Corn lignan compound as well as application and preparation method thereof

Abstract

The invention discloses a corn lignan compound as well as application and a preparation method thereof, and belongs to the field of natural medicines. The corn lignan compound extracted and separated from corn roots by a specific method can promote cell proliferation and has antioxidant and anti-inflammatory activity; the corn lignan compound disclosed by the invention is used as an Fyn partial agonist, and can enhance the combination of Fyn and a ligand thereof through an allosteric effect, so that the corn lignan compound has a strong anti-depression effect.

Description

Technical Field

[0001] The invention relates to the field of natural medicines, and in particular to a corn lignan compound and an application and a preparation method thereof. Background Art

[0002] Fyn is a non-receptor or cytoplasmic tyrosine kinase that participates in multiple transduction pathways in the central nervous system (CNS), including synaptic transmission, myelination, axon guidance, and oligodendrocyte formation. Pharmacological and molecular biological research results show that low Fyn expression levels will lead to abnormal neurological function, but its overactivation will also lead to oxidative stress, inflammatory damage, and excitotoxicity, thereby triggering diseases caused by oxidative stress, inflammatory damage, and excitotoxicity. In addition, excessive activation of Fyn is also associated with tumor cell proliferation and metastasis. In summary, Fyn is considered to be a therapeutic target for diseases related to oxidative stress, inflammatory damage, and excitotoxicity, as well as malignant tumors.

[0003] Fyn partial agonists have dual effects. When Fyn is activated by an agonist, they play an antagonistic role, inhibiting the over-activation of Fyn and bringing it close to normal physiological levels. When Fyn is over-inhibited by an inhibitor, Fyn partial agonists play an agonistic role, upregulating the kinase activity of Fyn and bringing it close to normal physiological levels. Therefore, Fyn partial agonists are expected to achieve safe regulation of Fyn, so that it will neither be over-inhibited, affecting its normal physiological function, nor be over-activated, leading to the occurrence of related diseases. At present, there are no reports of agonists and partial agonists of Fyn, and it is necessary to develop its partial agonists to promote the safe application of Fyn targeted drugs in the field of disease treatment.

[0004] Lignans are a class of naturally occurring organic compounds that are abundant in hundreds of plants and have a wide range of biological activities. Over the years, many studies have revealed the various pharmacological activities of lignans, including antioxidant, anti-inflammatory, antibacterial, antiviral, and antiplatelet aggregation activities. Although lignans are a class of natural compounds composed of two molecules of phenylpropanoid derivatives (i.e., C6-C3 monomers), their structures and activities vary greatly. Although the two lignan compounds have the same parent nucleus structure, the difference in their anti-tumor activity is huge due to the different substituents (see Journal of Natural Medicines, 2019, 73, 273-277); at the same time, the different compound substitution gene configurations lead to huge differences in the anti-inflammatory, neurological and other aspects of their activities (see Phytochemistry, 2022, 203, 113336 and Fitoterapia, 2023, 165, 105403). It can be seen that the parent nucleus structure of lignan compounds is diverse, and the position, type and configuration of substituents are complex, resulting in diverse activities. It is difficult to predict that other lignan compounds with the same parent nucleus or other different parent nucleus structures have the same biological activity based on the biological activity of one or several lignan structures. Therefore, the study of the structure and biological activity of lignan compounds has always received close attention from researchers in the field of natural medicinal chemistry.

[0005] At present, there are reports on extracting lignans from angelica, corn leaves, Litsea cubeba, Curculigo orchioides, etc., such as the preparation and use of new lignan compounds in angelica disclosed in Publication No. CN114380687A, by extracting new lignan compounds with antidepressant properties from angelica; the lignan compound parent nucleus and substituents of the new lignan compounds involved in the invention have large differences, resulting in different anti-tumor effects. The patent with Publication No. CN113149945A discloses an extract containing 8 lignan compounds separated and prepared from Litsea cubeba and its application in sedation, hypnosis, antidepression and / or antianxiety; the structure of the 8 lignan compounds in the invention is different from the lignan compound parent nucleus in the present invention. In addition, the invention does not measure the activity of the above 8 lignan compounds, but only provides the sedative, hypnotic, antidepressant and / or antianxiety activity of the total extract. In view of the fact that the activity of the main component after separation and purification of multiple active extracts decreases or even disappears, the real reason for the effect is the superposition of the activities of multiple trace compounds therein. Therefore, it is not clear whether the eight lignan compounds in Litsea cubeba have sedative, hypnotic, antidepressant and / or antianxiety activities and which specific lignan compounds have the above biological activities. The application of corn leaf extract disclosed in Publication No. CN1958048A for antidepressant and improving sexual function. Fresh corn leaves are cold-pressed to obtain juice and raw material residues, the raw material residues are extracted with water or alcohol, and the extract is concentrated and dried to obtain corn leaf extract. The parts and extraction methods of corn extracted in this invention are different, and the types of compounds obtained will also be very different. In addition, the chemical composition of the extract in this invention is still unclear, so it is impossible to determine whether there are lignan compounds in the extract, and even if there are lignan compounds, whether they are related to their antidepressant activity is still unknown. The lignan compounds in the above technical scheme are obtained by different preparation methods and different plant extracts, so their structures and biological activities may be very different. In addition, there is no report that lignan compounds exert antidepressant activity through the partial agonist Fyn. Therefore, the discovery of lignan compounds as Fyn partial agonists in the present invention has outstanding substantive characteristics, beneficial technical effects and remarkable progress. Summary of the invention

[0006] The present invention provides a corn lignan compound and an application and preparation method thereof. The corn lignan extracted from corn roots has a positive therapeutic effect on oxidative stress, inflammatory damage and depression.

[0007] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0008] A corn lignan compound has the following general structural formula:

[0009]

[0010] In the formula I, R1 is, R2 is, R3 is, R4 is, R5 is, R6 is, R7 is, and R8 is any one of hydrogen, hydroxyl, methyl, methoxy, ethyl, butyl, aromatic ring containing saturated or unsaturated substituents, and aromatic heterocycle containing saturated or unsaturated substituents.

[0011] In the formula II, R1 is, R2 is, R3 is, R4 is, R5 is, and R6 is any one of hydrogen, hydroxyl, methyl, methoxy, ethyl, butyl, aromatic ring containing saturated or unsaturated substituents, and aromatic heterocycle containing saturated or unsaturated substituents;

[0012] In the formula III, R1 is, R2 is, R3 is, R4 is, R5 is, R6 is, R7 is, and R8 is any one of hydrogen, hydroxyl, methyl, methoxy, ethyl, butyl, aromatic ring containing saturated or unsaturated substituents, and aromatic heterocyclic ring containing saturated or unsaturated substituents;

[0013] In the formula IV, R1 is, R2 is, R3 is, R4 is, and R5 is any one of hydrogen, hydroxyl, methyl, methoxy, ethyl, butyl, an aromatic ring containing a saturated or unsaturated substituent, and an aromatic heterocycle containing a saturated or unsaturated substituent.

[0014] Furthermore, the corn root lignan compound includes the following structural formula:

[0015]

[0016]

[0017] Another object of the present invention is to provide the use of the corn root lignan compounds in the prevention or common treatment drugs or target treatment drugs for diseases related to Fyn-related inflammation, oxidative stress and excitotoxicity injury;

[0018] The Fyn-related diseases include mental illness, nervous system diseases, inflammation, organ damage caused by oxidative stress and excitatory damage, respiratory system diseases, tumors and chronic inflammatory diseases;

[0019] The mental illnesses include depression, anxiety, schizophrenia, obsessive-compulsive disorder, mania, and bipolar disorder caused by oxidative stress, excitatory injury or inflammation.

[0020] The nervous system diseases include cerebrovascular diseases and neurodegenerative diseases caused by oxidative stress, excitatory injury or inflammation; the cerebrovascular diseases include cerebral infarction, cerebral hemorrhage and cerebral aneurysm; the neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis;

[0021] The organ damage includes oxidative stress, excitatory damage, and kidney damage, liver damage, heart damage, lung damage, osteoarthritis, etc. caused by acute or chronic inflammation.

[0022] Another object of the present invention is to provide a Fyn target partial agonist with antidepressant effect, comprising a corn lignan compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.

[0023] Furthermore, the antidepressant Fyn target partial agonist includes at least a compound of Formula 1 and / or a compound of Formula 2 and / or Formula 4 and / or Formula 6, and / or a pharmaceutically acceptable salt of a compound of Formula 1 and a pharmaceutically acceptable carrier or excipient, and / or a pharmaceutically acceptable salt of a compound of Formula 2 and a pharmaceutically acceptable carrier or excipient, and / or a pharmaceutically acceptable salt of a compound of Formula 4 and a pharmaceutically acceptable carrier or excipient, and / or a pharmaceutically acceptable salt of a compound of Formula 6 and a pharmaceutically acceptable carrier or excipient.

