Xanthophyll, xanthophyll composition and application of xanthophyll composition

Abstract

The invention discloses xanthophyll, a xanthophyll composition and application thereof, and belongs to the technical field of biological medicines.The preparation method of xanthophyll comprises the following steps that marigold particles are extracted with propane and butane as extracting solvents, then the obtained extracting solution is evaporated twice and then distilled and concentrated, the solvent of the obtained extract is removed, then centrifugation is conducted, and the xanthophyll is obtained; the xanthophyll is obtained. The invention also discloses a xanthophyll composition. The xanthophyll composition is prepared from the following raw materials: xanthophyll, zeaxanthin, curcumin, quercetin, fucoidin, a honeysuckle flower extract, casein, beeswax, soybean phospholipid, soybean oil, glycerol and hydroxypropyl methyl cellulose. The lutein composition disclosed by the invention has the advantages that the raw materials are natural, are food-grade or medicinal and edible components, have no side effects, are relatively low in production and manufacturing cost, are good in compliance of consumers or patients, can be used for a long time, and have double effects, so that the effects of conditioning and improving body inflammations to prevent and improve IBD and thrombus complications are achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to lutein, a lutein composition and applications thereof. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic nonspecific inflammatory disease with the characteristics of unknown etiology, chronic inflammation, and recurrent disease. It specifically includes two main disease types: Crohn's disease (CD) and ulcerative colitis (UC). It is worth noting that the extraintestinal complications of IBD are one of the important reasons for the recurrent disease and high mortality rate of patients.

[0003] In recent years, thromboembolism (TE), as an extraintestinal complication of IBD, has attracted more and more attention from scholars. It usually occurs during the outbreak of IBD disease activity. Thromboembolism mainly includes atherosclerosis, varicose veins, atrial fibrillation, pulmonary embolism, and acute myocardial infarction. It is a serious and potentially life-threatening event. Over the years, the risk of TE in patients with IBD has been increasing. It is reported that the overall incidence of systemic TE events in patients with IBD is 6.2%, and the risk of systemic TE events is three times higher than that in the general population. Evidence shows that inflammation and coagulation are interdependent physiological processes. The inflammatory response promotes coagulation and vice versa, and can trigger a vicious cycle. IBD is associated with a hypercoagulable state and an increased risk of thromboembolism. There is evidence that thrombosis occurs in both inflamed intestinal and extraintestinal tissues.

[0004] The hypercoagulable state and enhanced thrombosis observed in human IBD have been reproduced in animal models of colitis. Animal studies have also shown that increased levels of procoagulants (such as thrombin) and decreased levels of endogenous anticoagulants (such as activated protein C) contribute to the spread of intestinal inflammation in experimental IBD. There is also evidence that the expression of tissue factor and related proteins changes during the occurrence of experimental colitis-related thrombosis. IBD combined with venous thrombosis is closely related to abnormal activation of the procoagulant system, including the initiation of the coagulation system, downregulation of natural anticoagulant mechanisms, damage to fibrinolysis, increased platelet counts, and endothelial cell dysfunction. The relevant data show that the hypercoagulable state of blood in IBD is not caused by a specific mechanism, but by the interaction of a very large system.

[0005] The treatment and prevention of venous thrombosis in IBD patients is a key link in the effectiveness of IBD treatment. my country's guidelines suggest that preventive anticoagulation therapy mainly includes drug anticoagulation and mechanical prevention. Among them, anticoagulants mainly include low molecular weight heparin, pentosan heparin and (or) fondaparinux sodium, and mechanical prevention mainly uses graduated compression stockings (GCS), intermittent pneumatic pressure devices (IPS), and plantar pressure pumps (VFPs). However, drug treatment has adverse reactions such as skin bleeding, abdominal bleeding, hematuria, fever, headache, chills, nausea, vomiting, constipation, epistaxis, ecchymosis, mild hematuria, allergies, etc., and attention should be paid to drug interactions, resulting in poor patient compliance; compared with anticoagulant drug treatment, mechanical prevention is more acceptable to patients and the implementation process is simpler, but it is not suitable for patients with severe lower limb edema, pulmonary edema, dermatitis, etc., and its application is limited, the cost is high, and there is even a risk of serious infection and malignant tumors. Therefore, there is an urgent need to find an effective and inexpensive drug or method that can effectively relieve the symptoms of IBD and reduce thrombosis.

