Rehmannia pigment compound as well as preparation method and application thereof in additive

Abstract

The invention provides a rehmannia pigment compound as shown in a formula (I), and a stereoisomer or pharmaceutically acceptable salt thereof. The invention also provides a preparation method of the rehmannia pigment compound, the stereoisomer of the rehmannia pigment compound, or the pharmaceutically acceptable salt of the rehmannia pigment compound, and an application of the rehmannia pigment compound in an additive. The rehmannia pigment compound provided by the invention is clear in structure, has no obvious toxicity to cells under effective concentration, is proved to have good biological safety, and can be used as a multifunctional additive to be applied to the fields of foods, medicines or cosmetics.

Description

Technical Field

[0001] This application relates to the fields of natural product chemistry and applied technology, and in particular to a rehmannia flavonoid compound, its preparation method, and its application in additives. Background Technology

[0002] As consumers become increasingly health- and safety-conscious, naturally derived additives are gaining popularity in the food, pharmaceutical, and cosmetic industries as alternatives to synthetic additives with potential safety risks. Among the many natural additives, natural plant pigments are widely used due to their vibrant colors and biological origin. However, many natural pigments in existing technologies have relatively limited functions, primarily serving as colorants and lacking clearly defined, scientifically validated additional biological activities. For example, existing technologies have disclosed the preparation of a mixture of natural pigments from Rehmannia glutinosa for use as a colorant, but this technique only yields crude extracts with unclear compositions, without isolating and identifying the specific chemical monomers. Currently, the variety of existing natural pigments is limited, and the demand for safe, high-quality, and bioactive (e.g., anti-inflammatory) natural additives is growing in the pharmaceutical, food, and cosmetic sectors. Summary of the Invention

[0003] The purpose of this application is to provide a novel rehmannia flavonoid compound, its stereoisomer, or a pharmaceutically acceptable salt thereof, which has a well-defined structure, is safe and non-toxic, and has definite biological activity, and can be used as an additive in pharmaceuticals, food or cosmetics.

[0004] To achieve the above objectives, this application provides a rehmannia flavonoid compound of formula (I), its stereoisomer, or a pharmaceutically acceptable salt thereof:

[0005]

[0006] In formula (I), R1, R2, and R5 are independently hydroxyl, mercapto, or amino groups; R3 is oxygen or sulfur; R4 is independently oxygen, sulfur, or imine; the stereoconfigurations of 5-C and 6-C are independently R or S; the double bond configurations of 9-C and 13-C are independently E or Z; in formula (I), the brackets close to 13'-C are called a brackets, and the brackets far from 13'-C are called b brackets; the structural units within a bracket and the structural units within b brackets are independently optional structural units; a is 0 or 1; b is 0 or 1; when a is 0, the structural unit within a bracket does not exist; when a is 1, the structural unit within a bracket exists; when b is 0, the structural unit within b bracket does not exist; when b is 1, the structural unit within b bracket exists.

[0007] This embodiment provides novel and well-defined conjugated polyene carotenoid compounds (rehmannia pigment compounds). The chemical structures of these compounds have been fully confirmed by modern spectroscopic methods, filling the gap in the prior art for this type of compound.

[0008] The rehmannia glutinosa compound provided in this embodiment possesses both a vibrant orange-yellow coloring property and significant anti-inflammatory activity. Compared with single-function colorants or extracts of unknown composition in the prior art, the compound in this embodiment can serve as a multifunctional additive, providing additional health benefits to products while offering color. In particular, the compound in this embodiment exhibits no significant toxicity to cells at effective concentrations, demonstrating its good biocompatibility and suitability for use as an additive in food, pharmaceuticals, and cosmetics that come into direct contact with the human body.

[0009] Furthermore, the compound is a compound having the structure shown in formula (i), formula (ii), formula (iii), formula (iv), or formula (v):

[0010]

[0011]

[0012]

[0013]

[0014] in,

[0015] In equation (i), the 9-C double bond is of the E type, the 13-C double bond is of the E type, and a is 0 and b is 0;

[0016] In equation (ii), the 9-C double bond is of the Z type, the 13-C double bond is of the E type, and a is 0 and b is 0;

[0017] In equation (iii), the 9-C double bond is of the E type, the 13-C double bond is of the Z type, and a is 0 and b is 0;

[0018] In equation (iv), the 9-C double bond is of the E type, the 13-C double bond is of the E type, and a is 1 and b is 0;

[0019] In equation (v), the 9-C double bond is of type E, the 13-C double bond is of type E, and a is 1 and b is 1.

[0020] Furthermore, the compound is a compound having the structure shown in formula (ie), formula (ii-f), formula (iii-g), formula (iv-h), or formula (vi):

[0021]

[0022]

[0023]

[0024]

[0025] The specific compound structure provided in this embodiment indicates that the compound in this application can serve as a multifunctional additive, providing additional health benefits to the product while also offering color. In particular, the compound in this application exhibits no significant toxicity to cells at effective concentrations, demonstrating good biocompatibility and suitability for use as an additive in food, pharmaceuticals, and cosmetics that come into direct contact with the human body.

[0026] Furthermore, the stereo configurations of 5-C and 6-C are (5R, 6R) or (5S, 6R).

[0027] Furthermore, the 9-C double bond is of the Z type, and the 13-C double bond is of the E type.

[0028] Furthermore, R1 and R2 are independently hydroxyl or mercapto groups, R3 is oxygen, R4 is oxygen, and R5 is hydroxyl.

[0029] Furthermore, a is 0 and b is 0.

[0030] This application also provides a method for preparing the rehmannia flavonoid compound, its stereoisomer, or a pharmaceutically acceptable salt thereof as described in any one of the above claims, comprising the following steps: subjecting crude rehmannia flavonoid to normal-phase chromatography to obtain a preliminarily purified fraction containing the target component; subjecting the preliminarily purified fraction to reverse-phase chromatography to obtain a further purified subfraction; subjecting the subfraction to size exclusion chromatography to obtain a secondary fraction; and separating the secondary fraction by semi-preparative high-performance liquid chromatography and / or preparative high-performance liquid chromatography to obtain the rehmannia flavonoid compound.

[0031] This application also provides an additive composition comprising any of the above-described rehmannia flavonoid compounds, their stereoisomers, or pharmaceutically acceptable salts thereof, and food, pharmaceutical, or cosmetic excipients.

[0032] This application also provides the use of any of the foregoing rehmannia flavonoid compounds, their stereoisomers, or pharmaceutically acceptable salts thereof, or the foregoing additive compositions, in additives.

[0033] This application provides novel and well-defined conjugated polyene carotenoid compounds (rehmannia pigment compounds). The chemical structures of these compounds have been fully confirmed by modern spectroscopic methods, filling the gap in the prior art for this type of compound.

[0034] The rehmannia glutinosa compound provided in this application possesses both a vibrant orange-yellow coloring property and significant anti-inflammatory activity. Compared with existing single-function colorants or extracts of unknown composition, the compound in this application can serve as a multifunctional additive, providing additional health benefits to products while offering color. In particular, the compound in this application exhibits no significant toxicity to cells at effective concentrations, demonstrating its good biocompatibility and suitability for use as an additive in food, pharmaceuticals, and cosmetics that come into direct contact with the human body. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 The structural formula (I) of the thymol compound provided in the embodiments of this application is shown.

[0037] Figure 2 The structural formula (i) of the rehmannia pigment compound provided in the embodiments of this application is shown.

[0038] Figure 3 The structural formula (ii) of the rehmannia pigment compound provided in the embodiments of this application.

[0039] Figure 4 The structural formula (iii) of the thymol compound provided in the embodiments of this application is shown.

[0040] Figure 5 The structural formula (iv) of the rehmannia pigment compound provided in the embodiments of this application.

[0041] Figure 6 The structural formula (v) of the thymol compound provided in the embodiments of this application is shown.

[0042] Figure 7 The structure of the thymol compound E provided in the embodiments of this application is shown.

[0043] Figure 8 The structure of the thymol compound F provided in the embodiments of this application is shown.

[0044] Figure 9 The structure of the thymol compound G provided in the embodiments of this application is shown.

[0045] Figure 10 The structure of the thymol compound H provided in the embodiments of this application is shown.

[0046] Figure 11 The structure of the thymol compound I provided in the embodiments of this application is shown.

[0047] Figure 12 This is a flowchart illustrating a method for preparing a rehmannia pigment compound, as provided in an embodiment of this application. Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only for explaining this application and are not intended to limit this application.

[0049] It should be noted that the rehmannia pigment compounds mentioned in this application are not mixtures, but conjugated polyene carotenoid compounds having the structure described in this application.

[0050] In this application, those skilled in the art should understand that the numbers labeled in each molecular formula represent the relative positions of the carbon atoms in that formula, and have no substantial impact on the carbon atoms, molecular structure, or the corresponding compound. They are simply used to facilitate understanding or description of the relative positions of carbon atoms in the molecular structure. Figure 2 The numbers 6, 9, 13, 13', and 15' marked in the structure shown represent the carbon atoms at positions 6, 9, 13, 13', and 15', respectively, in the molecular structure. Figure 2 The carbon atoms, molecular structures, and corresponding compounds shown have no substantial impact.

[0051] It should be understood that those skilled in the art should be aware that the numbers in the methyl group (i.e., CH3-) in the molecular structural formula only represent the carbon atom in the methyl group being connected to three hydrogen atoms, and should be distinguished from the relative position numbering of the carbon atoms mentioned above.

[0052] Currently, the variety of natural pigments available is limited. In the pharmaceutical, food, and cosmetic industries, there is a growing demand for additives that are safe, have good coloring power, and possess biological activity (such as anti-inflammatory properties) with natural or similar natural characteristics. Limitations of existing additives in terms of performance or safety: Natural pigments or functional additives currently used in food, pharmaceuticals, and cosmetics (such as certain carotenoids) may have issues such as unsatisfactory color, insufficient stability, limited biological activity, or potential cytotoxicity.