[0024] The present invention also provides a method for preparing corn root lignan compounds, which is characterized by comprising the following steps:

[0025] (1) extracting corn roots with 50-70% anhydrous ethanol under reflux, and concentrating under reduced pressure to obtain a crude extract;

[0026] (2) adding a sufficient amount of pure water to the crude extract to uniformly disperse it in the water, extracting with petroleum ether to remove the remaining water extract of the high fat-soluble components, concentrating the water extract under reduced pressure to remove the residual petroleum ether, and then separating it through AB-8 macroporous resin column chromatography using ethanol-water solution as eluent, collecting the ethanol-water elution fraction, and concentrating under reduced pressure to obtain the extract;

[0027] (3) The extract obtained in (2) was dissolved in a dichloromethane / methanol mixed solution, mixed with silica gel, and then separated by silica gel column chromatography, and gradient eluted with dichloromethane / methanol to obtain a total of 6 fractions (A1 to A6);

[0028] (4) Separate and identify the corn root lignan compounds in the A2 to A5 fractions by silica gel column chromatography, ODS open column chromatography (methanol-water) elution, polyamide column chromatography (ethanol-water) elution, or cyclic preparative HPLC to obtain the corn root lignan compounds of formula 1 to formula 22 of the present invention.

[0029] Furthermore, after the water extract in step (2) is concentrated under reduced pressure to remove residual petroleum ether, 25% and 70% ethanol-water solutions are used as eluents, and the 70% ethanol-water eluate fraction is collected and concentrated under reduced pressure to obtain an extract.

[0030] Furthermore, the A2 fraction in step (3) is separated by silica gel column chromatography using a gradient solvent of dichloromethane / methanol as an eluent to obtain fractions A2-1 to A2-11, and the A2-2 is separated and purified by cyclic preparative HPLC to obtain compounds of formula 7, formula 9 and formula 12; the A2-3 is separated by polyamide column chromatography and cyclic preparative HPLC to obtain a compound of formula 4; the A2-4 is separated by cyclic preparative HPLC to obtain a compound of formula 6; the A2-6 is separated by cyclic preparative HPLC to obtain a compound of formula 20; the A2-8 is separated and purified by cyclic preparative HPLC to obtain a compound of formula 22;

[0031] The A3 fraction was separated by silica gel column chromatography using a gradient solvent of dichloromethane / methanol to obtain fractions A3-1 to A3-11, and the A3-2, A3-4 and A3-5 were separated and purified using cyclic preparative HPLC to obtain compounds of Formula 10, Formula 13, Formula 16 and Formula 17.

[0032] Furthermore, the A4 fraction was separated by ODS open column chromatography to obtain fractions A4-1 to A4-6, and the A4-1 to A4-6 fractions were separated and purified by cyclic preparative HPLC to obtain compounds of Formula 1, Formula 2, Formula 3, Formula 5, Formula 8, Formula 11, Formula 15 and Formula 18; the A5 fraction was separated by ODS open column chromatography to obtain fractions A5-1 to A5-6, and the A5-2, A5-3 and A5-5 fractions were separated and purified by cyclic preparative HPLC to obtain compounds of Formula 14, Formula 19 and Formula 21.

[0033] Furthermore, the ratio of the gradient solvent dichloromethane / methanol involved in the separation of the A2 and A3 fractions by silica gel column chromatography is 100:0→50:1→25:1→12.5:1→10:1→5:1→3:1→2:1→1:1→1:2→0:100; the ratio of the gradient ethanol / water involved in the polyamide column chromatography is 0:100→1:10→1:5→3:10→1:5→7:10→95:100.

[0034] Furthermore, the concentration ratio of methanol in the eluent methanol-water solution of the ODS open column chromatography used for separation of the A4 and A5 fractions is 10%, 20%, 40%, 60%, 80% and 100% respectively, and the ratio of methanol / water of the mobile phase of the cyclic preparative HPLC is 3:1, 3:2, 1:1, 1:2 or 2:3, or methanol / acetonitrile / water is 1:1:2.

[0035] The corn lignan compounds of the present invention and their application and preparation method have the following beneficial effects:

[0036] (1) The present invention adopts a specific method to extract and separate 22 lignan compounds from corn roots. The above compounds all have different degrees of antioxidant and anti-inflammatory activities. In particular, the compound of formula 1 exhibits significant antioxidant, anti-inflammatory and antidepressant effects in vivo by partially stimulating Fyn.

[0037] (2) The lignan compounds of the present invention can act on depression targets, affect the kinase activity of depression targets, and effectively and quickly achieve an antidepressant effect in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0039] Figure 1 The effect of the corn root lignan compounds of the present invention on the survival rate of SH-SY5Y cells treated with H2O2;

[0040] Figure 2 The free radical (ABTS and DPPH) scavenging activity of the corn root lignan compounds of the present invention;

[0041] Figure 3 The intracellular ROS scavenging activity of the corn root lignan compounds of the present invention;

[0042] Figure 4 The effect of the corn root lignan compounds of the present invention on the body membrane potential of SH-SY5Y cells treated with H2O2;

[0043] Figure 5 The corn root lignan compounds of the present invention are "partial + allosteric" dual agonists of Fyn;

[0044] Figure 6 The corn root lignan compounds of the present invention are directly combined with Fyn to enhance its thermal stability and enzymatic stability;

[0045] Figure 7 The corn root lignan compounds of the present invention target Fyn and BDNF / TrkB, PI3K / Akt and MAPK signaling pathways to exert antioxidant activity;

[0046] Figure 8 The in vivo antidepressant activity of the corn root lignan compounds of the present invention;

[0047] Fig. 9The in vivo antioxidant activity of the corn root lignan compounds of the present invention;

[0048] Fig.10 This is the in vivo anti-inflammatory activity of the corn root lignan compounds of the present invention. DETAILED DESCRIPTION

[0049] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0050] Example 1

[0051] A method for preparing corn root lignan compounds comprises the following steps:

[0052] (1) 50 kg of corn roots were crushed, and then refluxed and extracted with 55% anhydrous ethanol for three times, and the extracts were combined and concentrated under reduced pressure to obtain a crude extract;

[0053] (2) Add sufficient amount of pure water to the crude extract to make it uniformly dispersed in the water, and extract with petroleum ether to remove the high fat-soluble components. After the water extract is concentrated under reduced pressure to remove the residual petroleum ether, it is separated by chromatography on an AB-8 macroporous resin column using 25% and 70% ethanol-water solutions as eluents, and the 70% ethanol-water eluate is collected and concentrated under reduced pressure to obtain the extract;

[0054] (3) The extract obtained in (2) was dissolved in a dichloromethane / methanol mixed solution, separated by silica gel column chromatography, and gradient eluted with dichloromethane / methanol. The ratio of dichloromethane / methanol was 100:3, 100:7, 20:3, 10:3, 10:7 and 0:100, respectively, to obtain 6 fractions (A1-A6);

[0055] (4) fraction A2 obtained in step (3) was subjected to silica gel column chromatography using a gradient solvent of dichloromethane / methanol as an eluent, wherein the ratio of the gradient solvent of dichloromethane / methanol is 100:0→50:1→25:1→12.5:1→10:1→5:1→3:1→2:1→1:1→1:2→0:100, and fraction A2-1 to A2-11 were obtained in sequence;

[0056] (5) fraction A2-2 was separated and purified by cyclic preparative HPLC, wherein the ratio of methanol / water in the cyclic preparative HPLC mobile phase was 3:1, and the compounds of formula 7 and formula 9 were separated and purified;

[0057] Example 2

[0058] A method for preparing corn root lignan compounds comprises the following steps:

[0059] Steps (1) to (4) are the same as in Example 1;

[0060] Step 5: After separating the compounds of Formula 7 and Formula 9 from fraction A2-2 in step (4), the remaining fraction is further separated and purified by cyclic preparative HPLC, wherein the ratio of methanol / water in the mobile phase of the cyclic preparative HPLC is 2:1, to obtain a compound of Formula 12.

[0061] Example 3

[0062] A method for preparing corn root lignan compounds comprises the following steps:

[0063] Steps (1) to (4) are the same as in Example 1;

[0064] Step (5): The fraction A2-3 in step (4) is separated by polyamide column chromatography and cyclic preparative HPLC, wherein the gradient ethanol / water ratio involved in the polyamide column chromatography is 0:100→1:10→1:5→3:10→1:5→7:10→95:100, to obtain 7 fractions A2-3-1 to A2-3-7. The fraction A2-3-3 is further separated and purified by cyclic preparative HPLC, and the ratio of the mobile phase methanol / water is 3:1, to obtain the compound shown in formula 4.

[0065] Example 4

[0066] A method for preparing corn root lignan compounds comprises the following steps:

[0067] Steps (1) to (4) are the same as in Example 1;

[0068] Step (5): Fraction A2-4 is separated and purified by cyclic preparative HPLC to obtain a compound of formula 6, wherein the ratio of methanol / water in the mobile phase of the cyclic preparative HPLC is 1:3.

[0069] Example 5

[0070] A method for preparing corn root lignan compounds comprises the following steps:

[0071] Steps (1) to (4) are the same as in Example 1;

[0072] Step (5): Fraction A2-6 is separated and purified by cyclic preparative HPLC to obtain a compound of formula 20, wherein the ratio of methanol / water in the mobile phase of the cyclic preparative HPLC is 1:2.