[0006] Lutein is a xanthophyll carotenoid obtained from various foods, such as dark green leafy vegetables and egg yolk. Lutein has antioxidant activity and can scavenge reactive oxygen species such as singlet oxygen and lipid peroxyl radicals. Oxidative stress activates inflammatory mediators, leading to the development of metabolic and inflammatory diseases. Recent basic and clinical studies have investigated the anti-inflammatory effects of lutein based on its antioxidant activity and the regulation of oxidant-sensitive inflammatory signaling pathways. Therefore, it can prevent various inflammatory diseases, including neurodegenerative diseases, diabetic retinopathy, osteoporosis, cardiovascular diseases, skin diseases, liver damage, obesity, and colitis. In addition, lutein has tissue-specific effects, such as regulating lipid profiles in cardiovascular diseases, adipocyte differentiation in obesity, and skin function. Therefore, the consumption of foods rich in lutein may help prevent inflammatory diseases caused by oxidative stress.

[0007] Therefore, there is an urgent need to find an intervention plan that uses lutein to develop high universality, good patient compliance, low development cost, no side effects, and can simultaneously prevent and improve IBD and thrombotic complications, in order to prevent and improve IBD and thrombotic complications, improve the quality of life of IBD patients, and provide reference for the clinical development of related drugs. Summary of the invention

[0008] In order to solve the above technical problems, the present invention provides a lutein composition and a preparation method and application thereof.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] A method for preparing lutein comprises the following steps:

[0011] The marigold flower particles are extracted with propane and butane as extraction solvents, and then the obtained extract is evaporated twice and then distilled and concentrated, and then the obtained extract is centrifuged after removing the solvent to obtain the lutein.

[0012] Preferably, in the extraction solvent, the volume ratio of propane to butane is 1:(1.1-1.4).

[0013] Preferably, the mass ratio of the extraction solvent to the marigold flower particles is 1:(2-3).

[0014] Preferably, the extraction temperature is 40° C., the pressure is 0.8-0.9 MPa, and the time is 45-60 min.

[0015] Preferably, the evaporation temperature is 36°C ± 2°C and the pressure is 0.04-0.05MPa;

[0016] The temperature of the distillation concentration is 60° C. and the pressure is 0.04-0.05 MPa.

[0017] Beneficial effects: The lutein in the present invention is a natural extract component. Lutein is a heat-sensitive substance, afraid of light and heat, and very easy to oxidize and deteriorate. Its physical and chemical properties determine that the production process must be carried out in a closed and room temperature environment, so the entire leaching process is carried out in a pressure vessel. The present invention is prepared from fresh marigold flowers through low-temperature extraction technology. Under specific ratios of temperature, pressure and solvent conditions, the extraction rate and purity of natural lutein are improved, thereby improving the purity of lutein in the composition.

[0018] A method for preparing lutein obtains lutein.

[0019] A use of lutein in preparing a medicine for preventing and improving IBD and its thrombotic complications.

[0020] A lutein composition comprising the following raw materials in mass fractions:

[0021] The above-mentioned lutein 8-13.5%, zeaxanthin 5-12%, curcumin 8-16%, quercetin 3-21%, fucoidan 10-13%, honeysuckle extract 6-11.3%, casein 0.8-1.5%, beeswax 0.4-1.2%, soybean lecithin 0.4-1%, soybean oil 5.5-24.2%, glycerol 8-19% and hydroxypropyl methylcellulose 5-7.5%.

