredients, a new compound, namely triepoxyhexahydrochromone A (PFC-34), is found to ^{have significant effects on MPP +} induced PC12 cell damage Protective effects.

The purpose of the present invention is to provide a new compound triepoxyhexahydrochromone A (PFC-34) and its pharmaceutical composition and its application in the preparation of prevention and treatment of neurodegenerative diseases and/or Parkinson's disease.

In order to achieve the above objectives of the present invention, the present invention provides the following technical solutions:

The compound triepoxyhexahydrochromone A shown in the following structural formula,

The pharmaceutical composition consists of triepoxyhexahydrochromone A and a pharmaceutically acceptable carrier.

The pharmaceutical composition for preventing and treating neurodegenerative diseases and/or Parkinson's disease is composed of triepoxyhexahydrochromone A and a pharmaceutically acceptable carrier.

The dietary supplement composition is composed of triepoxyhexahydrochromone A and food supplements.

Application of triepoxyhexahydrochromone A in the preparation of drugs for preventing and/or treating neurodegenerative diseases.

Application of triepoxyhexahydrochromone A in the preparation of drugs for preventing and/or treating Parkinson's disease.

The preparation method of triepoxyhexahydrochromone A includes the following steps: pulverize dried agarwood, 90% ethanol at 60°C ultrasonic extraction for 30 minutes, finally obtain a concentrated crude extract extract, and then suspend the extract in water, Extract with equal volumes of petroleum ether, ethyl acetate and n-butanol in sequence, and recover the solvent to obtain petroleum ether extract, ethyl acetate extract, and n-butanol extract; use the same amount of 80-100 for the ethyl acetate extraction part Mix the sample with the mesh silica gel and divide it by normal phase silica gel column chromatography. The eluent is petroleum ether-ethyl acetate system (50:1, 30:1, 20:1, 10:1, 5:1, 3:1) , 2:1, 1:1, 0:1, v/v) for gradient elution, followed by ethyl acetate-methanol system for elution (5:1, 3:1, 2:1, 1:1, 0:1, v/v), the same components were combined by TLC detection to obtain 5 parts Fr.Y-1～Fr.Y-5; Fr.Y-4 was subjected to medium pressure RP-18 reversed-phase silica gel column chromatography (Methanol-water, 10%→100%), the same fractions were combined by TLC detection to obtain 4 parts Fr.Y-4-1～Fr.Y-4-4; Fr.Y-4-3 was tested by Sephadex LH- 20 gel column chromatography (methanol), normal phase silica gel column chromatography (300-400 mesh silica gel; petroleum ether-ethyl acetate, 50:1→1:1, v/v), to obtain 4 parts of Fr.Y- 4-3-1～Fr.Y-4-3-4; Fr.Y-4-3-2 was subjected to normal phase silica gel column chromatography (300～400 mesh silica gel; petroleum ether-ethyl acetate, 10:1) , And semi-preparative HPLC (Ultimate AQ-C ₁₈ chromatographic column, φ7.8×250mm; flow rate v=2mL/min, acetonitrile-water, 50:50) to obtain compound triepoxyhexahydrochromone A.

The above-mentioned pharmaceutically acceptable carrier refers to the conventional pharmaceutical carrier in the pharmaceutical field, for example, water, glucose, lactose, gum arabic, etc. and other suitable for use in the preparation of solid, semi-solid, liquid or aerosol formulations Carrier. The composition may additionally contain stabilizers, thickeners, and/or colorants and fragrances.

The composition prepared by the compound of the present invention and its pharmaceutically acceptable carrier can be administered orally or without oral administration. The dosage varies with different drugs. For adults, 1-100 mg per day is more appropriate. .

For oral administration, first mix the compound with conventional pharmaceutical adjuvants such as excipients, anti-oxidants, binders, lubricants, antioxidants, coating agents, colorants, fragrances, surfactants, etc., and mix It is administered in the form of granules, capsules, tablets, etc.: parenteral administration can be administered in the form of injections, infusions or suppositories. When preparing the above formulations, conventional formulation techniques can be used.

When the compound triepoxyhexahydrochromone A of the present invention is used as a medicine or health care product, it can be used directly or in the form of a composition. The pharmaceutical composition contains 0.1-99%, preferably 0.5-90% of the compound, and the rest are pharmaceutically acceptable, non-toxic and inert pharmaceutically acceptable carriers and/or excipients to humans and animals, or daily Use food additives and substrates.

detailed description

The following examples of the present invention are used to further illustrate the essential content of the present invention, but the present invention is not limited thereto.