[0073] Example 6

[0074] A method for preparing corn root lignan compounds comprises the following steps:

[0075] Steps (1) to (4) are the same as in Example 1;

[0076] Step (5): Fraction A2-8 is separated and purified by cyclic preparative HPLC to obtain the compound of formula 22, wherein the ratio of methanol / water in the mobile phase of the cyclic preparative HPLC is 3:1.

[0077] Example 7

[0078] A method for preparing corn root lignan compounds comprises the following steps:

[0079] Steps (1) to (3) are the same as in Example 1;

[0080] Step (4): fraction A3 obtained in step (3) is separated by silica gel column chromatography using a gradient solvent of dichloromethane / methanol, wherein the ratio of the gradient solvent of dichloromethane / methanol is 100:0→50:1→25:1→12.5:1→10:1→5:1→3:1→2:1→1:1→1:2→0:100, to obtain fractions A3-1 to A3-11;

[0081] In step (5), fraction A3-3 is separated and purified by cyclic preparative HPLC to obtain the compound of formula 10, wherein the ratio of methanol to water in the mobile phase is 3:1.

[0082] Example 8

[0083] A method for preparing corn root lignan compounds comprises the following steps:

[0084] Steps (1) to (4) are the same as in Example 7;

[0085] Step (5): Fraction A3-4 is further separated and purified by cyclic preparative HPLC to obtain a compound of formula 13, wherein the ratio of methanol to water in the mobile phase is 1:1.

[0086] Example 9

[0087] A method for preparing corn root lignan compounds comprises the following steps:

[0088] Steps (1) to (4) are the same as in Example 7;

[0089] Step (5): Fraction A3-5 is then separated and purified using circulating preparative HPLC with a mobile phase of methanol / water in a ratio of 3:1 to obtain the compound of formula 16.

[0090] Example 10

[0091] A method for preparing corn root lignan compounds comprises the following steps:

[0092] Steps (1) to (4) are the same as in Example 7;

[0093] Step (5): Fraction A3-6 is further separated and purified by cyclic preparative HPLC with a mobile phase of methanol / water in a ratio of 1:2 to obtain a compound represented by formula 17.

[0094] Embodiment 11

[0095] A method for preparing corn root lignan compounds comprises the following steps:

[0096] Steps (1) to (3) are the same as in Example 1;

[0097] Step (4): The A4 fraction obtained in step (3) was separated by ODS open column chromatography using an eluent methanol-water solution with methanol concentrations of 10%, 20%, 40%, 60%, 80% and 100% to obtain fractions A4-1 to A4-6.

[0098] Step (5): Fraction A4-1 was separated and purified using circulating preparative HPLC with a mobile phase of methanol / water in a ratio of 3:1 to obtain the compound represented by formula 1.

[0099] Example 12

[0100] A method for preparing corn root lignan compounds comprises the following steps:

[0101] Steps (1) to (4) are the same as in Example 11;

[0102] Step (5): Fraction A4-1 is further separated and purified using circulating preparative HPLC with a mobile phase of methanol / water in a ratio of 3:2 to obtain the compound of formula 2.

[0103] Example 13

[0104] A method for preparing corn root lignan compounds comprises the following steps:

[0105] Steps (1) to (4) are the same as in Example 11;

[0106] Step (5): Fraction A4-2 is then separated and purified using circulating preparative HPLC with a mobile phase of methanol / water in a ratio of 3:1 to obtain the compound of formula 3.

[0107] Embodiment 14

[0108] A method for preparing corn root lignan compounds comprises the following steps:

[0109] Steps (1) to (4) are the same as in Example 11;

[0110] Step (5): Fraction A4-2 was separated by circulating preparative HPLC using 3:2 methanol / water as the mobile phase to obtain the compound of formula 5.

[0111] Embodiment 15

[0112] A method for preparing corn root lignan compounds comprises the following steps:

[0113] Steps (1) to (4) are the same as in Example 11;

[0114] Step (5): The remaining fraction A4-2 obtained after preparing the compound of Formula 5 is separated and purified by cyclic preparative HPLC using 3:1 methanol / water as the mobile phase to obtain the compound of Formula 8.

[0115] Example 16

[0116] A method for preparing corn root lignan compounds comprises the following steps:

[0117] Steps (1) to (4) are the same as in Example 11;

[0118] Step (5): Fraction A4-3 was further separated and purified by circulating preparative HPLC using 1:1 methanol / water as the mobile phase to obtain the compound represented by formula 11.

[0119] Embodiment 17

[0120] A method for preparing corn root lignan compounds comprises the following steps:

[0121] Steps (1) to (4) are the same as in Example 11;

[0122] Step (5): Fraction A4-4 was separated and purified by cyclic preparative HPLC using 3:2 methanol / water as the mobile phase to obtain the compound represented by formula 15.

[0123] Embodiment 18

[0124] A method for preparing corn root lignan compounds comprises the following steps:

[0125] Steps (1) to (4) are the same as in Example 11;

[0126] Step (5): Fraction A4-5 was separated and purified by circulating preparative HPLC using 2:3 methanol / water as mobile phase to obtain the compound represented by formula 18;

[0127] Embodiment 19

[0128] A method for preparing corn root lignan compounds comprises the following steps:

[0129] Steps (1) to (3) are the same as in Example 1;

[0130] Step (4): The A5 fraction obtained in step (3) is further separated by ODS open column chromatography to obtain A5-1 to A5-6 fractions, wherein the concentration ratio of methanol in the eluent methanol-water solution of the ODS open column chromatography is 10%, 20%, 40%, 60%, 80% and 100% respectively;

[0131] Step (5): Fraction A5-2 is further separated and purified by cyclic preparative HPLC to obtain the compound of formula 14, with the mobile phase having a methanol / water ratio of 1:1.

[0132] Embodiment 20

[0133] A method for preparing corn root lignan compounds comprises the following steps:

[0134] Steps (1) to (4) are the same as in Example 19;

[0135] Step (5): Fraction A5-3 is further separated and purified by cyclic preparative HPLC to obtain the compound of formula 19, with the ratio of methanol to water being 3:2 as the mobile phase;

[0136] Embodiment 21

[0137] A method for preparing corn root lignan compounds comprises the following steps:

[0138] Steps (1) to (4) are the same as in Example 19

[0139] Step (5): Fraction A5-5 is further separated and purified by cyclic preparative HPLC to obtain the compound of formula 21, wherein the ratio of the mobile phase methanol / acetonitrile / water is 1:1:2.

[0140] The structures and NMR spectra of the lignan compounds of Formula 1 to Formula 22 isolated and identified from corn roots obtained in Examples 1 to 22 were analyzed, and the following four characteristic NMR hydrogen spectrum signal peaks were present:

[0141] 1) In the chemical shift region of 6.7-7.1, there are two groups of AA′BB′ coupling systems, ABX coupling systems or single-peak aromatic hydrogen signals.

[0142] 2) There are two trans double bond hydrogen signals in the chemical shift region of 6.2 to 6.7.

[0143] 3) There are two mutually coupled methylene hydrogen signals at chemical shifts around 4.6 and 5.1, or around 3.5 and 5.5.

[0144] 4) There is also a magnetically incompletely identical methylene hydrogen signal connected to oxygen between the chemical shifts of 3.7 and 3.9.

[0145] By performing nuclear magnetic resonance hydrogen spectrum analysis on the fractions A1 to A6 obtained in (3), it was found that the above four characteristic nuclear magnetic hydrogen spectrum signal peaks were obvious in A2 to A5, thereby concluding that the compounds of formula 1 to formula 22 extracted from the corn roots of the present invention are mainly four types of lignan compounds of formula (I), formula (II), formula (III) and formula (IV):

[0146]

[0147] Example 22 Structural Identification of Lignans of the Present Invention

[0148] 1.1. The structural formula of the compound of formula 1 and its structural identification data are:

[0149]

[0150] White powder (methanol), dark spots under UV 254nm, cyan color in 10% sulfuric acid-ethanol. HRESIMS ion peak m / z 729.2522 [M+H]+ (calc.mass 729.2542 for C 40 H 41 O 13 ) suggests that its molecular formula is C 40 H 40 O 13 , and the compound has 21 degrees of unsaturation. 1 In H NMR (600MHz, Methanold4): δ7.32 (2H,d,J=8.3Hz, B′2′ / 6′), 7.25 (2H,d,J=8.3Hz, A′2′ / 6′), 6.72–