[0022] Beneficial effects: The fucoidan in the present invention can form a complex with lutein, changing the physical form of lutein from a poorly soluble crystal form to an amorphous state that is easier to dissolve. This transformation increases the solubility of lutein in the gastrointestinal tract, thereby improving its absorption efficiency. At the same time, the complex formed by fucoidan and lutein is not stable enough due to weak interfacial interaction, thereby forming an oversaturated state in the aqueous solution, effectively inhibiting the precipitation of lutein and improving its solubility. Quercetin can interact with casein to form a complex with low hydrophobicity through covalent or non-covalent interactions, which plays a role in encapsulating and carrying lutein, thereby improving the bioaccessibility of lutein; hydroxypropyl methylcellulose forms a stable oversaturated system through multiple hydrogen bonds, which promotes the absorption and utilization of lutein. Glycerin plays the role of a binder and stabilizer in the preparation of lutein preparations. Curcumin and honeysuckle extract can effectively improve intestinal inflammation and thrombosis, and can synergize with lutein and zeaxanthin to improve IBD and extraintestinal thrombosis symptoms.

[0023] Preferably, the dosage form of the lutein composition includes one or more of powder, emulsion, capsule and soft candy dosage forms.

[0024] Beneficial effects: Starting from the mechanism of mutual influence between IBD and thrombosis, the present invention screens and optimizes a lutein composition that can effectively prevent and improve IBD and thrombotic complications, and explores the mechanism of action and possible pharmacological action signal pathways of the best formula lutein composition to improve IBD and thrombotic complications, confirming that the lutein composition of the present invention can effectively improve IBD and thrombotic complications by regulating inflammatory factors IL-6, IL-1β, TNF-α and thrombus flow time. The raw materials of the lutein composition of the present invention are natural, all of which are food-grade or medicinal and edible ingredients, without side effects, low production and manufacturing costs, good compliance of consumers or patients, and can be used for a long time, playing a dual effect, thereby achieving the effect of regulating and improving body inflammation to prevent and improve IBD and thrombotic complications.

[0025] A use of a lutein composition in the preparation of a medicine for preventing and improving IBD and its thrombotic complications.

[0026] Beneficial effects: The lutein composition provided by the present invention has an improving effect on intestinal inflammation and extraintestinal thrombosis, which is due to the fact that it regulates inflammatory factors in the inflammation-coagulation related signaling pathway, inhibits the expression of pro-inflammatory factors, reduces the inflammatory response and changes in the coagulation mechanism, and improves the flow of thrombus, thereby playing a good regulatory role on colitis and related extraintestinal thrombosis.

[0027] Compared with the prior art, the present invention has the following advantages and technical effects:

[0028] The present invention provides a lutein composition that can effectively prevent IBD and its intra- and extra-intestinal thrombotic complications, as well as a preparation method and application thereof. It is the first time that a lutein composition can simultaneously prevent and improve inflammatory bowel disease and corresponding thrombotic complications. The lutein of the present invention is obtained by preparing fresh marigold flowers through solvent subcritical low-temperature extraction technology. The present invention further confirms through animal experiments that the composition of the present invention can effectively regulate the symptoms of colitis and extra-intestinal thrombotic complications by regulating the inflammatory factors and thrombotic changes in the inflammation-coagulation system, has great clinical development and application value, and provides great reference significance for the clinical development of inflammation-coagulation drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The drawings constituting a part of the present application are used to provide a further understanding of the present application. The illustrative embodiments and descriptions of the present application are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0030] Figure 1 The results are for weight loss of mice in the experimental group;

[0031] Figure 2 The diarrhea results of mice in the experimental group are shown in Figure 2.

[0032] Figure 3 The results of blood in stool of mice in the experimental group are shown;

[0033] Figure 4 The macroscopic scores of mice in the experimental group are shown in Figure 2.

[0034] Figure 5 Results of vascular occlusion time of mice in the experimental group;

[0035] Figure 6 The results of peripheral blood cell analysis (neutrophils) of mice in the experimental group;

[0036] Figure 7 The results of peripheral blood cell analysis (platelets) of mice in the experimental group;

[0037] Figure 8 The thrombus weight results of mice in the experimental group;

[0038] Fig. 9 The results of thrombus length in mice of the experimental group;

[0039] Fig.10 The results of tail bleeding time of mice in the experimental group;

[0040] Fig.11 Results of hemoglobin levels in mice in the experimental group;

[0041] Fig.12 The results are the effects of DSS colitis on light / dye-induced thrombosis in the cremaster venules of mice;