Example 1:

The preparation process of compound PFC-34:

The dried Chinese medicine Aquilaria sinensis [namely the heartwood of Aquilaria sinensis (Lour.) Spreng.; 2.9kg] was crushed, and ultrasonically extracted with 90% ethanol at 60°C for 30 minutes, and finally a concentrated crude extract extract (459.3g) was obtained. Then the extract was suspended in water, extracted with equal volumes of petroleum ether, ethyl acetate, and n-butanol in sequence, and the solvent was recovered to obtain 0.7 g of petroleum ether extract, 374.8 g of ethyl acetate extract, and 52.5 g of n-butanol extract. .

The ethyl acetate extract (374.8g) was mixed with an equal amount of 80-100 mesh silica gel and segmented by normal phase silica gel column chromatography. The eluent was a petroleum ether-ethyl acetate system (50:1, 30:1). , 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, 0:1, v/v) for gradient elution, followed by ethyl acetate-methanol system for elution ( 5:1, 3:1, 2:1, 1:1, 0:1, v/v), the same components were combined by TLC detection to obtain 5 parts (Fr.Y-1～Fr.Y-5) .

Fr.Y-4 (35.9g) was subjected to medium pressure RP-18 reversed-phase silica gel column chromatography (methanol-water, 10%→100%), and the same fractions were combined by TLC detection to obtain 4 fractions (Fr.Y-4 -1～Fr.Y-4-4). Fr.Y-4-3 (2.3g) was subjected to Sephadex LH-20 gel column chromatography (methanol), normal phase silica gel column chromatography (300-400 mesh silica gel; petroleum ether-ethyl acetate, 50:1→1: 1, v/v), 4 parts (Fr.Y-4-3-1～Fr.Y-4-3-4) are obtained. Fr.Y-4-3-2 (889.7mg) was subjected to normal phase silica gel column chromatography (300-400 mesh silica gel; petroleum ether-ethyl acetate, 10:1), and semi-preparative HPLC (Ultimate AQ-C ₁₈ chromatography Column, φ7.8×250mm; flow rate v=2mL/min, acetonitrile-water, 50:50) to obtain compound PFC-34 (3.1 mg, t _R =17.256 min).

The chemical structure of triepoxyhexahydrochromone A (PFC-34) is shown below:

Spectral data of compound PFC-34:

Compound PFC-34, chemical name triepoxyhexahydrochromone A (triepoxyhexahydrochromoneA), yellow solid; [α] _D ²¹ +30.8(c 0.12,MeOH); UV(MeOH)λ _max (logε)260(3.81), 226(3.25)nm; ECD(c 0.020,MeOH)λ _max (Δε)320(+3.37),258(-5.19),213(-0.99),199(+0.09); IR v _max (KBr)3028, 1672,1624,1497,1455,1429,1391,1262,1169,963,940,862,755,701cm ^-1 ; ¹ H and ¹³ C NMR data are shown in Table 1; ESIMS m/z 321[M+Na] ⁺ ,619[2M+Na] ⁺ ; HRESIMS m/z 321.0733[M+Na] ⁺ (calcd for C ₁₇ H ₁₄ NaO ₅ ,321.0739).

^{Table 1. 1} H (500MHz) and ¹³ C NMR (126MHz) data of compound PFC-34 tested _{in CDCl 3}

no.	δ H (JinHz)	δ C
2	To	170.1
3	5.48(s)	104.5
4	To	185.5
5	3.82(d,3.8)	49.8
6	3.53(dd,3.8,3.6)	47.3

7	3.49(dd,3.6,3.5)	47.3
8	3.87(d,3.5)	47.0
9	To	86.6
10	To	61.1
1'	To	139.6
2′,6′	7.18(m)	128.4
3′,5′	7.31(m)	128.8
4′	7.23(m)	126.8
7′	2.91(t,8.0)	32.6
8'	2.62(m)	36.3

Example 2:

The neuroprotective activity test method of compound PFC-34:

1. PC12 poorly differentiated cells are cultured with DMEM high glucose + 10% FBS + 100 U/mL double antibody medium, the incubator temperature is 37 ℃, 5% CO ₂ ;

2. When PC12 poorly differentiated cells grow to 80-90%, trypsin digestion to prepare cell suspension;

3. Suction the cell suspension into a 15mL centrifuge tube, 800rpm, 5min;

4. After centrifugation, the centrifuge tube is disinfected with alcohol and taken into the ultra-clean table, and the supernatant is poured into the waste liquid tank;