[0151] 6.69 (4H, m, A′3′ / 5′, B′3′ / 5′) is the AA′BB′ coupling system of two benzene rings, 7.53 (d, J=15.9 Hz, 1H, B′7′), 6.24 (1H, d, J=15.9 Hz, B′8′), 7.21 (1H, d, J=15.9 Hz, A′7′), 6.06 (1H, d, J=15.9 Hz, A′8′) are two trans double bond hydrogen signals, according to the correlation signals in the HMBC spectrum: δH7.32 (2H, d, J=8.3 Hz, B′2′ / 6′) / δC 146.68 (CB′7′), δH 7.53 (1H, d, J=15.9 Hz, B′7′) / δC168.94 (CB′CO); δH 7.25 (2H, d, J = 8.3 Hz, A'2' / 6') / δC 146.61 (CA'7'), δH 7.21 (1H, d, J = 15.9 Hz, A'7') / δC 168.70 (CA'CO) It is known that there are two p-coumaric acid structures in the compound and its carboxyl group is converted into an ester group. δH 6.67 (2H, s, A2 / 6), 6.61 (2H, s, B2 / 6) and HMBC related signals: δH 6.67 (2H, s, A2 / 6) / δC 105.36 (C-A2 / 6), 148.97 (C-A3 / 5), 136.15 (CA4); 6.61 (2H, s, B2 / 6) / δC 104.90 (C-B2 / 6), 137.62 (C-B4), 154.25 (C-B3 / 5), indicating that the compound has two 1,3,4,5 symmetrically substituted benzene rings; δH 6.48 (1H, d, J = 15.7 Hz, B7), 6.19 (1H, dt, J = 15.8, 6.3 Hz, B8), 4.68 (2H, d, J = 6.4 Hz, B9), it can be seen that the trans double bond is connected to the oxymethylene. The related signal of HMBC: δH 6.48 (1H, d, J = 15.7 Hz, B7) / δC 104.90 (C-B2 / 6), confirming that the trans double bond is connected to C-B1.According to the coupling constants of δH 4.92 (1H, d, J = 6.6 Hz, A7), 4.38–4.32 (1H, m, A8), 4.26 (1H, dd, J = 12.0, 3.0 Hz, A9), 4.02 (1H, dd, J = 11.9, 5.2 Hz, A9) and HMBC related signals: δH 4.92 (1H, d, J = 6.6 Hz, A7) / δC 86.09 (C-A8), 105.36 (C-A2 / 6); δH 4.26 (1H, dd, J = 12.0, 3.0 Hz, A9) / δC 168.70 (C-ACO), it can be seen that the benzene ring of C-A1 and these three carbons form a phenylpropanoid structure and the A9 carbon is condensed with the carboxyl group in the above-mentioned p-coumaric acid structure to form an ester group. δH 3.70 (6H, s, BOCH3), 3.69 (6H, s, AOCH3) are hydrogen signals of methoxy. According to the hydrogen coupling constant of A7 and A8 of 6.6Hz, the relative configuration of the two was determined to be threo. After comparing the measured CD data with ECD and the literature [3], the configuration of compound A7 was determined to be R, and the configuration of compound A8 was determined to be S. Finally, the structure of the compound was determined to be a new compound, which was named zealignan H.

[0152] 1.2. The structural formula of the compound of formula 2 and its structural identification data are:

[0153]

[0154] White powder (methanol), dark spots under UV 254nm, cyan color in 10% sulfuric acid-ethanol. HRESIMS ion peak m / z 729.2540 [M+H]+ (calc.mass 729.2542 for C 40 H 41 O 13 ) suggests that its molecular formula is C 40 H 40 O 13 , and the compound has 21 unsaturations. Zealignan I and Zealignan H have the same planar structure, the difference is that the spatial configuration at A7 and A8 is different. 1In H NMR (600MHz, Methanol-d4), δ7.47 (2H, d, J = 8.8 Hz, HB'2' / 6'), 7.33 (2H, d, J = 8.8 Hz, HA'2' / 6'), 6.80 (4H, dd, J = 8.9, 7.0 Hz, HA'3' / 5', B'3' / 5') are AA'BB' coupling systems of two benzene rings, 7.66 (1H, d, J = 15.9 Hz, HB'7'), 6.38 (1H, d, J = 16.1 Hz, HB'8'), 7.18 (1H, d, J = 16.1 Hz, HA'7'), 6.08 (1H, d, J = 16.0 Hz, HA'8') are two trans double bond hydrogen signals. According to the correlation signals in the HMBC spectrum: δH 7.47 (2H, d, J = 8.8 Hz, HB′2′ / 6′) / δC 146.81 (CB′7′), δH 7.66 (1H, d, J = 15.9 Hz, HB′7′) / δC 169.04 (CB′CO); δH 7.33 (2H, d, J = 8.8 Hz, H-A2′ / 6′) / δC 146.46 (CA′7′), δH 7.18 (1H, d, J = 16.1 Hz, HA′7′) / δC 168.93 (CA′CO) It is known that there are two p-coumaric acid structures in the compound and its carboxyl group becomes an ester group. From δH 6.75 (4H, d, J = 2.4 Hz, H-B2 / 6, A2 / 6) and HMBC related signals: δH 6.75 (4H, d, J = 2.4 Hz, H-B2 / 6, A2 / 6) / δC 104.93 (C-B2 / 6, A2 / 6), 154.52 (C-B3 / 5), 149.05 (C-A3 / 5), 137.42 (C-B4), 135.85 (C-A4), indicating that the compound has two 1,3,4,5 symmetrically substituted benzene rings; from δH6.62 (1H, d, J=15.8 Hz, H-B7), 6.31 (1H, dt, J=15.8,6.1 Hz, H-B8), 4.81 (2H, d, J=6.4 Hz, H-B9), it can be seen that the trans double bond is connected to the oxymethylene group, and the related signal of HMBC is: δH 6.62 (1H, d, J=15.8 Hz, H-B7) / δC 104.93 (C-B2 / 6, A2 / 6) confirmed that the trans double bond was connected to C-B1.According to the coupling constants of δH 5.01 (1H, d, J = 4.3 Hz, H-A7), 4.61 (1H, p, J = 3.4 Hz, H-A8), 4.47 (1H, dd, J = 11.8, 7.1 Hz, H-A9), 4.34 (1H, dd, J = 11.8, 2.9 Hz, H-A9) and HMBC related signals: δH 5.01 (1H, d, J = 4.3 Hz, H-A7) / δC 84.77 (C-A8), 104.93 (C-B2 / 6, A2 / 6); δH 4.34 (1H, dd, J = 11.8, 2.9 Hz, H-A9) / δC 168.93 (CA′CO), it can be seen that the benzene ring of C-A1 and these three carbons form a phenylpropanoid structure and the A9 carbon has a phenylpropanoid structure with the above p-coumaric acid. The carboxyl group in the acid structure condensed into an ester group [2]. δH 3.85 (6H, s, H-BOMe), 3.81 (6H, s, H-AOMe) are hydrogen signals of the methoxy group. Based on the hydrogen spectrum coupling constants of A7 and A8, the relative configuration of the two was determined to be erythro. After comparing the measured CD data with ECD and the literature [3], it was determined that the configuration of compound A7 was S and the configuration of compound A8 was S. Finally, the structure of the compound was determined to be a new compound, which was named zealignan I.

[0155] 1.3, the structural formula of the compound of formula 4 and its structural identification data are:

[0156]

[0157] White powder (methanol), dark spots under UV254 nm, blue-gray in 10% sulfuric acid-ethanol. HRESIMS ion peak m / z 605.1996 [M+Na]+ (calc.mass 605.1999 for C 31 H 34 O 11 Na) suggests that its molecular formula is C 31 H 34 O 11 , and the compound has 15 degrees of unsaturation. 1In HNMR (600MHz, Methanol-d4), δ7.38 (2H, d, J = 8.6 Hz, A'2' / 6'), 6.79 (2H, d, J = 8.6 Hz, A'3' / 5') are a group of AA'BB' coupling systems of benzene rings, 7.33 (1H, d, J = 16.0 Hz, HA'7') and 6.17 (1H, d, J = 15.9 Hz, HA'8') are trans double bond hydrogen signals. According to the correlation signals in the HMBC spectrum: δH 7.38 (2H, d, J = 8.6 Hz, A'2' / 6') / δC 146.71 (CA'7'), δH 7.33 (1H, d, J = 16.0 Hz, HA'7') / δC 168.81 (C-CO), it is speculated that p-coumaric acid exists in the compound. The structure of the compound is acid and its carboxyl group is changed to an ester group. δH 6.75 (2H, s, A2 / 6), 6.73 (2H, s, B2 / 6) and HMBC related signals: δH 6.75 (2H, s, A2 / 6) / δC 105.35 (C-A2 / 6), 149.07 (C-A3 / 5), 136.24 (C-A4), 75.53 (C-A7); 6.73 (2H, s, B2 / 6) / δC 104.70 (C-B2 / 6), 129.84 (C-B8), 137.27 (C-B4), 154.39 (CB3 / 5) indicate that the compound has two 1,3,4,5 symmetrically substituted benzene rings and that the A benzene ring is substituted with the methine carbon δC 75.53 (C-A7), the B benzene ring is connected to another trans double bond carbon 129.84 (C-B8); from δH 6.52 (1H, dt, J = 15.8, 1.6 Hz, H-B7), 6.29 (1H, dt, J = 15.9, 5.6 Hz, B8), 4.21 (2H, dd, J = 5.6, 1.6 Hz, B9), it can be seen that the trans double bond is connected to the oxymethylene. According to the coupling constants of δH 4.98 (1H, d, J = 6.6 Hz, A7), 4.41 (1H, ddd, J = 6.5, 5.1, 3.1 Hz, A8), 4.31 (1H, dd, J = 11.9, 3.1 Hz, A9), 4.06 (1H, dd, J = 11.9, 5.2 Hz, A9) and HMBC related signals: δH 4.98 (1H, d, J = 6.6 Hz, A7) / δC 86.03 (C-A8), 105.35 (C-A2 / 6), 132.13 (C-A1); δH 4.31 (1H, dd, J = 11.9, 3.1 Hz, A9) / δC 75.53 (C-A7), 168.81 (C-CO), indicating that the benzene ring A and these three carbons form a phenylpropanoid structure and the Aγ carbon condenses with the carboxyl group in the above-mentioned p-coumaric acid structure to form an ester group [5].δH 3.82 (6H, s, AOCH3), 3.81 (6H, s, BOCH3) are hydrogen signals of methoxy. According to the coupling constants of 4.98 (1H, d, J = 6.6 Hz, A7), 4.41 (1H, ddd, J = 6.5, 5.1, 3.1 Hz, A8), the compound was judged to be of threo configuration. The measured CD data were compared with the literature [6, 7], and the positive cotton effect of 225 ± 5 nm determined that the configuration of compound A7 was R, and the configuration of compound A8 was S. Finally, the structure of the compound was determined to be a new compound, which was named zealignan F.