[0053] In one embodiment, this application provides a rehmannia flavonoid compound of formula (I), its stereoisomer, or a pharmaceutically acceptable salt thereof:

[0054]

[0055] In formula (I), R1, R2, and R5 are independently hydroxyl, mercapto, or amino groups; R3 is oxygen or sulfur; R4 is independently oxygen, sulfur, or imino; the stereoconfigurations of 5-C and 6-C are independently R or S; the double bond configurations of 9-C and 13-C are independently E or Z; in formula (I), the brackets close to 13'-C are called a brackets, and the brackets far from 13'-C are called b brackets; the structural units within a bracket and the structural units within b brackets are independently optional structural units; a is 0 or 1; b is 0 or 1; when a is 0, the structural unit within a bracket does not exist; when a is 1, the structural unit within a bracket exists; when b is 0, the structural unit within b bracket does not exist; when b is 1, the structural unit within b bracket exists.

[0056] In formula (I), the numbering of carbon atoms in the molecular structure has no effect on the atomic or molecular structure or its properties; it is merely used to describe the relative positions of carbon atoms. For example, 5 indicates the 5th carbon atom (i.e., 5-C), 6 indicates the 6th carbon atom (i.e., 6-C), 15' indicates the 15th carbon atom, 13' indicates the 13th carbon atom (i.e., 13'-C), 10' indicates the 10th carbon atom (i.e., 10'-C), 8' indicates the 8th carbon atom (i.e., 8'-C), and so on. Carbon atoms without explicit numbers can be deduced from the numbers on both sides, following the sequence. For example: the carbon atom between 9 and 11 is the carbon at position 10, the carbon atom between 15' and 13' is the carbon at position 14', the carbon between 13' and 10' is the carbon at position 12' and position 11' respectively (i.e., 12'-C and 11'-C respectively), and the carbon atom between 10' and 8' is the carbon at position 9' (i.e., 9'-C).

[0057] Regarding the brackets in equation (I), further description: when a=0, the structural unit inside bracket a does not exist, and the atoms before and after it are directly connected, that is, 13'-C is connected to 10'-C or 8'-C; when a=1, the structural unit inside bracket a exists, that is, 13'-C is connected to 12'-C, 12'-C is connected to 11'-C, and 11'-C is connected to 10'-C or 8'-C.

[0058] When b=0, the structural unit inside the b brackets does not exist, and the atoms before and after it are directly connected, that is, 11'-C or 13'-C is connected to 8'-C; when b=1, the structural unit inside the b brackets exists, that is, 10'-C is connected to 11'-C or 13'-C, 10'-C is connected to 9'-C, and 9'-C is connected to 8'-C.

[0059] If a=0 and b=0, it means that neither the structural unit inside the parentheses a nor the structural unit inside the parentheses b exists. In this case, 13'-C and 8'-C are directly connected.

[0060] If a=1 and b=1, it means that the structural unit inside the parentheses a and b both exist. The connection methods are as follows: 13'-C is connected to 12'-C, 12'-C is connected to 11'-C, 11'-C is connected to 10'-C; 10'-C is connected to 9'-C, and 9'-C is connected to 8'-C.

[0061] In this application, "connected" refers to the connection between atoms in a molecular structure by chemical bonds in a chemical sense.

[0062] This embodiment provides novel and well-defined compounds whose chemical structures have been fully confirmed by modern spectroscopic methods, filling a gap in the prior art for this type of structure.

[0063] Experimental data show that the specific compounds provided in this embodiment did not exhibit significant cytotoxicity at effective concentrations demonstrating significant biological activity. This proves that these compounds have a high safety window, laying the foundation for their application in the food, pharmaceutical, and other fields.

[0064] Furthermore, the compounds in this embodiment demonstrated a significant ability to inhibit nitric oxide (NO) release in a classic LPS-induced RAW264.7 cell inflammation model. For example, a specific compound showed an NO inhibition rate of approximately 66.8% at a concentration of 100 μM, indicating that it possesses a clear anti-inflammatory pharmacological effect, rather than being a result of nonspecific toxicity.

[0065] Furthermore, the compound in this embodiment exhibits a vibrant orange-yellow color, demonstrating its potential as a natural colorant. Simultaneously, it possesses the aforementioned safety and anti-inflammatory activity, enabling it to serve as a novel "function-color" integrated additive in pharmaceuticals, functional foods, health products, and cosmetics, providing a pleasing color while offering potential anti-inflammatory, antioxidant, and other health benefits.

[0066] By way of example, the compound may be a compound having the structure shown in formula (i), formula (ii), formula (iii), formula (iv), or formula (v):

[0067]

[0068]

[0069]

[0070]

[0071] In Equation (i), the 9-C double bond is of type E, the 13-C double bond is of type E, and a is 0 and b is 0; in Equation (ii), the 9-C double bond is of type Z, the 13-C double bond is of type E, and a is 0 and b is 0; in Equation (iii), the 9-C double bond is of type E, the 13-C double bond is of type Z, and a is 0 and b is 0; in Equation (iv), the 9-C double bond is of type E, the 13-C double bond is of type E, and a is 1 and b is 0; in Equation (v), the 9-C double bond is of type E, the 13-C double bond is of type E, and a is 1 and b is 1.

[0072] In equations (i), (ii), (iii), (iv), and (v), the numerical designations are the same as those in the explanation of equation (i). To maintain consistency with equation (i), the notation 13'-C, 8'-C, etc., are still used here.

[0073] This application is the first to isolate and identify a series of novel rehmannia flavonoid compounds with specific oxatricyclic core skeletons (as shown in formula (I) and formulas (i)-(v)). Their specific substitution modes, stereoconfigurations and double bond geometric isomers (such as 9-CZ and 13-CE) have not been reported in the prior art, enriching the chemical diversity of carotenoid derivatives and natural products.

[0074] In a preferred embodiment, the compound is a compound having the structure shown in formula (ie), formula (ii-f), formula (iii-g), formula (iv-h), or formula (vi):

[0075]

[0076]

[0077]

[0078]

[0079] The compound in this embodiment was isolated and clearly identified from Rehmannia glutinosa for the first time, and its structure was confirmed by modern analytical methods such as nuclear magnetic resonance spectroscopy, filling the gap in the prior art's understanding of the structure of this type of substance.

[0080] The compound in this embodiment exhibits a bright orange-yellow color and possesses the physical properties of a natural colorant. Simultaneously, its anti-inflammatory activity and high safety profile make it a versatile additive widely applicable in pharmaceuticals, functional foods, health products, and cosmetics requiring anti-inflammatory effects or natural coloring, achieving "multiple uses from one ingredient" and enhancing its application value.

[0081] The results confirm that the compounds in this embodiment can significantly inhibit the excessive release of nitric oxide (NO) in an LPS-induced macrophage inflammation model, exhibiting clear anti-inflammatory pharmacological activity. In particular, compounds with certain specific configurations (e.g., compounds with a Z-type 9-C double bond and an E-type 13-C double bond) show stronger activity, providing valuable candidate molecules for the development of novel anti-inflammatory drugs.

[0082] Through cytotoxicity experiments such as CCK-8, the compound in this embodiment showed no significant inhibition of cell growth and no cytotoxicity within the effective concentration range exhibiting anti-inflammatory activity, indicating that it has a high therapeutic index and good biosafety, meeting the requirements for low-side-effect drugs or functional additives.

[0083] In another preferred embodiment, the stereo configuration of 5-C and 6-C is (5R, 6R) or (5S, 6R).

[0084] For example, the 9-C double bond is of the Z type and the 13-C double bond is of the E type.

[0085] For example, R1 and R2 are independently hydroxyl or mercapto groups, R3 is oxygen, R4 is oxygen, and R5 is hydroxyl.

[0086] For example, a is 0 and b is 0.

[0087] For example, when the configuration of 5-C and 6-C is (5R, 6R), the following cases are included:

[0088] When R1 is hydroxyl, R2 is hydroxyl, mercapto, or amino; R5 is hydroxyl, mercapto, or amino; R3 is oxygen or sulfur; and R4 is oxygen, sulfur, or amino.

[0089] When R1 is a thiol group, R2 is a hydroxyl, thiol, or amino group; R5 is a hydroxyl, thiol, or amino group; R3 is oxygen or sulfur; and R4 is oxygen, sulfur, or amino group.

[0090] When R1 is amino, R2 is hydroxyl, mercapto, or amino; R5 is hydroxyl, mercapto, or amino; R3 is oxygen or sulfur; and R4 is oxygen, sulfur, or amino.

[0091] For example, when the configuration of 5-C and 6-C is (5S, 6R), the following cases are included:

[0092] When R1 is hydroxyl, R2 is hydroxyl, mercapto, or amino; R5 is hydroxyl, mercapto, or amino; R3 is oxygen or sulfur; and R4 is oxygen, sulfur, or amino.

[0093] When R1 is a thiol group, R2 is a hydroxyl, thiol, or amino group; R5 is a hydroxyl, thiol, or amino group; R3 is an oxygen or sulfur group; and R4 is an oxygen, sulfur, or amino group.

[0094] When R1 is amino, R2 is hydroxyl, mercapto, or amino; R5 is hydroxyl, mercapto, or amino; R3 is oxygen or sulfur; and R4 is oxygen, sulfur, or amino.

[0095] For example, when the configuration of 5-C and 6-C is (5R, 6S), the following cases are included:

[0096] When R1 is hydroxyl, R2 is hydroxyl, mercapto, or amino; R5 is hydroxyl, mercapto, or amino; R3 is oxygen or sulfur; and R4 is oxygen, sulfur, or amino.

[0097] When R1 is a thiol group, R2 is a hydroxyl, thiol, or amino group; R5 is a hydroxyl, thiol, or amino group; R3 is an oxygen or sulfur group; and R4 is an oxygen, sulfur, or amino group.