[0042] Fig.13 The results are the effects of DSS colitis on light / dye-induced thrombosis in mouse cremaster arterioles;

[0043] Fig.14 The changes of TNF-α inflammatory factors in the colon of the experimental group mice were compared with the changes in skeletal muscle;

[0044] Fig.15 The changes of IL-1β inflammatory factor in colon of experimental group mice were compared with those in skeletal muscle;

[0045] Fig.16 The changes of IL-6 inflammatory factors in the colon of the experimental group mice were compared with the changes in skeletal muscle;

[0046] Fig.17 The results of the lutein formula obtained in Examples 1-9 on the effect of the onset and stop time of thrombosis;

[0047] Fig.18 The level of TNF-α, a pro-inflammatory factor, in the colon of mice with colitis after intervention with the lutein composition obtained in Example 1-9;

[0048] Fig.19 The level of pro-inflammatory factor IL-1β in the colon of mice with colitis after intervention with the lutein composition obtained in Example 1-9;

[0049] Fig. 20 The level of IL-6, an inflammatory factor in the colon of mice with colitis, after the lutein composition obtained in Example 1-9 intervened in the mice with colitis;

[0050] Fig.21 The results of arterial thrombus flow time in mice after the lutein composition obtained in Examples 2, 4 and 7 intervened in colitis mice;

[0051] Fig. 22 The results of the neutrophil count in mice after the lutein compositions obtained in Examples 2, 4 and 7 were used to intervene in colitis mice;

[0052] Fig.23 The thrombus weight results of mice after the lutein composition obtained in Examples 2, 4 and 7 intervened in colitis mice;

[0053] Fig.24 The thrombus length results of mice after the lutein composition obtained in Examples 2, 4 and 7 intervened in colitis mice;

[0054] Fig.25 The results of the tail bleeding time of mice after the lutein composition obtained in Examples 2, 4 and 7 intervened in colitis mice;

[0055] Fig.26 The level of TNF-α, a pro-inflammatory factor, in the colon of mice with colitis after intervention with the lutein composition obtained in Examples 2 and 4;

[0056] Fig. 27 The level of pro-inflammatory factor IL-1β in the colon of mice with colitis after intervention with the lutein composition obtained in Examples 2 and 4;

[0057] Fig.28 The level of IL-6, an inflammatory factor in the colon of mice with colitis, after the lutein composition obtained in Examples 2 and 4 was used to intervene in the mice with colitis;

[0058] Fig.29 The thrombus weight results of mice after the lutein composition obtained in Examples 2 and 4 intervened in colitis mice;

[0059] Fig.30 The results of arterial thrombus flow time in mice after the lutein composition obtained in Examples 2 and 4 intervened in colitis mice;

[0060] Fig.31 The effect of the lutein composition obtained in Examples 2 and 4 on the expression level of EPCR in mouse microvascular endothelial cells;

[0061] Fig.32 The effect of the lutein composition obtained in Examples 2 and 4 on the expression level of TM in mouse microvascular endothelial cells;

[0062] Fig.33 The effect of the lutein composition obtained in Examples 2 and 4 on the expression level of EPCR mRNA in mouse microvascular endothelial cells;

[0063] Fig.34 The effects of the lutein compositions obtained in Examples 2 and 4 on the expression level of TM mRNA in mouse microvascular endothelial cells are shown in FIG. DETAILED DESCRIPTION

[0064] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of 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.

[0065] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0066] Unless otherwise specified, the raw materials in the examples of the present invention were purchased from commercial sources.

[0067] Among them, the marigold flower granules were purchased from the marigold flower planting base in Xihe County, Longnan City, Gansu Province.

[0068] The boiling points of the components in liquefied butane and propane are mostly below 0°C, among which the boiling point of propane is -42.07°C and the boiling point of butane is -0.5°C. They are gases at normal temperature and pressure of 37°C and become liquids after pressurization.

[0069] Unless otherwise specified, the room temperature or normal temperature in the embodiments of the present invention refers to 25±3°C.