[0158] 1.4. The structural formula of the compound of formula 6 and its structural identification data are:

[0159]

[0160] White powder (methanol), dark spots under UV254 nm, blue-gray in 10% sulfuric acid-ethanol. HRESIMS ion peak m / z 765.2527 [M+Na]+ (calc.mass 765.2523 for C 41 H 42 O 13 Na) suggests that its molecular formula is C 41 H 42 O 13 , and the compound has 21 degrees of unsaturation. 1In H NMR (600MHz, Methanol-d4): δ7.30 (4H, t, J = 8.4 Hz, A′2′ / 6′, B′2′ / 6′), 6.72–6.66 (5H, m, A6, A′3′ / 5′, B′3′ / 5′) are AA′BB′ coupling systems of benzene rings, 7.51 (1H, d, J = 16.0 Hz, A′7′), 6.26 (1H, d, J = 15.9 Hz, A′8′), 7.48 (1H, d, J = 15.9 Hz, B′7′), 6.23 (1H, d, J = 15.9 Hz, B′8′) are trans double bond hydrogen signals. According to the correlation signals in the HMBC spectrum: δH 7.30 (4H, t, J=8.4 Hz, A′2′ / 6′, B′2′ / 6′) / δC 146.57 (CA′7′), 146.84 (CB′7′), δH 7.51 (1H, d, J=16.0 Hz, A′α′) / δC 169.59 (CA′CO), δH 6.23 (1H, d, J=15.9 Hz, B′8′) / δC 169.16 (CB′CO) It is known that there are two p-coumaric acid structures in the compound and its carboxyl group is changed into an ester group. δH 6.51 (s, 2H, B2 / 6) and HMBC related signals: δH 6.51 (s, 2H, B2 / 6) / δC 107.27 (C-B2 / 6), 134.56 (CB4), 140.12 (C-B1), 148.94 (C-B3 / 5) indicate that the compound has 1,3,4,5 symmetrically substituted benzene rings. From δH 4.52–4.44 (2H, m, A9, B9), 4.34 (1H, dd, J = 11.3, 6.5 Hz, A9), 4.32 (1H, d, J = 2.0 Hz, A7), 2.22 (1H, dtd, J = 12.4, 6.3, 2.0 Hz, A8); δH 4.52–4.44 (2H, m, A9, B9), 4.28 (1H, dd, J = 11.6, 3.3 Hz, B9), 3.96 (1H, d, J = 8.6 Hz, B7), 2.46 (1H, ddt, J = 11.8, 8.6, 3.0 Hz, B8) and the related signal of HMBC: δH 4.32 (1H, d, J = 2.0 Hz, A7) / δC 42.74 (C-B8), 67.19 (C-A9), 109.40 (C-A6), 127.62 (C-A1); δH 2.46 (1H, ddt, J=11.8, 8.6, 3.0 Hz, B8) / δC 41.74 (C-A8), 44.83 (C-B7), 80.10 (C-A7), 140.12 (C-B1). It can be seen that the compound has the structural skeleton of aryl tetralinolone [8].From the HMBC related signals: δH 4.34 (dd, J = 11.3, 6.5 Hz, 1H, A9) / δC 169.59 (CA′CO), it can be seen that the A9 carbon forms an ester group with the above-mentioned p-coumaric acid; δH 4.32 (d, J = 2.0 Hz, 1H, A7) / δC 56.38 (A7-OCH3), it can be seen that the methoxy A7-OCH3 is connected to the A7 carbon; from the HMBC related signals: δH 3.86 (s, 3H, A5-OCH3) / δC 148.14 (C-A5), δH 3.73 (s, 6H, B3 / 5-OCH3) / δC 148.94 (C-B3 / 5), δH 3.11 (s, 3H, A3-OCH3) / δC 147.66 (C-A3) was assigned to the methoxy group. The coupling constants of δH 4.32 (d, J = 2.0 Hz, 1H, A7), 2.22 (dtd, J = 12.4, 6.3, 2.0 Hz, 1H, A8) showed that A7 and A8 were erythroforms, δH 3.96 (d, J = 8.6 Hz, 1H, B7), 2.46 (ddt, J = 11.8, 8.6, 3.0 Hz, 1H, B8), B7 and B8 were threoforms. According to the measured CD data 270nm (-7.34), 290nm (+1.67) and compared with the literature [3], it was determined that the configuration of compound A7 was R, the configuration of A8 was R, the configuration of B7 was R, and the configuration of B8 was S. The structure of the compound was a new compound and was named Zealignan G.

[0161] Example 23 Cytotoxic Activity Test of Corn Lignans Compounds of the Present Invention

[0162] Experimental method: SH-SY5Y cells were purchased from the cell bank of the Type Culture Collection Committee of the Chinese Academy of Sciences and cultured in complete DMEM / F12 (Gibco, USA) medium containing 10% FBS at 37°C and 5% CO2. After the cells adhered to the wall, 2.5 μM to 20 μM of the corn lignan compounds of the present invention were added to treat the cells for 72 hours, and the effects of the compounds on the cell survival rate were calculated by measuring the OD value to evaluate the cytotoxicity of the compounds on SH-SY5Y cells.

[0163] Experimental results: As shown in Table 1, the tested corn lignan compounds Formula 1-Formula 22 had no obvious cytotoxicity to SH-SY5Y cells within 20 μM. In addition, most of the compounds also have a certain activity in promoting cell proliferation and regeneration, especially Formula 4, Formula 8, Formula 15, Formula 21 and Formula 22.

[0164] Table 1 Effects of corn root lignans on the cell proliferation activity of SH-SY5Y cells

[0165]

[0166]

[0167] Example 24 Antioxidant Activity Test of Corn Lignans of the Present Invention

[0168] Experimental method: 22 corn lignan compounds shown in Formula 1 to Formula 22 obtained in the present invention are dissolved in DMSO with an initial concentration of 20mM, and the maximum content of DMSO added to the cells is far less than 0.1%. Cells are treated with a specified concentration of corn lignan compounds. After the treated cells are incubated with 200μM H2O2 and the corn lignan compounds of the present invention for 2 hours, they are further cultured with a complete medium containing the corresponding concentration of corn lignan compounds for 24 hours. The absorbance at 450nm is measured by CCK8 method to calculate the survival rate of the cells.

[0169] Experimental results: The effects of the corn lignan compounds obtained in the present invention on the oxidative stress of SH-SY5Y cells induced by H2O2. Figure 1 As shown, 200 μM H2O2 treatment of SH-SY5Y cells resulted in a decrease in cell survival rate to about 60%. However, after treatment with the compounds of Formula 1 to Formula 22 of the present invention, the survival rate of SH-SY5Y cells after H2O2 treatment can be improved to varying degrees, showing a certain antioxidant activity. Among them, the cell survival rates of the groups treated with Formula 1, Formula 3, Formula 4, Formula 5, Formula 7, Formula 9, Formula 10, Formula 11, Formula 14, Formula 17 and Formula 18 all exceeded 70%. In particular, the cell survival rate (75.60%) of the compound of formula 1 after treating SH-SY5Y cells was significantly higher than that of the H2O2-treated group (58.09%). It should be noted that the compound of formula 2 is only different from the compound of formula 1 in configuration, but its antioxidant activity is significantly weaker than that of the compound of formula 1, indicating that the configuration difference of lignan compounds has a great influence on their biological activity. Therefore, the corn lignan compounds of the present invention, especially the compound of formula 1, have in vitro antioxidant activity and are worth further studying in vivo efficacy. Therefore, we selected the compound of formula 1 as a representative compound for subsequent research on target and antidepressant efficacy.