[0098] When R1 is amino, R2 is hydroxyl, mercapto, or amino; R5 is hydroxyl, mercapto, or amino; R3 is oxygen or sulfur; and R4 is oxygen, sulfur, or amino.

[0099] For example, when the configuration of 5-C and 6-C is (5S, 6S), the following cases are included:

[0100] When R1 is hydroxyl, R2 is hydroxyl, mercapto, or amino; R5 is hydroxyl, mercapto, or amino; R3 is oxygen or sulfur; and R4 is oxygen, sulfur, or amino.

[0101] When R1 is a thiol group, R2 is a hydroxyl, thiol, or amino group; R5 is a hydroxyl, thiol, or amino group; R3 is an oxygen or sulfur group; and R4 is an oxygen, sulfur, or amino group.

[0102] When R1 is amino, R2 is hydroxyl, mercapto, or amino; R5 is hydroxyl, mercapto, or amino; R3 is oxygen or sulfur; and R4 is oxygen, sulfur, or amino.

[0103] For example, physiologically acceptable salts in this application are inorganic or organic acid salts of compounds having the structures shown in formula (I), (i), (ii), (iii), (iv), (v), (ie), (ii-f), (iii-g), (iv-h), or (vi).

[0104] Preferably, the inorganic acid salt may be hydrochloride, sulfate, phosphate, hydrobromide, or hydroiodide.

[0105] For example, the organic acid salts are tartrates, citrates, formates, acetates, oxalates, butates, oxalates, maleates, succinates, adipates, alginates, citrates, aspartates, benzenesulfonates, camphorates, camphor sulfonates, diglucuronates, cyclopentanepropionates, dodecyl sulfates, ethanesulfonates, glucohepanoates, glycerol phosphates, hemisulfates, heptahydrates, hexanoates, fumarates, 2-hydroxyethanesulfonates, lactates, maleates, methanesulfonates, nicotinates, 2-naphthalenesulfonates, pyrates, pectin esters, 3-phenylpropionates, picrates, neopentanoates, propionates, succinates, tartrates, thiocyanates, p-toluenesulfonates, or undecanoates.

[0106] For example, the organic acid salt is tartrate, citrate, formate, acetate, oxalate, or butyrate.

[0107] For example, the organic acid salt is glycerol phosphate, hemisulfate, heptanate, hexanoate, fumarate, 2-hydroxyethanesulfonate, or lactate.

[0108] For example, the organic acid salt is 3-phenylpropionate, picrate, neopentanoate, propionate, succinate, tartrate, and thiocyanate.

[0109] In a preferred embodiment, an additive composition is also provided, comprising any of the above-described rehmannia flavonoid compounds, their stereoisomers, or pharmaceutically acceptable salts thereof, and food-, pharmaceutically, or cosmetically acceptable excipients.

[0110] For example, excipients that are acceptable in food science, pharmaceutical science, or cosmetic science include one or more of diluents, binders, disintegrants, lubricants, pH adjusters, solubilizers, and antioxidants.

[0111] Preferably, excipients that are acceptable in food science, pharmaceutical science or cosmetic science include diluents, binders and disintegrants.

[0112] For example, in the additive composition, the acceptable salt is an inorganic or organic acid salt of a compound having the structure shown in formula (I), (i), (ii), (iii), (iv), (v), (ie), (ii-f), (iii-g), (iv-h), or (vi).

[0113] For example, in the additive composition, the inorganic acid salt is a hydrochloride, sulfate, phosphate, hydrobromide, or hydroiodide.

[0114] For example, in the additive composition, the organic acid salt is tartrate, citrate, formate, acetate, oxalate, butyrate, oxalate, maleate, succinate, adipate, alginate, citrate, aspartate, benzenesulfonate, camphorate, camphorsulfonate, disglucuronate, cyclopentanepropionate, dodecyl sulfate, ethanesulfonate, glucono-p-ethylhexanoate, glyceryl phosphate, hemisulfate, heptaate, hexanoate, fumarate, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, pyrate, pectin ester, 3-phenylpropionate, picrate, neopentanoate, propionate, succinate, tartrate, thiocyanate, p-toluenesulfonate, or undecanoate.

[0115] The additive composition comprises additives in the form of emulsifiable concentrates, suspensions, wettable powders, powders, granules, aqueous solutions, poisoned baits, mother liquors, and master powders.

[0116] In a preferred embodiment, the use of any of the rehmannia flavonoid compounds described in this application, their stereoisomers, or pharmaceutically acceptable salts thereof, or the above-described additive compositions, in an additive is provided.

[0117] For example, the dosage form of the additive may be an emulsifiable concentrate, suspension concentrate, wettable powder, powder, granule, aqueous solution, poison bait, mother liquor or master powder.

[0118] The rehmannia glutinosa compound in this embodiment possesses both a vibrant orange-yellow coloring property and significant anti-inflammatory activity. Compared to single-function colorants or extracts with unknown composition in the prior art, this compound can serve as a multifunctional additive, providing additional health benefits to products while offering color. Furthermore, the compound in this application exhibits no significant toxicity to cells at effective concentrations, demonstrating its good biocompatibility and suitability for use in food, pharmaceuticals, and cosmetics that come into direct contact with the human body.

[0119] In one embodiment, such as Figure 12 As shown, a method for preparing any of the above-described rehmannia flavonoid compounds, their stereoisomers, or pharmaceutically acceptable salts thereof:

[0120] S1: The crude rehmannia pigment was subjected to normal phase chromatography to obtain a preliminary purified fraction containing the target component;

[0121] S2: Perform reverse phase chromatography on the preliminarily purified component to obtain a further purified subcomponent;

[0122] S3: Perform size exclusion chromatography on the subcomponent to obtain the secondary component;

[0123] S4: and separating the secondary components by semi-preparative high-performance liquid chromatography and / or preparative high-performance liquid chromatography to obtain the rehmannia glutinosa compound.

[0124] The crude rehmannia glutinosa pigment can be an unpurified extract obtained using currently known methods. Alternatively, it can be an unpurified extract obtained using optimized methods. Purification here can be understood as any one or more chromatographic treatments.

[0125] Of course, the crude rehmannia pigment can be an extract obtained by currently known methods, after preliminary extraction and vacuum concentration.

[0126] An exemplary crude rehmannia glutinosa extract may be an unpurified initial extract obtained from the fresh or dried rhizomes of Rehmannia glutinosa Libosch. after preliminary extraction and vacuum concentration. The preliminary extraction may be performed using ethanol or other organic solvents of appropriate concentration, followed by vacuum concentration to obtain the unpurified extract. For example, the ethanol concentration may be 70-80%, and the vacuum concentration may be performed by concentrating to 40-70% of the original volume, followed by filtration through a 0.1-1 μm microporous membrane.

[0127] For example, it can also be understood that crude rehmannia pigment is a mixture mainly composed of medium polar to non-polar pigment components, such as non-cyclohexene ethers and non-phenylethanol glycosides.

[0128] In this application, the rehmannia glutinosa compounds are not a mixture, but rather different conjugated polyene compounds extracted from crude rehmannia glutinosa. For example, they may be any of the rehmannia glutinosa compounds AG mentioned in this application.

[0129] Normal-phase chromatography of crude rehmannia pigment can remove impurities with significantly different polarities from the target yellow pigment compound. These impurities include chlorophyll degradation products, plant waxes, long-chain alkanes, triglycerides, some terpenes, residual polysaccharides, inorganic salts, amino acids, organic acids, glycosylated pigments, or oligomers.

[0130] The preliminary purified components of the target component will contain the target yellow pigment compound, its homologues and direct analogues, as well as lipid-soluble impurities.

[0131] For example, if normal phase chromatography is used for fractional elution, preliminary purified fractions containing different target components can be collected separately. For example: preliminary purified fractions with relatively small molecular weights or more hydroxyl groups; preliminary purified fractions with larger molecular weights, longer alkyl chains, or fewer hydroxyl groups.

[0132] For example, after step S1 and before S2, the process further includes analyzing the pre-purified fractions containing different target components using analytical liquid chromatography, thereby selecting one or more of the pre-purified fractions containing the target components to facilitate subsequent separation.

[0133] The reversed-phase chromatography of the preliminarily purified components can remove residual water-soluble impurities (polysaccharides, proteins), homologue impurities, trace amounts of lipid-soluble residues, etc.

[0134] The subcomponent may contain the target yellow pigment compound, and may also contain structurally similar analogs or isomers.

[0135] Size exclusion chromatography can remove some small molecule residues and salts, as well as impurities that are similar in hydrophobicity to the target analyte but have different molecular sizes / configurations. Examples include: organic salts, solvent residues (such as methanol and water), monosaccharides, amino acids and small molecule organic acids, homologues of the target pigment (dimers or oligomers), and some structurally highly similar isomers.

[0136] The secondary components may contain the target yellow pigment compound, and may also contain enantiomers or diastereomers of the target yellow pigment compound, as well as small molecule impurities, solvent residues, etc. At this point, the target yellow pigment compound is usually already of high purity, reaching 80-90%.

[0137] The secondary components are separated by semi-preparative high-performance liquid chromatography (HPLC) and / or preparative HPLC, which typically removes diastereomers or positional isomers of the target compound, structural analogs with retention behaviors extremely similar to the target compound, and trace amounts of polymers or dimers that may be present. This step yields a single rehmannia flavonoid compound with extremely high purity, reaching or exceeding 99%.

[0138] Therefore, the method of this embodiment can isolate and confirm a series of novel rehmannia glutinosa compounds (rehmannia glutinosa compound AG) from Rehmannia glutinosa, filling a technological gap in this field. This application's purification of crude rehmannia glutinosa using multi-step chromatography effectively addresses the challenges of similar structures, similar polarities, and difficult separation of rehmannia glutinosa compounds. The process is stable and reproducible, representing a significant improvement over existing crude extraction methods. The structurally defined rehmannia glutinosa compounds obtained by this invention provide a high-quality material basis and key technical support for the in-depth development of rehmannia glutinosa and its precise application in the food, pharmaceutical, and cosmetic fields, particularly laying the foundation for its anti-inflammatory, antioxidant, and other pharmacological activities.