[0070] Example 1

[0071] 1. Lutein extraction and preparation

[0072] (1) Pretreatment of marigold particles: The marigold particles are first sent to the pretreatment workshop for screening, magnetic separation, stone removal, classification and other pretreatment processes. After removing the sand, gravel, broken particles and metal debris, they are used in the leaching workshop.

[0073] (2) Leaching: Using liquefied butane and propane (the volume ratio of liquefied butane to liquefied propane is 1:1.28) as the extraction solvent, the treated marigold flower particles are transported to the leacher through a scraper conveyor, and mixed with the extraction solvent at a material-liquid mass ratio of 1:2.5. The solvent is continuously leached through 6 solvent pumps, wherein the leaching temperature is 40°C, the pressure is 0.87 MPa, and the leaching time is 56 minutes. Lutein is continuously countercurrently leached by the extraction solvent and dissolved into one. The appearance of the solution is clear, and the solution becomes turbid after adding lutein, indicating that the lutein has exceeded the solubility of the solvent at the current temperature, and the solution is in a supersaturated state, forming a saturated solution; after leaching, the residue is separated from the extract by filtering through a 150-mesh net in the silo;

[0074] (3) The obtained filter residue is evaporated by a residue evaporator at 124°C under negative pressure steam. The residue evaporator evaporates the extraction solvent in the filter residue and enters a refrigeration device for cooling and recovery for reuse. The residue is transported to a residue storage bin by an auger.

[0075] (4) Evaporation and concentration: The obtained extract is pumped into a cyclone for separation, and then the obtained saturated solution is added to an evaporator for evaporation, wherein the vacuum pressure in the evaporator is 0.048 MPa. Under the separation of the flash box in the evaporator, the vapor phase solvent obtained by evaporation enters a refrigerated separator for cooling, recovery and recycling; the liquid phase enters another evaporator for secondary evaporation, and again under the separation of the flash box, the vapor phase solvent obtained by the secondary evaporation enters a refrigerated separator for cooling, recovery and recycling; the liquid phase enters a distillation tower with a vacuum degree of 0.048 MPa and is distilled and concentrated at a temperature of 60°C to obtain lutein extract.

[0076] (5) Removal of disability

[0077] The evaporated extract is further used to remove the residual solvent, and the concentrated extract is treated with pigments using a high-speed centrifuge to further reduce the insoluble content, thereby obtaining lutein.

[0078] A lutein composition comprising the following raw materials in weight fractions:

[0079] Lutein 8%, zeaxanthin 5%, curcumin 4%, quercetin 3%, fucoidan 10%, honeysuckle extract 5%, casein 1%, beeswax 1%, soybean lecithin 1%, soybean oil 29%, glycerin 23% and hydroxypropyl methylcellulose 10%.

[0080] A method for preparing a lutein composition (soft capsule), comprising the following steps:

[0081] 1. Capsule shell preparation (gelatinization): gelatin: glycerin: water = 1: 0.35: 0.8. Add glycerin and water into the gelatinization tank, heat to 68℃±2℃, then add gelatin. Sol for about 1 hour, vacuum to remove bubbles and moisture, so that the gelatin is completely dissolved without particles. The particle size index can reach 15,000 to 25,000 cp, thus meeting the product requirements.

[0082] 2. Preparation of molten suspending and dispersing agent R1: Mix soybean oil, beeswax and soybean lecithin and heat them in a water bath to 70℃±3℃ to melt to obtain R1 melt.

[0083] 3. Content preparation (ingredients): weigh the raw materials of the lutein composition mentioned above, grind and mix them evenly, then mix them evenly with the R1 melt, stir them in a high shear dispersing emulsifier for 2 minutes, heat them to 90-95°C, grind and mix them evenly, and obtain the capsule content liquid for use.

[0084] 4. Pill pressing: Add the contents into the hopper of the pill press, control the spray temperature within the range of 38-45°C, the glue temperature within the range of 55-70°C, the cooling drum temperature within the range of 15-26°C, the liquid temperature not higher than 42°C, and press the pill press into shape.

[0085] 5. Drying: Place the freshly pressed soft capsules in a rotating cage and air dry them dynamically during the rotation process. The setting time is about 2 to 4 hours, and the moisture content is reduced to between 25 and 30%. The soft capsule shell becomes elastic and does not deform.