[0170] Example 25 In vitro ABTS scavenging activity test of corn lignan compounds of the present invention

[0171] In vitro ABTS free radical scavenging experimental method: The compound of formula 1 obtained by the present invention is respectively prepared into a certain concentration with an extract, and the required solution and 10mmol / L vitamin C solution are prepared into 0.4, 0.2, 0.1, 0.05, 0.025, and 0.0125mmol / L vitamin C solutions with the extract according to the reagent instructions for standby use. Preheat the spectrophotometer / enzyme reader for more than 30 minutes, adjust the wavelength to 405nm, and zero the spectrophotometer with distilled water. Add the prepared reagents to the 96-well plate according to the operation table, mix well, and stand at room temperature in the dark for 6 minutes. Measure the absorbance at 405nm.

[0172] Calculation of ABTS free radical scavenging rate:

[0173] 1. The free radical scavenging rate calculation formula of the positive control is: ABTS free radical scavenging rate DVC% = [(A blank - A positive control) ÷ A blank] × 100%;

[0174] 2. The free radical scavenging rate calculation formula of the sample: ABTS free radical scavenging rate D% = [A blank - (A measurement - A control)] ÷ A blank × 100%.

[0175] Experimental results: The ABTS scavenging activity of the compound of formula 1 is shown in the attached Figure 2 As shown in Figure A, at different concentrations from 2.5 to 40 μM, the ABTS scavenging activities of the compound of formula 1 were 13.09%, 14.56%, 16.37%, 19.76% and 27.18%, respectively. Figure 2 ), with good dose dependence.

[0176] Example 26 In vitro DPPH scavenging activity test of corn lignan compounds of the present invention

[0177] DPPH removal experimental method: The compound of formula 1 obtained by the present invention is respectively prepared into a certain concentration with an extract, and the required solution and 10mmol / L vitamin C solution are prepared into 0.3, 0.25, 0.125, 0.0625, 0.03125, 0.015625mg / mL vitamin C solution with an extract according to the reagent instructions for standby use. Preheat the spectrophotometer / enzyme reader for more than 30 minutes, adjust the wavelength to 515nm, and zero the spectrophotometer with anhydrous ethanol. Add the prepared reagents to the 96-well plate according to the operation table, vortex mix, stand at room temperature and avoid light for 30 minutes, and measure the absorbance at 515nm.

[0178] 1. Calculation formula of free radical scavenging rate of positive control: DPPH free radical scavenging rate DVC% = [(A blank - A positive control) ÷ A blank] × 100%;

[0179] 2. The free radical scavenging rate of the sample is calculated as follows: DPPH free radical scavenging rate D% = [[A blank - (A assay - A control)] ÷ A blank] × 100%.

[0180] Experimental results: The DPPH scavenging activity of the compound of formula 1 of the present invention is shown in the attached Figure 2 As shown in Figure B, at different concentrations from 2.5 μM to 40 μM, the compound of formula 1 exhibited a strong DPPH scavenging ability, with scavenging rates of 18.63%, 21.67%, 27.62%, 31.82% and 42.46%, respectively. Figure 3 ).

[0181] Example 27 Intracellular ROS scavenging activity test of corn lignan compounds of the present invention

[0182] Experimental method for intracellular accumulation of ROS: After treating the cells with the compound of formula 1 obtained by the present invention, dilute DCFH-DA with serum-free culture medium at a ratio of 1:1000 to a final concentration of 10 micromol / L. After collecting the cells, suspend them in the diluted DCFH-DA at a cell concentration of 1 million to 20 million / ml and incubate them in a cell culture incubator at 37°C for 20 minutes. Invert and mix every 3-5 minutes to allow the probe and cells to fully contact. Wash the cells three times with serum-free cell culture medium to fully remove the DCFH-DA that has not entered the cells, and observe the fluorescence changes by flow cytometry or fluorescence microscopy.

[0183] Experimental results: Figure 3 As shown, the results of flow cytometry analysis showed that after co-treatment with the compound of formula 1, the level of ROS in SH-SY5Y cells treated with H2O2 was significantly decreased. At the same time, the green fluorescence representing ROS was significantly reduced in a dose-dependent manner, which was close to the ROS level of the control group cells ( Figure 4 ). The above results show that the compound of formula 1 has good antioxidant ability and can protect cells from oxidative stress damage.

[0184] Example 28 Test on the effect of corn lignan compounds of the present invention on mitochondrial function

[0185] Mitochondrial membrane potential test method: After treating the cells with the compound of formula 1 obtained by the present invention, prepare the JC-1 staining working solution according to the experimental instructions and collect the cells, add 1 ml of JC-1 staining working solution, and mix thoroughly. Incubate in a cell culture incubator at 37°C for 20 minutes. During the incubation period, prepare an appropriate amount of JC-1 staining buffer (1X) according to the ratio of adding 4 ml of distilled water to every 1 ml of JC-1 staining buffer (5X), and place it in an ice bath. After the incubation at 37°C, remove the supernatant and wash twice with JC-1 staining buffer (1X). Add 2 ml of cell culture fluid, which may contain serum and phenol red. Observe under a fluorescence microscope or a laser confocal microscope.

[0186] Experimental results: Mitochondrial dysfunction may lead to increased oxidative stress in patients with depression. Therefore, the function of the compound of formula 1 to protect mitochondrial function and reduce oxidative stress is beneficial to depression. Figure 4 As shown, the red fluorescence intensity in the control group cells was very high, indicating that the mitochondrial △ψm was high. The red fluorescence intensity in the H2O2-treated group cells was significantly reduced, and the corresponding green intensity was significantly increased, indicating that △Ψm was depolarized and the cells had mitochondrial dysfunction. However, after pretreatment with the compound of formula 1, the red fluorescence intensity in the cells increased in a dose-dependent manner (23.41%, 26.62%, 32.19%), indicating that the compound of formula 1 can protect SH-SY5Y cells from mitochondrial dysfunction caused by H2O2 treatment and inhibit oxidative damage.

[0187] Example 29 Test on the antioxidant activity of corn lignan compounds in the present invention by partially stimulating Fyn

[0188] (1) Screening of inhibitory activity of corn lignans on targets related to nervous system diseases

[0189] Experimental method: Using Eurofins KinaseProfiler TM The kinase activity assay method is used to detect the effects of the preferred compounds of formula 1 of the present invention on the kinase activities of targets related to nervous system diseases.

[0190] As shown in Table 2 and Figure 5 As shown in A, the compound of formula 1 can significantly enhance the kinase activity of Fyn. Except for a weak activation effect on TrkB, it has no significant effect on the kinase activity of other related enzymes.

[0191] Table 2 Effects of corn lignan H on the kinase activity of kinases related to the treatment of nervous system diseases

[0192]

[0193] (2) Effect of the corn lignan compounds of the present invention on the binding of Fyn to its ligands

[0194] Test method: KINOMEscan by Eurofins TM The kinase screening and analysis technology platform tests the effect of the corn lignan compounds of the present invention on the binding of Fyn and its ligand, and analyzes whether the corn lignan compounds of the present invention are orthosteric agonists, taking the compound of formula 1 as an example.

[0195] Experimental results: Figure 5 B shows KINOMEscan TM Competitive binding analysis shows that the compound of formula 1 can significantly increase the binding of Fyn with its small molecule ligand, indicating that there is no competitive binding relationship between the compound of formula 1 and the ligand of Fyn, and it does not act on the orthosteric site of Fyn, but binds to the allosteric site of Fyn and promotes the binding of Fyn with its small molecule ligand. Therefore, it can be seen that the compound of formula 1 is an allosteric agonist of Fyn.

[0196] Example 30 Test on the effect of corn lignan compounds of the present invention on enzyme kinetic parameters

[0197] Experimental method: Follow the instructions of the kit. Dilute the enzyme, endogenous substrate, ATP and inhibitor or (and) compound of formula 1 with kinase buffer. Add: (1) 1 μl inhibitor or 5% DMSO; (2) 2 μl enzyme; (3) 2 μl substrate / ATP mixture to 384 small volume plate wells, then incubate at room temperature for 60 minutes, add 5 μl ADP-Glo TM Reagent, incubate at room temperature for 40 min, add 10 μl kinase detection reagent, incubate at room temperature for 30 min, and record luminescence (integration time 0.5-1 s).

[0198] Experimental results: Figure 5 As shown in C, unlike the endogenous substrate of Fyn, the compound of formula 1 can only partially stimulate Fyn, E max The value of E is 36.32%, indicating that the compound of formula 1 is a partial agonist of Fyn. When the endogenous substrate of Fyn is present, the compound of formula 1 will inhibit the kinase activity of Fyn. On the contrary, when there is a Fyn inhibitor, the agonist effect of the compound of formula 1 on Fyn is enhanced. As the concentration of the compound of formula 1 increases, its E max The above results show that the compound of formula 1 exhibits an antagonistic effect when Fyn is activated, and exhibits an enhancing effect on Fyn when Fyn is inhibited. In view of the characteristics of the compound of formula 1 that simultaneously promotes the binding affinity of Fyn with its endogenous substrate or small molecule inhibitor and its partial agonist effect on Fyn, the compound of formula 1 is a new type of partial agonist acting on the allosteric site of Fyn.