[0139] For example, the stationary phase of the normal phase chromatography can be alumina, polyamide, or silica-bonded phase, preferably a normal phase silica column. The mobile phase of the normal phase chromatography can be dichloromethane-ethanol, ethyl acetate-methanol, or n-hexane-methanol, typically a dichloromethane-methanol system.

[0140] For example, the stationary phase of the reversed-phase chromatography can be a C8 (octylsilane) column, a phenyl column, a cyano column (-CN), or a pentafluorophenyl column, preferably a reversed-phase ODS column; the mobile phase of the reversed-phase layer can be water-acetonitrile, a water-methanol / acetonitrile mixture, water-methanol / acetonitrile with added modifier, or water-tetrahydrofuran, preferably a water-methanol system as the mobile phase.

[0141] In another embodiment, the normal phase chromatography is normal phase silica gel column chromatography, and the normal phase silica gel column chromatography uses a dichloromethane-methanol system as the mobile phase; the reverse phase chromatography is reverse phase ODS column chromatography, and the reverse phase ODS column chromatography uses a water-methanol system as the mobile phase.

[0142] ODS stands for Octadecyl-bonded Silica.

[0143] The dichloromethane-methanol system refers to a mixture of dichloromethane and methanol in a specific ratio; the water-methanol system refers to a mixture of water and methanol in a specific ratio. In other embodiments, the use of "-" as a connector also refers to a mixture of two or more compounds.

[0144] Among them, normal phase silica gel column chromatography and dichloromethane-methanol gradient system are based on the polarity difference of compounds. Therefore, polar target compounds (such as iridoid glycosides) can be specifically separated through the polarity matching mechanism. This can efficiently remove lipid-soluble impurities in crude rehmannia glutinosa, achieving a purity of >90% and a recovery rate of >85%.

[0145] Among them, the reversed-phase ODS column, being nonpolar, forms a reverse partitioning mechanism with the water-methanol system, which can accurately separate nonpolar / moderately polar compounds.

[0146] For example, silica gel column chromatography can be performed using a dry loading method, wherein the crude rehmannia pigment is mixed with 60-100 mesh silica gel at a mass ratio of 1:0.5-1:2, and the stationary phase of the silica gel column can be 100-200 mesh silica gel.

[0147] In this embodiment, after normal-phase silica gel column chromatography and dichloromethane-methanol gradient system chromatography, a preliminary purified component containing the target component is obtained. The dichloromethane-methanol is easy to evaporate completely and can be completely dissolved in the starting mobile phase (water-methanol) of the subsequent reversed-phase column, avoiding solvent compatibility issues and ensuring sample loading uniformity and chromatographic peak shape.

[0148] For example, the stationary phase of the size exclusion chromatography can be dextran gel LH-60, polymethacrylate gel, or polystyrene sulfonic acid ion exchange resin, etc.; preferably, it can be a dextran gel LH-20 column. The mobile phase of the size exclusion chromatography can be water-ethanol (a mixture of water and ethanol), water-acetone, chloroform-methanol, tetrahydrofuran-water, or dichloromethane-methanol-water mixed solvent, etc., preferably methanol; another preferred option is a dichloromethane-methanol mixed solvent (a mixture of dichloromethane and methanol).

[0149] In another embodiment, the size exclusion chromatography is performed on a dextran gel LH-20 column, using methanol or a dichloromethane-methanol mixed solvent as the mobile phase.

[0150] In this embodiment, size exclusion chromatography, specifically a dextran gel LH-20 column and a methanol / dichloromethane-methanol mobile phase, is used. Based on molecular size, it can remove small molecule impurities (such as polysaccharides and organic acids). Therefore, it can effectively remove silica gel microparticles, filler degradation products, or solid impurities.

[0151] In this embodiment, the subcomponent can be purified into a simpler and purer secondary component, reducing the difficulty of subsequent separation and significantly improving the yield and purity of the final product.

[0152] A dextran gel LH-20 column, also known as a Sephadex LH-20, is a liquid chromatography column filled with dextran gel LH-20. It refers to a dextran-based gel chromatography medium with a large pore size (approximately 200–300 Å) and a hydrophobic surface.

[0153] For example, the secondary components can be separated using semi-preparative high-performance liquid chromatography (HPLC). The stationary phase can be a C8 (octyl) column, a C4 (butyl) column, a phenyl column, a pentafluorophenyl column, or a cyano column, etc.; a reversed-phase column (usually C18) is preferred; the mobile phase can be a water-methanol system, a water-ethanol system, a water-tetrahydrofuran system, or an acetonitrile-methanol-water ternary system; preferably an acetonitrile-water system (a mixed solvent composed of acetonitrile and water).

[0154] In a preferred embodiment, the semi-preparative high-performance liquid chromatography (HPLC) and / or preparative HPLC separation includes: separating the secondary components using semi-preparative HPLC, wherein the semi-preparative HPLC uses a reversed-phase column and an acetonitrile-water mobile phase; wherein the volume percentage concentration of acetonitrile in the aqueous solution in the acetonitrile-water mobile phase is 57%-74%; and the semi-preparative HPLC uses isocratic or gradient elution.

[0155] For example, the volume percentage concentration of acetonitrile in the aqueous solution is 57%, 58%, 60%, 68%, or 74%, etc.

[0156] In this embodiment, a reversed-phase column (typically C18) and an acetonitrile-water system can separate moderately to weakly polar natural products. Compared to methanol, acetonitrile has lower viscosity, stronger elution power, and better UV transmittance, enabling baseline separation of structurally very similar pigment homologues or isomers under high-concentration aqueous phase conditions (57%-74% acetonitrile corresponds to 26%-43% water).

[0157] Exemplarily, the semi-preparative high-performance liquid chromatography (HPLC) and / or preparative HPLC separation can employ chiral supercritical fluid chromatography (SCLC) or chiral capillary electrophoresis; preferably, chiral column semi-preparative HPLC is used. The mobile phase of chiral column semi-preparative HPLC can be a heptane-ethanol mixture, a cyclohexane-methanol mixture, or a heptane-tetrahydrofuran mixture; preferably, a hexane-isopropanol mixture.

[0158] As a preferred embodiment of this application, the semi-preparative high-performance liquid chromatography (HPLC) and / or preparative HPLC separation includes: separating the secondary components using chiral column semi-preparative HPLC, wherein the mobile phase of the chiral column semi-preparative HPLC is a hexane-isopropanol mixed solvent, and gradient elution is used.

[0159] For example, the gradient elution procedure is as follows: the volume fraction of isopropanol in n-hexane is increased from 10% to 13%, held for 10-30 minutes, and then increased from 13% to 30%. Preferably, it can be held for 15-25 minutes.

[0160] In this embodiment, the stationary phase of the chiral column has chiral recognition sites that can separate enantiomers or diastereomers; and in the hexane-isopropanol mixed solvent, hexane provides a low polarity background, while isopropanol, as a polarity modifier, can work synergistically with the stationary phase.

[0161] For example, before performing normal-phase chromatography on the crude rehmannia glutinosa, the method further includes: mixing the crude rehmannia glutinosa with 60-100 mesh silica gel to obtain the mixed crude rehmannia glutinosa; and the stationary phase of the normal-phase silica gel column is 100-200 mesh silica gel; the use of a dichloromethane-methanol system as the mobile phase is to use a dichloromethane-methanol mixed solvent to perform gradient elution on the mixed crude rehmannia glutinosa, wherein the gradient elution of the dichloromethane-methanol mixed solvent starts from pure dichloromethane and ends with pure methanol.

[0162] Using coarser silica gel (60-100 mesh) mixed with the crude sample allows for the formation of a uniformly dispersed solid mixture before loading. 100-200 mesh silica gel used for column packing provides a larger specific surface area and a higher theoretical plate number.

[0163] The gradient begins with pure dichloromethane, which initially elutes lipid-soluble impurities from the crude pigment, achieving early impurity removal. The proportion of methanol is gradually increased until pure methanol is reached, enabling systematic elution of compounds across the entire polarity range. The pre-purified fraction containing the target component is selectively eluted and collected.

[0164] Furthermore, the reversed-phase ODS column chromatography employs a water-methanol system for gradient elution of the initially purified components, wherein the gradient elution volume ratio of the water-methanol system varies from 9:1 to pure methanol.

[0165] In this embodiment, a high aqueous phase ratio (water:methanol = 9:1) is started to ensure that all components are strongly adsorbed at the column head and strongly polar impurities are preferentially eluted. Increasing the methanol ratio to pure methanol allows for selective elution based on subtle differences in hydrophobicity between target molecules.

[0166] For example, the gradient elution volume ratio of the water-methanol system varies from 8:1 to pure methanol, from 8.5:1 to pure methanol, etc.

[0167] In another preferred embodiment, before performing normal-phase chromatography on the crude rehmannia glutinosa pigment, the method further includes: pretreating the rehmannia glutinosa raw material to obtain the phloem; extracting the phloem using a first organic solvent to obtain an extract; concentrating the extract to obtain an extract; dispersing the extract in a polar solvent and performing liquid-liquid extraction using a second organic solvent that is immiscible with the polar solvent to separate the second organic solvent phase containing rehmannia glutinosa pigment, and removing the solvent to obtain the crude rehmannia glutinosa pigment.

[0168] In this embodiment, crude rehmannia pigment was obtained, the main components of which are moderately polar to non-polar pigments. This greatly reduces the separation burden on the subsequent normal-phase silica gel column, avoiding rapid column head contamination and severe peak overlap.

[0169] For example, the first organic solvent is ethyl acetate, the polar solvent is water, and the second organic solvent is dichloromethane.

[0170] In this embodiment, an efficient and directional polarity transfer pathway is formed from raw materials to crude products, ultimately obtaining a medium-polarity target pigment concentrate.