[0086] Example 2

[0087] A method for preparing lutein is the same as that in Example 1.

[0088] A lutein composition comprising the following raw materials in weight fractions:

[0089] Lutein 8%, zeaxanthin 12%, curcumin 8%, quercetin 3%, fucoidan 13%, honeysuckle extract 8%, casein 0.8%, beeswax 1.2%, soybean lecithin 1.8%, soybean oil 20.2%, glycerin 19% and hydroxypropyl methylcellulose 5%.

[0090] A method for preparing a lutein composition is the same as that in Example 1.

[0091] Example 3

[0092] A method for preparing lutein is the same as that in Example 1.

[0093] A lutein composition comprising the following raw materials in weight fractions:

[0094] Lutein 0, zeaxanthin 21%, curcumin 19%, quercetin 5%, fucoidan 14%, honeysuckle extract 11.3%, casein 1.5%, beeswax 0.8%, soybean lecithin 0.4%, soybean oil 8.5%, glycerin 11% and hydroxypropyl methylcellulose 7.5%.

[0095] A method for preparing a lutein composition is the same as that in Example 1.

[0096] Example 4

[0097] A method for preparing lutein is the same as that in Example 1.

[0098] A lutein composition comprising the following raw materials in weight fractions:

[0099] Lutein 10.5%, zeaxanthin 5%, curcumin 8%, quercetin 9%, fucoidan 10%, honeysuckle extract 9.8%, casein 0.8%, beeswax 0.6%, soybean lecithin 0.6%, soybean oil 24.2%, glycerin 14% and hydroxypropyl methylcellulose 7.5%.

[0100] A method for preparing a lutein composition is the same as that in Example 1.

[0101] Example 5

[0102] A method for preparing lutein is the same as that in Example 1.

[0103] A lutein composition comprising the following raw materials in weight fractions:

[0104] Lutein 10.5%, zeaxanthin 12%, curcumin 16%, quercetin 9%, fucoidan 13%, honeysuckle extract 11%, casein 1.5%, beeswax 0.8%, soybean lecithin 0.7%, soybean oil 7.5%, glycerin 11% and hydroxypropyl methylcellulose 7%.

[0105] A method for preparing a lutein composition is the same as that in Example 1.

[0106] Example 6

[0107] A method for preparing lutein is the same as that in Example 1.

[0108] A lutein composition comprising the following raw materials in weight fractions:

[0109] Lutein 0%, zeaxanthin 21%, curcumin 6.5%, quercetin 14, fucoidan 14%, honeysuckle extract 6%, casein 1%, beeswax 0.5%, soybean lecithin 0.5%, soybean oil 20%, glycerin 11% and hydroxypropyl methylcellulose 5.5%.

[0110] A method for preparing a lutein composition is the same as that in Example 1.

[0111] Example 7

[0112] A method for preparing lutein is the same as that in Example 1.

[0113] A lutein composition comprising the following raw materials in weight fractions:

[0114] Lutein 13.5%, zeaxanthin 5%, curcumin 16%, quercetin 21%, fucoidan 10%, honeysuckle extract 11.3%, casein 1.5%, beeswax 0.8%, soybean lecithin 0.4%, soybean oil 5.5%, glycerin 8% and hydroxypropyl methylcellulose 7%.

[0115] A method for preparing a lutein composition is the same as that in Example 1.

[0116] Example 8

[0117] A method for preparing lutein is the same as that in Example 1.

[0118] A lutein composition comprising the following raw materials in weight fractions:

[0119] Lutein 13.5%, zeaxanthin 12%, curcumin 4%, quercetin 21%, fucoidan 13%, honeysuckle extract 6%, casein 1%, beeswax 0.4%, soybean lecithin 0.6%, soybean oil 9%, glycerin 14% and hydroxypropyl methylcellulose 5.5%.

[0120] A method for preparing a lutein composition is the same as that in Example 1.

[0121] Example 9

[0122] A method for preparing lutein is the same as that in Example 1.