[0199] Example 31 Direct combination of corn lignan compounds of the present invention with Fyn enhances the thermal stability and enzymatic stability of Fyn

[0200] Experimental method: The total protein lysate of mouse brain tissue was collected and incubated with the corresponding concentration of the compound of formula 1 to examine the thermal stability and enzymatic stability of Fyn and to detect whether the compound of formula 1 directly binds to Fyn.

[0201] Experimental results: Figure 6 As shown in A, the compound of formula 1 significantly increased the cellular thermal stability of Fyn protein. Figure 6 As shown in Figure B, the effect of the compound of formula 1 on the cellular thermal stability of Fyn protein is significantly concentration-dependent. Figure 6 As shown in C, in the DARTS assay, the enzymatic degradation of Fyn was significantly reduced after treatment with the compound of Formula 1, indicating that the compound of Formula 1 directly targets Fyn.

[0202] Example 32 Test on the antioxidant activity of corn lignan compounds in the present invention by partially stimulating Fyn

[0203] Experimental method: In order to investigate the effect of other lignan compounds in the present invention on the kinase activity of Fyn, the KinaseProfiler of Eurofins was used. TM The kinase activity assay method was used to detect the agonistic activity of the 22 lignan compounds of the present invention on the kinase activity of Fyn. The results are shown in Table 3.

[0204] Experimental results: The 22 lignan compounds of the present invention have different degrees of agonistic effects on Fyn. Among them, Formula 1, Formula 3, Formula 9, Formula 10, Formula 11, Formula 12, Formula 14, Formula 15, Formula 17 and Formula 18 have significant agonistic activity. Compared with the control group, the kinase activity of Fyn in the lignan compound treatment group is greater than 120%, indicating that the above compounds are similar to the compound of Formula 1 and are all partial agonists of Fyn.

[0205] Table 3 Effect of the lignan compounds of the present invention on the kinase activity of Fyn

[0206]

[0207]

[0208] Example 33 The corn lignan compounds of the present invention exert antioxidant effects by regulating PI3K / Akt / mTOR, MAPKs, BDNF / TrkB / CREB and Nrf2 / HO-1 signaling pathways

[0209] Experimental method: Western blotting assay was used to detect the role of PI3K / Akt / mTOR, MAPKs, BDNF / TrkB / CREB and Nrf2 / HO-1 signaling pathways in the anti-oxidative stress injury of the compound of formula 1.

[0210] Experimental results: Fyn can phosphorylate Nrf2, promote its export to the cytoplasm, and inhibit the transcription of its downstream genes. Western blot analysis was used to further study whether the cell protection activity of the compound of formula 1 of the present invention is related to the activation of the Nrf2 / HO-1 signaling pathway. Figure 7 As shown in A, the results of Western blot analysis showed that H2O2 treatment significantly increased the protein expression of Fyn in SH-SY5Y cells. However, after treatment with the compound of formula 1, the changes in Fyn protein induced by H2O2 were reversed in a dose-dependent manner. H2O2 treatment reduced the total protein levels of Nrf2 and HO-1 in SH-SY5Y cells. After co-treatment with the compound of formula 1, the protein levels of Nrf1 and HO-1 were restored to a certain extent, and there was an obvious dose-dependent relationship.

[0211] Fyn kinase enhances the neuroinflammatory response of microglia through the mitogen-activated protein kinase (MAPK) pathway, which is related to the pathogenesis of depression. Figure 7 As shown in B, H2O2 treatment significantly reduced the levels of p-ERK and p-P38 in SH-SY5Y cells. In addition, H2O2 treatment significantly activated JNK, thereby inducing cell apoptosis. The compound of formula 1 restored the levels of p-ERK and p-P38 in SH-SY5Y cells in a dose-dependent manner and reduced the protein level of p-JNK. PI3K / Akt / mTOR can inhibit Fyn nuclear translocation and reduce oxidative stress.

[0212] according to Figure 7 As shown in Figure C, H2O2 inhibits the activation of the PI3K / Akt pathway by reducing the levels of phosphoproteins PI3K, Akt, mTOR and GSK3β. However, treatment with the compound of formula 1 of the present invention offsets the inhibition of the PI3K / Akt pathway caused by H2O2 in a dose-dependent manner. The PI3K / Akt / CREB pathway, as a downstream of BDNF / Fyn, plays an important role in neuronal survival and synaptic plasticity in cerebral hypoperfusion injury. The CREB / BDNF / TrkB pathway has been widely studied for its antioxidant properties because it can significantly enhance the upregulation of Nrf2 / HO-1.

[0213] like Figure 7As shown in D, H2O2 reduced the levels of p-CREB, BDNF and TrkB, and treatment with the compound of formula 1 of the present invention reversed this effect in a dose-dependent manner. These results indicate that the compound of formula 1 promotes cell viability through these signaling pathways and exerts antioxidant activity against H2O2-induced oxidative stress.

[0214] Example 34 Tissue distribution characteristics of corn lignan compounds of the present invention

[0215] Experimental method: The existence of the blood-brain barrier severely limits the development of neurological drugs, with a success rate of only about 8%. After a single intraperitoneal administration of the compound of formula 1 of the present invention (4 mg / kg), samples of the heart, liver, spleen, lung, kidney and brain of C57 were collected at 5 hours.

[0216] Experimental results: As shown in Table 4, the compound of formula 1 was found in spleen, liver, kidney and lung, followed by a relatively high level of the compound of formula 1 found in the brain. In addition, the level of the compound of formula 1 in the heart area was low. Therefore, the compound of formula 1 of the present invention has good blood-brain barrier permeability and antidepressant effect in vivo.

[0217] Table 4 Distribution of corn lignan H in mouse tissues

[0218]

[0219] The above data are the average values ​​of the corresponding data of 6 mice.

[0220] Example 35 The corn lignan compounds of the present invention significantly alleviate the depressive-like behavior caused by CUMS. Experimental method: C57 / J male mice were randomly divided into 4 groups, sham group (Sham), model group (Model), 50mg / kg Formula 1 compound 2 groups, 100mg / kg Formula 1 compound 2 groups. Except for the control group, the other three groups were modeled according to the experimental conditions of Tables 5 and 6. In the third week of modeling, gavage was started, and the model group was gavaged with a corresponding volume of physiological saline. After the modeling was completed, the open field test, tail suspension test, forced swimming and sugar water preference behavior tests were carried out in sequence, and the drug administration continued during the behavioral experiment until the animals were killed.

[0221] Table 5. Stimulation methods of the CUMS model

[0222]

[0223]

[0224] Table 6. Stimulation daily schedule of the CUMS model

[0225]

[0226] Open field experiment: The experiment is conducted in a quiet environment. The animal is placed in the center of the bottom of the box, and video and timing are performed simultaneously. Video recording is stopped after 8 minutes of observation. The experimental results are analyzed according to the parameters set by the computer software design.

[0227] Tail suspension test: The software used is debugged to adjust the experimental conditions. The mouse is suspended in the middle of the compartment. The width and depth are large enough so that it cannot touch the wall. After the suspension, the distance between the mouse nose tip and the equipment floor is about 20 cm. The tape used for suspension should be fixedly attached to the mouse tail and the suspension rod, and it should be strong enough to bear the weight of the mouse being tested. The swinging and struggling behaviors can be accurately distinguished and identified by calculating and analyzing the activity. After the experiment, the animal is returned to the mouse cage.

[0228] Forced swimming: Place mice in a cylindrical glass tank 20 cm high and 14 cm in diameter, one mouse in each tank. Put warm water in the tank in advance for swimming for 3 min 30 s, and record the cumulative immobility time of the mice.

[0229] Sugar water preference behavior: On the first day of the experiment, two bottles of drinking water were placed in each cage. On the second day, a bottle of 1% sucrose water and a bottle of drinking water were replaced; on the third day, the two bottles were swapped to adapt to the sugar water; on the fourth day, the mice were deprived of food and water; on the fifth day, a bottle of sugar water and drinking water were placed in each cage; on the sixth day, the amount of remaining water was weighed and calculated: sugar water preference is sucrose water / (drinking water + sucrose water) × 100%.

[0230] Experimental results: The efficacy of the compound of formula 1 of the present invention in depression induced by the CUMS model is as follows Figure 8 Behavioral tests (open field test, tail suspension test, forced swimming test, and sucrose preference test) were used to detect the effects of drugs on the depressive behaviors induced by the CUMS program. Figure 8 As shown in Figures B and 8C, in the open field test (OFT), the time that the CUMS model mice spent passing through the central area was significantly reduced compared with the control group, and they showed a slower average speed and a longer immobility time. However, after treatment with the compound of Formula 1, the time that the CUMS model mice spent passing through the central area was significantly increased, and the total distance and average speed of movement were also significantly improved. Figure 8 E and Figure 8 As shown in Figure F, compared with the control group, the immobility time of the CUMS model group mice in the tail suspension test (TST) and forced swimming test (FST) was significantly increased. After treatment with the compound of formula 1, the immobility time of the CUMS mice was significantly reduced. In addition, compared with the control mice, the model mice had a lower preference for sugar water ( Figure 8 G). However, treatment with the compound of formula 1 significantly increased the sugar water consumption of CUMS model mice. The above behavioral study results show that the compound of formula 1 of the present invention can effectively improve the depressive-like symptoms of CUMS model mice.