[0171] Among them, ethyl acetate with moderate polarity was selected as the first solvent, which can efficiently dissolve the target components while minimizing the dissolution of large amounts of highly polar impurities and non-polar lipids, thus achieving selective enrichment for the first time.

[0172] In the embodiments provided in this application, a series of novel compounds were isolated and identified for the first time from Rehmannia glutinosa. These compounds have clearly defined components and controllable quality, providing a material basis for standardized production and application, and overcoming the shortcomings of traditional plant extracts, such as complex composition and large batch-to-batch variations. The implementation of this application also expands the application fields of the traditional Chinese medicine Rehmannia glutinosa, extending it from its traditional medicinal uses to functional foods and cosmetic additives, thereby enhancing its economic value.

[0173] The following detailed description is provided in conjunction with specific examples:

[0174] Example 1

[0175] This embodiment provides a method for extracting and preliminarily separating rehmannia pigment compounds from Rehmannia glutinosa, which lays the foundation for obtaining the compounds described in this application. The overall process of this method mainly includes a raw material processing step S101, an organic solvent extraction step S102, and a normal-phase column chromatography separation step S103.

[0176] In the raw material processing step S101, 60 kg of commercially available fresh Rehmannia glutinosa root is purchased as the starting material. The peeled fleshy root is cut into small pieces with a size of about 0.5 cm to 2 cm square. This increases the contact area with the extraction solvent, thereby significantly improving the efficiency of subsequent extraction steps.

[0177] In the organic solvent extraction step S102, all the processed Rehmannia glutinosa roots from the previous step are transferred to a corrosion-resistant extraction vessel made of stainless steel or glass. Ethyl acetate is added to the vessel as the extraction solvent, with the amount of solvent added enough to completely submerge all the Rehmannia glutinosa roots. It is understood that ethyl acetate, as a moderately polar solvent, has excellent solubility and extraction ability for the compound involved in this application. The Rehmannia glutinosa roots are soaked and extracted at room temperature (approximately 20-25°C), with each soak lasting 24 hours. To ensure that the target compound is fully extracted, this soaking process is repeated three times. After each extraction, the supernatant extract is collected, and the three extracts are combined. The combined ethyl acetate extract is concentrated under reduced pressure using a rotary evaporator. To avoid decomposition of the target compound due to excessive temperature, the water bath temperature is controlled at 40°C. The concentration process continues until the solvent is completely evaporated, finally yielding a brownish-yellow to orange-red paste-like substance, namely the total extract of Rehmannia glutinosa pigment, weighing a total of 30.9 grams.

[0178] Subsequently, in normal-phase column chromatography step S103, the total extract was initially separated and purified. First, 30.9 g of the obtained total extract was mixed with approximately 46.4 g (by weight, approximately 1:1.5) of silica gel powder with a particle size of 60-100 mesh in a small amount of dichloromethane. The solvent was then removed by rotary evaporation under reduced pressure to obtain dry silica gel powder adsorbed with the sample. A large glass chromatography column was then wet-packed with approximately 464 g (approximately 15 times the weight of the total extract) of silica gel with a particle size of 100-200 mesh as the stationary phase. After the column was uniformly packed and stabilized, the sample silica gel powder obtained from the previous mixing was evenly spread at the top of the column. Gradient elution was performed using a dichloromethane-methanol solvent system. Gradient elution utilizes the differences in adsorption forces of different compounds on the stationary phase and the differences in solubility in mobile phases of different polarities to achieve separation. Elution was initially performed using pure dichloromethane (volume ratio 1:0), followed by a gradual increase in the proportion of methanol. The volume ratios of the elution gradient were set sequentially to 200:1, 100:1, 80:1, 60:1, 40:1, 30:1, 20:1, 10:1, and 5:1, and finally washed with pure methanol (volume ratio 0:1). Throughout the elution process, the eluent was monitored in real time using thin-layer chromatography. Specifically, a certain volume of eluent was collected, sampled onto a silica gel plate, developed with a suitable developing solvent (e.g., dichloromethane:methanol = 50:1), and the spots were observed under visible and ultraviolet light. Based on the color, position, and number of spots on the thin-layer chromatogram, eluents with similar chemical compositions were merged. Through this step, the total extract was finally separated into 18 different subfractions, named Fr-1 to Fr-18. Subsequent analysis confirmed that subfractions Fr-8 and Fr-9 are key components rich in the target compound of this application and can be used for further purification.

[0179] To further separate homologues with similar molecular weights, a size exclusion chromatography step can be added before reversed-phase high-performance liquid chromatography (RP-HPLC) purification. For example, the Fr-8 or Fr-9 fraction can be dissolved in methanol, loaded onto a chromatography column packed with dextran gel (such as Sephadex LH-20), and eluted with methanol as the mobile phase. Since larger molecules elute first, followed by smaller molecules, this step effectively separates homologues of different chain lengths, thereby improving the efficiency and purity of subsequent purification steps.

[0180] Example 2: Preparation of Rehmannia glutinosa pigment compound E

[0181] This embodiment describes in detail the process of separating and purifying the monomeric thymosin compound E from the subfraction prepared in Example 1 and confirming its structure, namely, purification step S104 and structure confirmation step S105 by reversed-phase high-performance liquid chromatography.

[0182] Fr-8-27, a subfraction of Fr-8 obtained in Example 1, was taken. Preliminary analysis showed that this fraction contained a high concentration of the target compound. An appropriate amount of Fr-8-27 sample was dissolved in chromatographically pure methanol to prepare a sample solution for separation. To prevent insoluble particles from clogging the chromatographic column, the sample solution was filtered through an organic phase filter membrane with a pore size of 0.22 micrometers before use.

[0183] The filtered sample solution was separated and purified using a semi-preparative high-performance liquid chromatography (HPLC) system (i.e., step S104). This system utilizes the difference in partition coefficients between the nonpolar stationary phase and the polar mobile phase for separation. Specific chromatographic conditions were set as follows: a YMC-Pack ODS-A reversed-phase column (250 mm × 10 mm, 5 μm particle size) was used, which exhibits good separation performance for structurally similar carotenoid isomers; the mobile phase was a mixture of acetonitrile and water, eluted isocratically, with acetonitrile and water comprising 74% and 26% by volume, respectively; the flow rate was set to 2 mL / min. Under UV detection (e.g., monitoring near the characteristic absorption wavelength of carotenoids at 420 nm), a single, symmetrical chromatographic peak eluted at a retention time of 32.5 min was collected.

[0184] The corresponding fractions collected from multiple injections were combined and transferred to a rotary evaporator. To preserve the native configuration of the compound to the greatest extent possible and to avoid isomerization or degradation during post-processing, the water bath temperature was strictly controlled at a low 20°C for vacuum evaporation to gently remove the mobile phase. After the solvent was completely evaporated, the purified target compound was obtained and named Rehmannia glutinosa compound E (compound E for short).

[0185] To definitively confirm its chemical structure, compound E underwent nuclear magnetic resonance (NMR) spectroscopy analysis. Specifically, the NMR methods (1D and 2D NMR) confirmed the structure of compound E with the following data parameters:

[0186] 1 H-NMR (500MHz, CDCl3) δ1.28(m,H-2),1.62(m,H-2),1.46(m,H-3),1.82(m,H-3),1.49(m,H-4),1.83(m,H-4),6.23(d,J=16.0Hz,H-7),6.39(d,J=16.0H z, H-8), 6.22 (d, J=11.5Hz, H-10), 6.69 (dd, J=15.0, 11.5Hz, H-11), 6.36 (d, J=15.0Hz, H-12), 6.25 (d, J=11.5Hz, H-14), 6.85 (dd, J=11.5, 14.5Hz, H-15), 6.49 (dd, J=14.5, 11.5Hz, H-15'), 7.09 (d, J=11.5Hz, H-14'), 0.84 (s, CH3-16), 1.18 (s, CH3-17), 1.14 (s, CH3-18), 1.98(s, CH3-19), 1.98(s, CH3-20), 1.99(s, CH3-20'), 3.52(m, H-1''), 3.77(t, J=5.0Hz, H-2''), 6.31(t, J=5.5Hz, NH).

[0187] 13 C-NMR (125MHz, CDCl3) δ38.6 (C-1), 36.4 (C-2), 17.9 (C-3), 36.4 (C-4), 75.3 (C-5), 79.7 (C-6), 131.3 (C-7), 134.6 (C-8), 135.8 (C-9), 130.5 (C-10), 126.3 (C-11), 137.2 (C-12), 139.2 (C-13), 131.4 (C-14), 135.0 (C-15), 127.7 (C-15'), 135.0 (C-14'), 128.4 (C-13'), 170.3 (C-12'), 26.6 (C-16), 25 .3 (C-17), 27.2 (C-18), 13.5 (C-19), 13.2 (C-20), 13.1 (C-20'), 43.1 (C-1''), 62.8 (C-2'').

[0188] Those skilled in the art should be fully able to understand the above spectral data, which is cited here to demonstrate the actual experimental process and results.

[0189] Liquid chromatography analysis conditions: Sample chamber temperature: 20℃, column temperature: 35℃, column: AcquityUPLCBEHC18 (2.1×100mm, 1.7μm, WatersCorp., Milford, USA), mobile phase: acetonitrile / water, gradient setting: 5-75% (acetonitrile), run time: 20min, flow rate: 0.3mL / min.

[0190] Physical observation revealed that compound E was an orange-yellow powdery solid. High-resolution electrospray ionization mass spectrometry analysis of a small sample showed a quasi-molecular ion peak detected in positive ion mode. Its mass-to-charge ratio is 444.3156. Based on this precise molecular weight, its molecular formula is deduced to be... (The theoretical value is 444.3114). The measured value is within the allowable error range of the theoretical value, which preliminarily confirms its elemental composition.