[0123] A lutein composition comprising the following raw materials in weight fractions:

[0124] Lutein 17.5%, zeaxanthin 0%, curcumin 11.5%, quercetin 21%, fucoidan 14%, honeysuckle extract 20%, casein 0.8%, beeswax 0.9%, soybean lecithin 0.6%, soybean oil 4.2%, glycerin 6% and hydroxypropyl methylcellulose 3.5%.

[0125] A method for preparing a lutein composition is the same as that in Example 1.

[0126] Technical effect:

[0127] Animal model construction

[0128] (1) Materials and methods

[0129] The present invention uses male C57BL / 6 mice (Jackson Laboratories, ME) as experimental animals. All mice were kept in standard cages under specific pathogen-free conditions and fed with standard laboratory food and water until the desired age (6-8wk). All animal procedures were reviewed and approved by the Animal Care and Use Committee, and experimental animals were managed in strict accordance with the requirements of the AAALAC guidelines and the 3R principles of animal welfare.

[0130] DSS-induced colitis: The mice in the experimental group were fed with a solution of 3% (wt / vol) DSS (mol wt, 40,000) dissolved in filtered drinking water. The first day of DSS feeding was defined as day 0, and the mice were maintained on DSS feeding until day 6.

[0131] The mice in the control group were given drinking saline (without DSS).

[0132] Assessment of colitis progression: During the period of DSS treatment, body weight, fecal status, presence of occult blood in feces, and perianal bleeding were observed and recorded daily. The disease activity index (DAI) is a measure of disease severity from 0 to 4 and is calculated based on the collected data of fecal consistency, presence of blood in feces, and weight loss. The DAI is monitored to ensure that the clinical response induced by DSS is consistent with colitis disease activity.

[0133] Cremaster muscle preparation: On day 6 of DSS (experimental group) or water (control)-induced colitis, mice were anesthetized intraperitoneally with 50 mg / kg sodium pentobarbital and supplementary doses (12.5 mg / kg) were given as needed. The right internal jugular vein was cannulated for intravenous administration of FITC-dextran. Body temperature was maintained at 35.5-36.5°C using a homeothermic blanket throughout the experiment and monitored with a rectal temperature probe. Cremaster muscle was prepared for intravital fluorescence microscopy as described previously. The exposed cremaster muscle surface was continuously filled with bicarbonate-buffered saline, pH 7.35-7.45. The cremaster muscle preparation was moved to the stage of an upright fluorescence microscope and allowed to stabilize for 20-30 minutes before the start of the experiment.

[0134] An upright microscope (BX51WI; Olympus, Tokyo, Japan) equipped with a 40× water immersion objective (LUMPlanFI / IR40× / 0.80W) was used. Microscopic images were projected onto a monitor (SonyTRINITRON PVM-2030) by a color video camera (Hitachi VK-C150) and recorded using a DVD recorder (JVC SR-MV50). A video timer (Panasonic Time-Date Generator WJ-810) was connected to the monitor to record time and date. The diameter of the cremaster blood vessels was measured by analysis software (ImageJ 1.37v, NIH, Public Domain software) on a personal computer (G4 Macintosh, Apple). After the preparation was stabilized, 10 ml / kg 5% FITC-dextran (150,000 MW; Sigma, St. Louis, MO) was slowly injected intravenously. Let it circulate for 10 min, and then the diameter was selected to be 30-50 μm for study.

[0135] (2) Experimental plan

[0136] Grouping and treatment of mice in the light / dye endothelial injury model:

[0137] 1) Control group mice (no DSS in drinking water), 2) Experimental group DSS-treated (colitis) mice, 3) Experimental group mice were gavaged and fed with the lutein composition obtained in Example 1-9 5 minutes before vascular epi-irradiation, with a feeding amount of 5 mg / kg.

[0138] Wherein, the gavage feeding method comprises the following steps:

[0139] Dilute the lutein soft capsule solution 100 times (with saline) and gavage at 5 mg / kg body weight. Aim the gavage needle vertically at the mouse's mouth. Insert the needle vertically along the corner of the mouth close to the mouse's upper jaw. Fix the gavage needle without shaking it and slowly inject the drug solution into the mouse's stomach. Pull out the needle and put the mouse back into the original cage. Wash the needle and keep it for next use.