[0231] Example 36 Corn lignan compounds of the present invention significantly reduce oxidative stress caused by CUMS

[0232] Experimental methods: Oxidative stress is a typical pathological feature of depression. Therefore, basic markers such as malondialdehyde (MDA) and GSH-PX were measured to evaluate oxidative stress in serum and brain tissue.

[0233] Experimental results: Fig. 9 As shown, depressive brain damage can cause an increase in MDA content or a decrease in GSH-PX content in serum and brain tissue. The group treated with the compound of formula 1 can restore the levels of MDA and GSH-PX ( Fig. 9 ), indicating that the compound of formula 1 of the present invention can significantly reduce the oxidative damage in patients with depression.

[0234] Example 37 The corn lignan compounds of the present invention significantly reduce the inflammatory response caused by CUMS

[0235] Experimental method: Since oxidative stress can induce inflammatory damage, the levels of inflammatory factors TNF-α, IL-1β and IL-6 in brain tissue and serum were further measured by ELISA kits.

[0236] Experimental results: Fig.10 As shown, compared with the control group, the production of proinflammatory factors TNF-α, IL-1β and IL-6 in the serum and brain tissue of mice in the CUMS model group increased. However, the levels of TNF-α, IL-1β and IL-6 were significantly reduced after treatment with the compound of formula 1 of the present invention. These data indicate that the compound of formula 1 of the present invention can alleviate oxidative stress and inflammatory damage in patients with depression.

[0237] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present invention. The appearance of the phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0238] The embodiments disclosed in the present invention are only preferred embodiments of the present invention, which are only used to illustrate the technical solutions of the present invention rather than to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it should be understood by those skilled in the art that the technical solutions described in the aforementioned embodiments can still be modified, or some of the technical features therein can be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A corn root lignan compound, characterized in that: It has the following general structural formula: In the formula I, R1 is, R2 is, R3 is, R4 is, R5 is, R6 is, R7 is, and R8 is any one of hydrogen, hydroxyl, methyl, methoxy, ethyl, butyl, aromatic ring containing saturated or unsaturated substituents, and aromatic heterocycle containing saturated or unsaturated substituents. In the formula II, R1 is, R2 is, R3 is, R4 is, R5 is, and R6 is any one of hydrogen, hydroxyl, methyl, methoxy, ethyl, butyl, aromatic ring containing saturated or unsaturated substituents, and aromatic heterocycle containing saturated or unsaturated substituents; In the formula III, R1 is, R2 is, R3 is, R4 is, R5 is, R6 is, R7 is, and R8 is any one of hydrogen, hydroxyl, methyl, methoxy, ethyl, butyl, aromatic ring containing saturated or unsaturated substituents, and aromatic heterocyclic ring containing saturated or unsaturated substituents; In the formula IV, R1 is, R2 is, R3 is, R4 is, and R5 is any one of hydrogen, hydroxyl, methyl, methoxy, ethyl, butyl, an aromatic ring containing a saturated or unsaturated substituent, and an aromatic heterocycle containing a saturated or unsaturated substituent.

2. The corn root lignan compound according to claim 1, characterized in that: Including the following structural formula:

3. An application of the corn root lignan compound according to claim 1 or 2, characterized in that: Application of the corn root lignan compounds in the prevention or common treatment of Fyn-related inflammation, oxidative stress and excitotoxicity-related diseases or target treatment drugs; The Fyn-related diseases include mental illness, nervous system diseases, inflammation, organ damage caused by oxidative stress and excitatory damage, respiratory system diseases, tumors and chronic inflammatory diseases; The mental illnesses include depression, anxiety, schizophrenia, obsessive-compulsive disorder, mania, and bipolar disorder caused by oxidative stress, excitatory injury or inflammation. The nervous system diseases include cerebrovascular diseases and neurodegenerative diseases caused by oxidative stress, excitatory injury or inflammation; the cerebrovascular diseases include cerebral infarction, cerebral hemorrhage and cerebral aneurysm; the neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis; The organ damage includes oxidative stress, excitatory damage, and kidney damage, liver damage, heart damage, lung damage and osteoarthritis caused by acute or chronic inflammation.

4. A Fyn target partial agonist for antidepressant, characterized in that: It comprises the corn root lignan compounds of formula 1 to formula 22 as described in claim 1 or 2 or their pharmaceutically acceptable salts and a pharmaceutically acceptable carrier or excipient.

5. The Fyn target partial agonist for antidepressant according to claim 4, characterized in that: At least including a compound of Formula 1 and / or a compound of Formula 2 and / or a compound of Formula 4 and / or a compound of Formula 6, and / or a pharmaceutically acceptable salt of a compound of Formula 1 and a pharmaceutically acceptable carrier or excipient, and / or a pharmaceutically acceptable salt of a compound of Formula 2 and a pharmaceutically acceptable carrier or excipient, and / or a pharmaceutically acceptable salt of a compound of Formula 4 and a pharmaceutically acceptable carrier or excipient, and / or a pharmaceutically acceptable salt of a compound of Formula 6 and a pharmaceutically acceptable carrier or excipient.

6. A method for preparing corn root lignan compounds according to claim 1, characterized in that: The following steps are involved: (1) extracting corn roots with 50-70% anhydrous ethanol under reflux, and concentrating under reduced pressure to obtain a crude extract; (2) adding a sufficient amount of pure water to the crude extract to uniformly disperse it in the water, extracting with petroleum ether to remove the remaining water extract of the high fat-soluble components, concentrating the water extract under reduced pressure to remove the residual petroleum ether, and then separating it through AB-8 macroporous resin column chromatography using ethanol-water solution as eluent, collecting the ethanol-water elution fraction, and concentrating under reduced pressure to obtain the extract; (3) dissolving the extract obtained in (2) in a dichloromethane / methanol mixed solution, mixing the sample with silica gel, and performing silica gel column chromatography, and performing gradient elution with dichloromethane / methanol to obtain fractions; (4) Separating and identifying the lignan compounds in the fraction obtained in step (3) by silica gel column chromatography, ODS open column chromatography (methanol-water) elution, polyamide column chromatography (ethanol-water) elution, or circulating preparative HPLC to obtain the corn root lignan compounds of the present invention.

7. The method for preparing corn root lignan compounds according to claim 6, characterized in that: The A2 fraction in step (3) is separated by silica gel column chromatography using a gradient solvent of dichloromethane / methanol as an eluent to obtain fractions A2-1 to A2-11, and the fractions A2-1 to A2-11 are separated by cyclic preparative HPLC and / or purified by polyamide column chromatography to obtain compounds represented by Formula 4, Formula 6, Formula 7, Formula 9, Formula 12, Formula 20 and Formula 22; The A3 fraction was separated by silica gel column chromatography using a gradient solvent of dichloromethane / methanol to obtain fractions A3-1 to A3-11, and the A3-3, A3-4, A3-5 and A3-6 were then separated and purified using cyclic preparative HPLC to obtain compounds of Formula 10, Formula 13, Formula 16 and Formula 17.

8. The method for preparing corn root lignan compounds according to claim 6, characterized in that: The A4 fraction was separated by ODS open column chromatography to obtain fractions A4-1 to A4-6, and the A4-1 to A4-5 fractions were separated and purified by cyclic preparative HPLC to obtain compounds of Formula 1, Formula 2, Formula 3, Formula 5, Formula 8, Formula 11, Formula 15 and Formula 18; the A5 fraction was separated by ODS open column chromatography to obtain fractions A5-1 to A5-6, and the A5-2, A5-3 and A5-5 fractions were separated and purified by cyclic preparative HPLC to obtain compounds of Formula 14, Formula 19 and Formula 21.

9. The method for preparing corn root lignan compounds according to claim 7, characterized in that: The ratio of the gradient solvent dichloromethane / methanol involved in the separation of the A2 and A3 fractions by silica gel column chromatography is 100:0→50:1→25:1→12.5:1→10:1→5:1→3:1→2:1→1:1→1:2→0:100; the ratio of the gradient ethanol / water involved in the polyamide column chromatography is 0:100→1:10→1:5→3:10→1:5→7:10→95:

100.

10. The method for preparing corn root lignan compounds according to claim 6 or 8, characterized in that: The concentration ratio of methanol in the methanol-water solution of the ODS open column chromatography eluent used for separation of the A4 and A5 fractions is 10%, 20%, 40%, 60%, 80% and 100% respectively; the ratio of methanol / water of the mobile phase of the cyclic preparative HPLC is 3:1, 3:2, 1:1, 1:2 or 2:3, or methanol / acetonitrile / water is 1:1:2.

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