[0191] The obtained proton and carbon spectrum data, after analysis, fully support... Figure 1 or Figure 2 The structure of the compound shown is specifically illustrated in Figure 7. Specifically, its structure is: (2E,4E,6E,8E,10E,12E)-13-((1R,2R)-1,2-dihydroxy-2,6,6-trimethylcyclohexyl)-N-(2-hydroxyethyl)-2,7,11-trimethyltrideca-2,4,6,8,10,12-hexaenamide, which is named (2E,4E,6E,8E,10E,12E)-13-[(1R,2R)-1,2-dihydroxy-2,6,6-trimethylcyclohexyl]-N-(2-hydroxyethyl)-2,7,11-trimethyltrideca-2,4,6,8,10,12-hexaenamide. .

[0192] This structure belongs to Figure 1 The structure shown falls within the scope of the general structural formula (I), where substituents R1 and R2 are both hydroxyl groups, R3 is oxygen, the carbon atom at position R4 forms a double bond with an oxygen atom to constitute an amide carbonyl group, and R5 is a hydroxyl group. The 5-C and 6-C stereoconfigurations of its cyclohexyl moiety are (5R, 6R). The length parameters a and b of the conjugated polyene chain are both 0, and the 9-C and 13-C double bonds on its conjugated polyene chain are both of E configuration. Therefore, compound E is... Figure 2 A specific example of the structural skeleton shown in equation (i) is also Figure 7The specific compound shown is an example. This embodiment successfully obtained monomeric compound E with a purity exceeding 95% and confirmed its novel chemical structure, providing a reliable material basis for subsequent activity and performance evaluation.

[0193] Figure 7 The numbers in the molecular formula represent the relative positions of the carbon atoms in that formula. They have no substantial impact on the carbon atoms, molecular structure, or the corresponding compound; they are simply used to facilitate understanding or description of the relative positions of carbon atoms in the molecular structure. Examples of such numbers include: 1, 5, 7, 16, 17, 18, 20, 13' and 1', 2', etc.

[0194] Example 3: Preparation of rehmannia pigment compounds F and G

[0195] This embodiment describes the isolation of two other monomeric compounds from the same source that are geometric isomers of compound E, namely, rehmannia pigment compound F (e.g. Figure 8 (as shown) and rehmannia pigment compound G (such as Figure 9 This demonstrates that the preparation method disclosed in this application can effectively separate a variety of cis-trans isomers and provides examples to support the diversity of the compound family protected in this application.

[0196] The starting materials used in this embodiment are exactly the same as those in Example 2, namely the subfraction Fr-8-27 obtained in Example 1. The separation equipment and most of the chromatographic conditions used are also the same as those in Example 2, namely, using a YMC-Pack ODS-A semi-preparative reversed-phase column (250 × 10 mm, 5 μm), with 74% acetonitrile aqueous solution as the mobile phase and a flow rate of 2 mL / min.

[0197] The key difference between this embodiment and Example 2 lies in the chromatographic peaks collected. In Example 2, a chromatographic peak with a retention time of 32.5 minutes was collected to obtain compound A. In this embodiment, however, chromatographic monitoring continued, and two additional well-separated chromatographic peaks with longer retention times were collected. Specifically, chromatographic peaks with retention times of 42.0 minutes and 49.5 minutes were collected.

[0198] The two newly collected fractions were subjected to the same post-treatment as in Example 2, namely, solvent removal by rotary evaporation in a 20°C water bath. The purified compound, named thymol compound F, was obtained from the fraction with a retention time of 42.0 minutes; the purified compound, named thymol compound G (abbreviated as compound G), was obtained from the fraction with a retention time of 49.5 minutes.

[0199] The structure of compound F was confirmed by nuclear magnetic resonance (1D and 2D NMR), and the data parameters are as follows:

[0200] 1 H-NMR (500MHz, CDCl3) δ1.28(m,H-2),1.62(m,H-2),1.47(m,H-3),1.84(m,H-3),1.48(m,H-4),1.84(m,H-4),6.26(d,J=16.0Hz,H-7),6.93(d,J=16 .0Hz, H-8), 6.08 (d, J=11.5Hz, H-10), 6.86 (dd, J=11.5, 14.5Hz, H-11), 6.29 (d, J=14.5Hz, H-12), 6.24 (d, J=11.5Hz, H-14), 6. 86 (dd, J=11.5, 14.5Hz, H-15), 6.49 (dd, J=14.5, 11.5Hz, H-15'), 7.09 (d, J=11.5Hz, H-14'), 0.87 (s, CH3-16), 1.19 (s, CH3-17 ), 1.16(s, CH3-18), 1.98(s, CH3-19), 1.99(s, CH3-20), 2.00(s, CH3-20'), 3.54(m, H-1''), 3.79(t, J=5.0Hz, H-2''), 6.24(br s, NH).

[0201] 13 C-NMR (125MHz, CDCl3) δ38.5 (C-1), 36.4 (C-2), 18.0 (C-3), 36.4 (C-4), 75.3 (C-5), 79.9 (C-6), 132.5 (C-7), 126.7 (C-8), 134.5 (C-9), 129.6 (C-10), 125.1 (C-11), 136.5 (C-12), 139.3 (C-13), 131.2 (C-14), 135.0 (C-15), 127.6 (C-15'), 135.1 (C-14'), 128.4 (C-13'), 170.5 (C-12'), 26.5 (C-16), 25 .2 (C-17), 27.1 (C-18), 21.4 (C-19), 13.0 (C-20), 13.0 (C-20'), 43.0 (C-1''), 62.9 (C-2'').

[0202] Those skilled in the art should be fully able to understand the above spectral data, which is cited here to demonstrate the actual experimental process and results.

[0203] Liquid chromatography analysis conditions: Sample chamber temperature: 20℃, column temperature: 35℃, column: AcquityUPLCBEHC18 (2.1×100mm, 1.7μm, WatersCorp., Milford, USA), mobile phase: acetonitrile / water, gradient setting: 5-75% (acetonitrile), run time: 20min, flow rate: 0.3mL / min.

[0204] The structure of compound G was confirmed by nuclear magnetic resonance (1D and 2D NMR), and the data parameters are as follows:

[0205] 1 H-NMR (500MHz, CDCl3) δ1.28(m,H-2),1.62(m,H-2),1.45(m,H-3),1.82(m,H-3),1.48(m,H-4),1.84(m,H-4),6.24(d,J=15.0Hz,H-7),6.42(d,J=15 .0Hz, H-8), 6.26 (d, J=11.5Hz, H-10), 6.70 (dd, J=11.5, 15.0Hz, H-11), 6.86 (d, J=15.0Hz, H-12), 6.11 (d, J=11.5Hz, H-14), 7. 01 (dd, J=11.5, 10.0Hz, H-15), 6.43 (dd, J=10.0, 11.5Hz, H-15'), 7.08 (d, J=11.5Hz, H-14'), 0.84 (s, CH3-16), 1.19 (s, CH3-17 ), 1.14(s, CH3-18), 1.98(s, CH3-19), 2.00(s, CH3-20), 1.99(s, CH3-20'), 3.53(m, H-1''), 3.78(t, J=5.0Hz, H-2''), 6.25(br s, NH).

[0206] 13C-NMR (125MHz, CDCl3) δ38.9 (C-1), 36.7 (C-2), 18.4 (C-3), 36.7 (C-4), 75.8 (C-5), 79.9 (C-6), 131.2 (C-7), 134.9 (C-8), 136.9 (C-9), 131.7 (C-10), 127.8 (C-11), 129.3 (C-12), 138.1 (C-13), 130.1 (C-14), 134.1 (C-15), 127.4 (C-15'), 135.4 (C-14'), 128.9 (C-13'), 170.6 (C-12'), 26.9 (C-16), 25 .6 (C-17), 27.5 (C-18), 13.8 (C-19), 21.2 (C-20), 13.4 (C-20'), 43.4 (C-1''), 63.2 (C-2'').

[0207] Those skilled in the art should be fully able to understand the above spectral data, which is cited here to demonstrate the actual experimental process and results.

[0208] Liquid chromatography analysis conditions: Sample chamber temperature: 20℃, column temperature: 35℃, column: AcquityUPLCBEHC18 (2.1×100mm, 1.7μm, WatersCorp., Milford, USA), mobile phase: acetonitrile / water, gradient setting: 5-75% (acetonitrile), run time: 20min, flow rate: 0.3mL / min.

[0209] The structures of compounds F and G were confirmed. Both compounds were orange-yellow powders. High-resolution mass spectrometry analysis showed quasi-molecular ion peaks in compounds F and G. The mass-to-charge ratios of both compounds are 444.3156, exactly the same as compound E, indicating that they have the same molecular formula. They are isomers.

[0210] The structure of compound F was confirmed as (2E,4E,6E,8E,10Z,12E)-13-((1R,2R)-1,2-dihydroxy-2,6,6-trimethylcyclohexyl)-N-(2-hydroxyethyl)-2,7,11-trimethyltrideca-2,4,6,8,10,12-hexaenamide, which can be named in Chinese as: (2E,4E,6E,8E,10Z,12E)-13-[(1R,2R)-1,2-dihydroxy-2,6,6-trimethylcyclohexyl]-N-(2-hydroxyethyl)-2,7,11-trimethyltrideca-2,4,6,8,10,12-hexaenamide. , specifically Figure 8 As shown. This structure belongs to Figure 1 The general structural formula (I) shown falls within the category of, and is Figure 3 A specific example of the structural skeleton shown in (ii). Figure 8 The numbers in the molecular formula represent the relative positions of the carbon atoms in that formula. They have no substantial impact on the carbon atoms, molecular structure, or the corresponding compound; they are simply used to facilitate understanding or description of the relative positions of carbon atoms in the molecular structure. Examples of such numbers include: 1, 5, 8, 16, 17, 19, 20' and 1', 2', etc.