5. Add 5 mL of new complete medium and pipette ten times to disperse the cells as much as possible, but not too hard;

6. Take 0.02mL cell suspension, add it to the cell counting plate, and count;

7. Adjust the cell concentration to 1×10 ⁵ cells/mL, add it to a 96-well plate, 0.1 mL per well, and place it in a cell incubator for culture;

8. After 23 hours, aspirate the original medium, then add a new medium (same formula as in 1), add the test compound, and 1 hour later, add MPP ⁺ (MPP ⁺ in the system with a final concentration of 750 μM);

9. Experimental design: 3 replicates for each group design.

Blank group: only medium;

Model group (MPP ⁺ ): medium plus final concentration of 750μM MPP ⁺ ;

Positive control group (Vitamin E): The medium is supplemented with a final concentration of 0.2μM Vitamin E, and then a final concentration of 750μM MPP ⁺ .

Compound group (group 1 to group 5): add compound PFC-34 at a final concentration of 5, 2, 1, 0.2, 0.1 μM to the medium, and then add MPP ^{+ at a} final concentration of 750 μM.

10. After adding MPP ⁺ 24 hours, adding MTS, 2 hours later, reading and testing.

Example 3:

The effect of the neuroprotective activity of compound PFC-34:

The neuroprotective activity data of compound PFC-34 is shown in Table 2. When the compound concentration is 1 μM and 2 μM, it ^{has significant protective activity against MPP +} -induced PC12 cell injury (P<0.001).

Table 2 Compound Table 2 Protective effect of ^{PFC-34 on MPP +-induced PC12 injury}

Note to Table 2: Compared with the model group, **P<0.01, ***P<0.001

Example 4:

To prepare drugs for the treatment of neurological diseases, mix according to the following weight percentages: PFC-34 accounts for 20 to 80%, dispersant 2 to 20%, disintegrant 3 to 5%, emulsifier 3 to 8%, binder 0.2-2%, wetting agent 0.5-10%, the rest is filler. Then, the therapeutic drug for neurological diseases with the active ingredient PFC-34 is prepared according to the conventional drug preparation method.

Example 5:

Preparation of oral liquid preparations:

With PFC-34 as the active ingredient, the oral liquid is prepared according to the conventional oral liquid preparation method.

Example 6:

Preparation of capsules, granules, or granules:

Add excipients in a ratio of 5:1 by weight of PFC-34 to excipients to make capsules, granules or granules.

Example 7:

The preparation of the dietary supplement composition: mixing according to the following weight percentages: PFC-34 accounts for 20 to 80%, and commonly used food supplements 80 to 20%.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications are also It should be regarded as the protection scope of the present invention.

Claims (24)

1. The compound triepoxyhexahydrochromone A shown in the following structural formula,

   
2. The pharmaceutical composition consists of the triepoxyhexahydrochromone A described in claim 1 and a pharmaceutically acceptable carrier.
3. The pharmaceutical composition for preventing and/or treating neurodegenerative diseases and Parkinson's disease is composed of the triepoxyhexahydrochromone A according to claim 1 and a pharmaceutically acceptable carrier.
4. The pharmaceutical composition according to claim 2 or 3, wherein the pharmaceutically acceptable carrier is water, glucose, lactose, gum arabic and suitable for preparing preparations in solid, semi-solid, liquid or aerosol form Other vectors used in the.
5. The pharmaceutical composition according to claim 4, wherein the pharmaceutical composition additionally contains stabilizers, thickeners, and/or coloring agents and perfumes.
6. The pharmaceutical composition according to claim 2 or 3, wherein the pharmaceutical composition contains 0.1-99% triepoxyhexahydrochromone A.
7. The pharmaceutical composition according to claim 6, wherein the pharmaceutical composition contains 0.5-90% triepoxyhexahydrochromone A.
8. The pharmaceutical composition according to claim 2 or 3, wherein the dosage form of the pharmaceutical composition is a preparation for oral administration or a preparation for parenteral administration;

   The preparations for oral administration are granules, capsules, tablets or granules;