[0211] The structure of compound G was confirmed as: (2E,4E,6Z,8E,10E,12E)-13-((1R,2R)-1,2-dihydroxy-2,6,6-trimethylcyclohexyl)-N-(2-hydroxyethyl)-2,7,11-trimethyltrideca-2,4,6,8,10,12-hexaenamide, which can be named in Chinese as: (2E,4E,6Z,8E,10E,12E)-13-[(1R,2R)-1,2-dihydroxy-2,6,6-trimethylcyclohexyl]-N-(2-hydroxyethyl)-2,7,11-trimethyltrideca-2,4,6,8,10,12-hexaenamide, the structure of which is shown in [Figure / Image]. Figure 3 This structure also belongs to Figure 1 The general structural formula (I) shown falls within the category of, and is Figure 3 This is a specific example of the structural skeleton shown in equation (iii), and also Figure 9 The specific compound shown. Figure 9The numbers in the molecular formula represent the relative positions of the carbon atoms in that formula. They have no substantial impact on the carbon atoms, molecular structure, or the corresponding compound; they are simply used to facilitate understanding or description of the relative positions of carbon atoms in the molecular structure. Examples of such numbers include: 1, 5, 12, 16, 19, 20' and 1', 2', etc.

[0212] Example 4: Rehmannia pigment compound H (referred to as compound H)

[0213] This embodiment aims to describe two homologues with different chain lengths from the compounds in Examples 2 and 3—the rehmannia pigment compound H (such as...). Figure 10 The separation, purification, and structural confirmation process (shown in the figure) demonstrates that by separating the different subfractions obtained in Example 1, a series of compounds with different conjugated polyene chain lengths can be obtained, thereby providing... Figure 1 The chain length variation defined by parameters a and b in the general structure provides specific instance support.

[0214] In this embodiment, the subfraction Fr-9 obtained in Example 1 was used as the starting material. Fr-9 was dissolved in methanol, filtered through a 0.22-micron filter membrane, and then separated by semi-preparative high-performance liquid chromatography (HPLC). A gradient elution method was used for better separation of complex components. The chromatographic conditions were as follows: a YMC-Pack ODS-A column (250 × 10 mm, 5 μm), a gradient mixture of acetonitrile (A) and water (B) as the mobile phase, with the gradient program set to linearly increase the proportion of acetonitrile in the mobile phase from 58% to 100% over a certain period, and a flow rate of 2 mL / min. The peak with a retention time of 16.0 min was collected under UV detection. The collected fraction was rotary evaporated to dryness at 20 °C to obtain the purified compound H.

[0215] The structure of compound H was confirmed by nuclear magnetic resonance (1D and 2D NMR), with the following parameters:

[0216] 1H-NMR (500MHz, CDCl3) δ1.27 (m, H-2), 1.61 (m, H-2), 1.47 (m, H-3), 1.82 (m, H-3), 1.49 (m, H-4), 1.83 (m, H-4), 6.22 (d, J=16.0Hz, H-7), 6.40 (d, J=16.0Hz, H-8), 6. 22 (d, J=11.5Hz, H-10), 6.69 (dd, J=11.5, 15.0Hz, H-11), 6.36 (d, J=15.0Hz, H-12), 6.26 (d, J=11.5Hz, H-14), 6.26 (dd, J=11.5, 11.5Hz, H-1 5), 6.59 (dd, J=11.5, 11.5Hz, H-15'), 6.50 (d, J=11.5Hz, H-14'), 7.35 (d, J=15.0Hz, H-12'), 5.89 (d, J=15.0Hz, H-11'), 0.84 (s, CH3-16), 1 .18(s, CH3-17), 1.14(s, CH3-18), 1.98(s, CH3-19), 1.99(s, CH3-20), 1.91(s, CH3-20'), 3.55(m, H-1''), 3.79(t, J=5.0Hz, H-2''), 6.04(br s, NH).

[0217] 13 C-NMR (125MHz, CDCl3) δ38.6 (C-1), 36.4 (C-2), 17.9 (C-3), 36.4 (C-4), 75.4 (C-5), 79.6 (C-6), 130.3 (C-7), 134.6 (C-8), 135.6 (C-9), 131.4 (C-10), 125.9 (C-11), 137.4 (C-12), 138.6 (C-13), 131.9 (C-14), 133.4 (C-15), 1 28.9 (C-15'), 138.7 (C-14'), 133.3 (C-13'), 145.9 (C-12'), 118.1 (C-11'), 167.8 (C-10'), 26.5 (C- 16), 25.1 (C-17), 27.1 (C-18), 13.4 (C-19), 12.7 (C-20), 12.9 (C-20'), 42.8 (C-1''), 62.8 (C-2'').

[0218] Those skilled in the art should be fully able to understand the above spectral data, which is cited here to demonstrate the actual experimental process and results.

[0219] Liquid chromatography analysis conditions: Sample chamber temperature: 20℃, column temperature: 35℃, column: AcquityUPLCBEHC18 (2.1×100mm, 1.7μm, WatersCorp., Milford, USA), mobile phase: acetonitrile / water, gradient setting: 5-75% (acetonitrile), run time: 20min, flow rate: 0.3mL / min.

[0220] Compound H is an orange-red powder. High-resolution mass spectrometry analysis shows its quasi-molecular ion peak. The mass-to-charge ratio is 470.3256, and the calculated molecular formula is: ( The theoretically calculated value is 470.3270. Compared to compound AC, its molecular weight has increased by 26, corresponding to an increase of one. Unit. Nuclear magnetic resonance spectroscopy data further confirmed its detailed structure, such as Figure 10 The structure shown is (2E,4E,6E,8E,10E,12E,14E)-15-((1R,2R)-1,2-dihydroxy-2,6,6-trimethylcyclohexyl)-N-(2-hydroxyethyl)-4,9,13-trimethylpentadeca-2,4,6,8,10,12,14-heptaenamide. This structure belongs to the […]. Figure 1 The category of general structural formulas, where parameters a=1 and b=0, corresponds to Figure 5 The structural skeleton of formula (iv) in the middle, and is Figure 10 The specific compounds shown.

[0221] Example 5: Preparation of Rehmannia glutinosa pigment compound I (hereinafter referred to as compound I)

[0222] In this embodiment, another subfraction, Fr-8-30, obtained in Example 1, was used as the starting material. Separation was performed using semi-preparative high-performance liquid chromatography (HPLC), similar to Example 4, with an acetonitrile-water gradient elution as the mobile phase. Under optimized chromatographic conditions, the peak with a retention time of 49.7 minutes was collected. Following the same post-processing and structural confirmation steps, purified compound I was obtained.

[0223] The structure of compound I was confirmed by nuclear magnetic resonance (1D and 2D NMR), with the following parameters:

[0224] 1 H-NMR(500MHz,CDCl3) δ1.27(m,H-2),1.62(m,H-2),1.45(m,H-3),1.84(m,H-3),1.49(m,H-4),1.83(m,H-4),6.21(d,J=16.0Hz,H-7),6.40(d,J=16.0Hz,H-8),6.23(d,J=11.0Hz,H-10),6.67(dd,J=11.0,11.0Hz,H-11),6.37(d,J=11.0Hz,H-12),6.27(d,J=11.5Hz,H-14),6.71(dd,J=11.5,11.5Hz,H-15),6.63(dd,J=11.5,11.0Hz,H-15'),6.34(d,J=11.0Hz,H-14'),6.58(d,J=15.0Hz,H-12'),6.48(d,J=11.0,15.0Hz,H-11'),7.08(d,J=11.0Hz,H-10'),0.84(s,CH3-16),1.18(s,CH3-17),1.14(s,CH3-18),1.97(s,CH3-19),1.98(s,CH3-20),1.96(s,CH3-20'),2.03(s,CH3-19'),3.54(br s,H-1''),3.79(t,J=5.0Hz,H-2''),6.23(br s,NH)。

[0225] 13 C-NMR(125MHz,CDCl3) δ38.7(C-1),36.5(C-2),18.1(C-3),36.5(C-4),75.6(C-5),79.6(C-6),130.2(C-7),134.8(C-8),135.6(C-9),131.7(C-10),125.6(C-11),137.7(C-12),137.6(C-13),132.5(C-14),131.8(C-15),129.7(C-15'),135.5(C-14'),135.4(C-13'),143.3(C-12'),122.9(C-11'),135.4(C-10'),128.1(C-9'),170.4(C-8'),26.7(C-16),25.2(C-17),27.3(C-18),13.5(C-19),13.0(C-20),12.9(C-20'),13.3(C-19'),43.2(C-1''),63.1(C-2'')。

[0226] Those skilled in the art should be fully able to understand the above spectral data, which is cited here to demonstrate the actual experimental process and results.

[0227] Liquid chromatography analysis conditions: Sample chamber temperature: 20℃, column temperature: 35℃, column: AcquityUPLCBEHC18 (2.1×100mm, 1.7μm, WatersCorp., Milford, USA), mobile phase: acetonitrile / water, gradient setting: 5-75% (acetonitrile), run time: 20min, flow rate: 0.3mL / min.

[0228] The compound is an orange-red powder. High-resolution mass spectrometry analysis shows its quasi-molecular ion peak. The mass-to-charge ratio is 510.3579, and the calculated molecular formula is: (Theoretical calculated value is 510.3583). Nuclear magnetic resonance spectroscopy data confirms its structure as follows: Figure 11 The structure shown is (2E,4E,6E,8E,10E,12E,14E,16E)-17-((1R,2R)-1,2-dihydroxy-2,6,6-trimethylcyclohexyl)-N-(2-hydroxyethyl)-2,6,11,15-tetramethylheptadeca-2,4,6,8,10,12,14,16-octaenamide, which is translated into Chinese as (2E,4E,6E,8E,10E,12E,14E,16E)-17-[(1R,2R)-1,2-dihydroxy-2,6,6-trimethylcyclohexyl]-N-(2-hydroxyethyl)-2,6,11,15-tetramethylheptadeca-2,4,6,8,10,12,14,16-octaenamide. This structure belongs to... Figure 1 The category of general structural formulas, where parameters a=1, b=1, corresponds to Figure 6 The structural skeleton of formula (v) in the middle, and is Figure 11 The specific compounds shown.

[0229] Example 6

[0230] CCK8 detection of compound EI:

[0231] Experimental procedure: RAW264.7 cells were... Cells were seeded at a density of 1 cell / well in 96-well plates. After treatment with a gradient concentration of compounds (4, 20, 100 μM) for 48 hours, 10 μL of CCK-8 reagent was added to the original culture plate, and the plates were incubated for 90 minutes. The absorbance at 450 nm was measured to determine the cell growth inhibition rate. Results are expressed as mean ± standard deviation (n=3).

[0232] Table 1. Inhibition rate of compound AI on the growth of RAW264.7 cells (24 h)

[0233]

[0234] Experimental results: As shown in Table 1, the growth inhibition rate of RAW264.7 cells increased in a concentration-dependent manner. The compound did not exhibit significant cytotoxicity.

[0235] The conclusion of this embodiment is that compound EI has good biocompatibility, which provides important safety evidence for its application as an additive in food, pharmaceuticals, or cosmetics that can come into direct contact with the human body.

[0236] Example 7

[0237] This example demonstrates the inhibitory effect of various compounds (EI) on LPS-induced NO secretion in RAW264.7 cells:

[0238] Experimental procedure: RAW264.7 cells in logarithmic growth phase were digested with trypsin and resuspended in DMEM medium containing 10% fetal bovine serum. Cell density was adjusted to... Inoculate at a rate of 100 cells / mL into a 96-well plate, with each well inoculated with 100 cells / mL. Cells were used. The experimental design included the following groups: untreated control group, LPS-induced model control group, and compound treatment group (LPS + compound). The test compound was diluted with culture medium and added to the wells of the treatment groups at final concentrations of 4, 20, and 100 μM. After incubation for 30 minutes, LPS at a final concentration of 1 μg / mL was added to the model group and treatment groups, and stimulation continued for 24 hours. After stimulation, 100 μL of supernatant was collected, and nitrite was quantitatively detected using Griess' reagent (R1 and R2, each reacting for 5 minutes, protected from light, at room temperature). The absorbance at 540 nm was measured, and the NO inhibition rate was calculated. Quercetin was used as a positive control. All experiments were repeated three times.

[0239] Table 2. Inhibition rate (%) of NO release from RAW264.7 cells. Results are expressed as mean ± standard deviation (n=3).

[0240]

[0241] Experimental results: The results showed that compound E–I (at a concentration of 20 μM) significantly inhibited NO production in LPS-induced RAW264.7 cells. These results suggest that these compounds possess anti-inflammatory properties.

[0242] The results of this embodiment strongly demonstrate that compound EI possesses significant in vitro anti-inflammatory activity. This bioactivity, combined with its vibrant color and high safety profile, makes it a highly promising multifunctional additive capable of providing additional health benefits to products while delivering coloring effects, such as for soothing and anti-inflammatory purposes in cosmetics or for assisting in the regulation of inflammatory levels in functional foods.

[0243] Example 8

[0244] This embodiment provides an example of preparing the rehmannia pigment compound described in this application into a specific additive composition to demonstrate its processability and applicability in actual product formulations.

[0245] Example 1: Preparation of a cosmetic lotion with anti-inflammatory and soothing effects

[0246] This example aims to prepare a skin lotion containing compound F. Compound F not only gives the lotion a delicate orange-yellow appearance, but also provides a skin-soothing effect through its anti-inflammatory activity.

[0247] The formulation is as follows (by weight percentage): Oil phase: - White oil: 10.0% - Stearic acid: 5.0% - Tween-80: 3.0% Aqueous phase: - Glycerin: 5.0% - Compound F: 0.1% - Phenoxyethanol: 0.5% - Deionized water: to 100%

[0248] Preparation Process: 1. Oil Phase Preparation: Accurately weigh white oil, stearic acid, and Tween-80, place them in a beaker, and heat and stir in a 75°C water bath until all components are completely dissolved, forming a homogeneous and transparent oil phase. 2. Aqueous Phase Preparation: In another beaker, weigh glycerol, compound B, phenoxyethanol, and deionized water, and heat and stir in a 75°C water bath until all components are completely dissolved, forming a clear orange-yellow aqueous solution. 3. Emulsification: Under stirring in a high-speed shear homogenizer (e.g., 5000 rpm), slowly and continuously add the aqueous phase, maintained at 75°C, to the oil phase at the same temperature. Continue high-speed homogenization for 5 minutes to shear the oil phase into tiny droplets and uniformly disperse them in the aqueous phase, forming a stable oil-in-water emulsion. 4. Cooling: Stop heating, reduce the homogenizer speed to a slow stirring state, and continue stirring until the emulsion system cools naturally to room temperature.

[0249] The final product is a stable emulsion with a uniform appearance, fine texture, and a light orange-yellow color. This emulsion feels moisturizing but not greasy when applied to the skin, and due to the presence of active ingredient compound F, it can be used as a skincare product with triple benefits of moisturizing, coloring, and anti-inflammatory soothing.

[0250] Example 2: Preparation of functional food coloring liquid

[0251] This example aims to prepare a water-soluble colorant containing compound H, which can be used for coloring beverages, jellies, candies, and other foods. Compound H not only has a more orange-red color but also exhibits significant anti-inflammatory activity, providing added health value to food products.

[0252] The formula is as follows (by weight percentage): - Compound H: 0.05% - Citric acid: 0.20% - Vitamin C: 0.10% - Purified water: to 100%

[0253] Preparation Process: 1. Dissolving: Weigh purified water into a clean container, and add citric acid, vitamin C, and compound 4 sequentially while stirring. Continue stirring until all solids are completely dissolved, forming a clear, transparent orange-red aqueous solution. The acidity is weakened, and the solution is stable at pH 7-9. Vitamin C acts as an antioxidant, protecting the pigment from oxidation and fading during storage and use. 2. Sterilization and Filling: Filter the prepared solution through a sterile membrane with a 0.22-micron pore size for sterilization. Under aseptic conditions, fill the filtered coloring solution into sterilized glass or plastic bottles and seal for storage.

[0254] The final product is a clear, transparent, and bright orange-red liquid. This liquid can be used as a natural and safe functional food additive, added directly to various foods to give them an appealing color, while utilizing the bioactivity of compound 4 to provide consumers with potential health benefits.

[0255] The above description is merely a preferred embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A rehmannia flavonoid compound of formula (I), its stereoisomer, or a pharmaceutically acceptable salt thereof: ； in, R1, R2, and R5 are independently hydroxyl, mercapto, or amino groups; R3 is oxygen or sulfur; R4 can be an oxygen, sulfur, or imine group independently; The stereochemical configurations of 5-C and 6-C are independently R or S; The double bond configurations of 9-C and 13-C are independently either E-type or Z-type; Wherein: brackets close to 13'-C are called brackets a, and brackets far from 13'-C are called brackets b; the structural units within brackets a and brackets b are independently optional structural units; a is 0 or 1; b is 0 or 1; When a is 0, the structural unit inside the parentheses a does not exist; when a is 1, the structural unit inside the parentheses a exists. When b is 0, the structural unit inside the parentheses b does not exist; when b is 1, the structural unit inside the parentheses b exists.

2. The rehmannia glutinosa compound, its stereoisomer, or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The compound is a compound having the structure shown in formula (i), formula (ii), formula (iii), formula (iv) or formula (v): ； ； ； ； In equation (i), the 9-C double bond is of the E type, the 13-C double bond is of the E type, and a is 0 and b is 0; In equation (ii), the 9-C double bond is of the Z type, the 13-C double bond is of the E type, and a is 0 and b is 0; In equation (iii), the 9-C double bond is of the E type, the 13-C double bond is of the Z type, and a is 0 and b is 0; In equation (iv), the 9-C double bond is of the E type, the 13-C double bond is of the E type, and a is 1 and b is 0; In equation (v), the 9-C double bond is of type E, the 13-C double bond is of type E, and a is 1 and b is 1.

3. The rehmannia glutinosa compound, its stereoisomer, or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that, The compound is a compound having the structure shown in formula (ie), formula (ii-f), formula (iii-g), formula (iv-h), or formula (vi): ； ； ； 。 4. The rehmannia glutinosa compound, its stereoisomer, or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The stereo configurations of 5-C and 6-C are (5R, 6R) or (5S, 6R).

5. The rehmannia glutinosa compound, its stereoisomer, or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The 9-C double bond is of the Z type, and the 13-C double bond is of the E type.

6. The rehmannia glutinosa compound, its stereoisomer, or a pharmaceutically acceptable salt thereof according to claim 1 or 2, characterized in that, R1 and R2 are independently hydroxyl or mercapto groups, R3 is oxygen, R4 is oxygen, and R5 is hydroxyl.

7. The rehmannia glutinosa compound, its stereoisomer, or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, a is 0 and b is 0.

8. A method for preparing the rehmannia glutinosa compound, its stereoisomer, or a pharmaceutically acceptable salt thereof according to any one of claims 1-7, characterized in that, Includes the following steps: The crude rehmannia pigment was subjected to normal phase chromatography to obtain a preliminary purified fraction containing the target component. The preliminarily purified fraction was subjected to reverse-phase chromatography to obtain a further purified subfraction; Size exclusion chromatography was performed on the sub-components to obtain secondary components; The secondary components were separated by semi-preparative high-performance liquid chromatography and / or preparative high-performance liquid chromatography to obtain the rehmannia glutinosa pigment compound.

9. An additive composition, characterized in that, The compound comprising any one of claims 1 to 7, its stereoisomers, or pharmaceutically acceptable salts thereof, and food, pharmaceutical, or cosmetic excipients.

10. The use of the rehmannia glutinosa compound, its stereoisomer, or a pharmaceutically acceptable salt thereof, or the additive composition of claim 9, as an additive in any one of claims 1 to 7.

Images