   The preparations for parenteral administration are injections, infusions or suppositories.
9. The pharmaceutical composition according to claim 8, wherein the pharmaceutical adjuvants for oral administration include excipients, desolvents, binders, lubricants, antioxidants, coating agents, coloring agents, and fragrances. Agent, surface active agent.
10. The pharmaceutical composition according to claim 9, wherein the pharmaceutical adjuvant in the capsule, granule or granule is an excipient, and the weight ratio of PFC-34 to the excipient is 5:1.
11. The pharmaceutical composition according to claim 2 or 3, characterized in that it is mixed in the following weight ratios: PFC-3420～80%, dispersant 2-20%, disintegrant 3～5%, emulsifier 3. ～8%, adhesive agent 0.2～2%, wetting agent 0.5～10%, the rest is filler.
12. The method of administration of the pharmaceutical composition according to any one of claims 2 to 11, characterized in that it is administered orally or without oral administration, and for an adult, the dosage is 1-100 mg per day.
13. The dietary supplement composition is composed of the triepoxyhexahydrochromone A described in claim 1 and food supplements.
14. The dietary supplement composition according to claim 13, characterized in that it is mixed in the following weight percentages: PFC-3420-80%, food supplements 80-20%.
15. The use of the triepoxyhexahydrochromone A of claim 1 in the preparation of drugs for preventing and/or treating neurodegenerative diseases.
16. The use of the triepoxyhexahydrochromone A according to claim 1 in the preparation of a medicine for preventing and/or treating Parkinson's disease.
17. The preparation method of triepoxyhexahydrochromone A according to claim 1, which comprises the following steps: crushing dried agarwood, 90% ethanol and 60°C ultrasonic extraction for 30min, finally obtaining concentrated crude extract extract, and then The concentrated crude extract extract is suspended in water, extracted with equal volumes of petroleum ether, ethyl acetate, and n-butanol in sequence, and the solvent is recovered to obtain petroleum ether extract, ethyl acetate extract, and n-butanol extract; The ethyl acetate extract was mixed with an equal amount of 80-100 mesh silica gel, and segmented by normal phase silica gel column chromatography. The eluent was a petroleum ether-ethyl acetate system for gradient elution, followed by ethyl acetate- The methanol system was eluted, and the same components were combined by TLC detection to obtain 5 fractions Fr.Y-1～Fr.Y-5; Fr.Y-4 was chromatographed on a medium-pressure RP-18 reversed-phase silica gel column, and then TLC The same fractions were detected and combined to obtain 4 parts Fr.Y-4-1～Fr.Y-4-4; Fr.Y-4-3 was obtained by Sephadex LH-20 gel column chromatography and normal phase silica gel column chromatography. Four parts Fr.Y-4-3-1～Fr.Y-4-3-4; Fr.Y-4-3-2 was subjected to normal phase silica gel column chromatography and semi-preparative HPLC to obtain the compound triepoxyhexahydro Chromone A.
18. The preparation method according to claim 17, wherein the volume ratio of petroleum ether and ethyl acetate in the petroleum ether-ethyl acetate system is 50:1, 30:1, 20:1, 10:1, 5 :1, 3:1, 2:1, 1:1, 0:1.
19. The preparation method according to claim 17, wherein the volume ratio of ethyl acetate and methanol in the ethyl acetate-methanol system is 5:1, 3:1, 2:1, 1:1, 0: 1.
20. The preparation method according to claim 17, wherein the mobile phase of the medium-pressure RP-18 reversed-phase silica gel column chromatography is methanol-water, 10%→100%.
21. The preparation method according to claim 17, wherein the mobile phase of the Sephadex LH-20 gel column chromatography is methanol.
22. The preparation method according to claim 17, wherein the stationary phase of the normal phase silica gel column chromatography of Y-4-3 is silica gel, and the particle size of the silica gel is 300-400 mesh;

    The mobile phase of the normal phase silica gel column chromatography of Y-4-3 is petroleum ether-ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 50:1→1:1.
23. The preparation method according to claim 17, wherein the stationary phase of the normal phase silica gel column chromatography of the Fr.Y-4-3-2 is silica gel, and the particle size of the silica gel is 300-400 mesh;

    The mobile phase of Fr. Y-4-3-2 subjected to normal phase silica gel column chromatography is petroleum ether-ethyl acetate, 10:1.
24. The preparation method according to claim 17, wherein the semi-preparative HPLC column of Fr.Y-4-3-2 is UltimateAQ-C ₁₈ chromatographic column, φ7.8×250mm;

    The flow rate of the mobile phase of Fr.Y-4-3-2 by semi-preparative HPLC is v=2mL/min;

    The mobile phase of Fr. Y-4-3-2 by semi-preparative HPLC is acetonitrile-water, 50:50.