[0140] Light / dye-induced thrombosis:

[0141] Second-order or third-order venules and arterioles (1-3 per mouse) with a diameter of 35-50 μm, a length of at least 100 μm, and a wall shear rate of ≥500 / s were randomly selected in each cremaster muscle to study thrombosis. Then, 10 ml / kg of 5% FITC-dextran (150,000 MW) (Sigma chemicals, St. Louis, MO) was slowly injected into the intravenous cannula and allowed to circulate for 10 minutes. Photoactivation of FITC-dextran (excitation: 495 nm, emission: 519 nm) within the microvasculature was achieved by epi-illumination using a 175 W xenon lamp (Lambda LS, Sutter, CA) and a fluorescein filter cube (HQ-FITC, Chroma Technology Company, VT). The excitation power density was measured daily (ILT 1700 radiometer, SED033 detector, International Light, MA) and maintained within 1% of 0.74 W / cm. Epi-illumination was applied continuously to the vessels, and thrombus formation was quantified by determining: 1) the onset of platelet deposition / aggregation within the microvessels (onset time), and 2) the time required for ≥60 seconds to completely stop blood flow (stop time). Epi-illumination was stopped once blood flow in the study vessel had ceased. Results for each vessel type (venules, arterioles) were averaged from 2-4 thrombi generated per mouse.

[0142] Mesenteric artery thrombosis modeling steps:

[0143] FeCl3 induced thrombosis in mouse mesenteric arterioles by in vivo microscopy: After mice were anesthetized with 1.5% isoflurane (RWD), rhodamine 6G (Sigma, 83697) was injected into the mouse blood via the tail vein for 10 min to label platelets and leukocytes. 1×1 mm filter paper soaked in 5% FeCl3 (MACKLIN, I809489) was used to treat superior mesenteric arteries with a diameter of ≈100 μm. Five minutes after the mice were injured, the filter paper was removed and the residual FeCl3 was washed away with PBS. Vascular occlusion was monitored under a Leica inverted microscope (CTR7000 HS) and retimed for 15 min, and the body temperature of the mice was maintained during the experiment. Among them, only one arteriole was selected for each mouse.

[0144] Inferior vena cava (IVC) stenosis:

[0145] Stenosis of the inferior vena cava was used to induce deep vein thrombosis. After the mice were anesthetized with isoflurane, the skin and peritoneum were incised along the midline of the abdomen to expose the abdominal cavity, and the intestines were removed and wrapped with gauze soaked in 37°C saline. All branches of the IVC were ligated, and the IVC was carefully separated from the aorta below the angle between the left renal vein and the IVC. The inferior vena cava was then ligated with polypropylene sutures and a 30-gauge needle and then removed to avoid endothelial detachment. The peritoneum and skin were sutured continuously, and the thrombus was removed 48 hours later to measure length and body weight.

[0146] Tail bleeding assay:

[0147] Isoflurane-anesthetized mice were placed on a heating plate to maintain body temperature, a 5 mm cut was made from the tip of the tail to cause a coherent injury, and the tail was immediately immersed in 15 mL of 37 ° C saline, and the bleeding time was recorded until the bleeding stopped or until 10 minutes. Hemostasis > 1 min was considered to be the end of bleeding, and the tail was removed in time. The hemoglobin content was measured using a spectrophotometer to indicate the amount of blood loss of the mouse. After removing the supernatant, 900 μL of water was added to resuspend and lyse the red blood cells. After 17 s, 100 μL of PBS was added to restore the osmotic pressure, and the cells and debris were removed by centrifugation (10000 rpm, 5 min), and the supernatant containing hemoglobin was retained for 550 nm optical density detection (OD550).

[0148] 2. Confirmation of successful DSS modeling

[0149] Clinical and Macroscopic Analysis of Colitis in Mice:

[0150] Mice were monitored daily for clinical analysis of colitis, including body weight, diarrhea, and bleeding. Weight loss was calculated as the percentage difference relative to initial body weight. Mice were euthanized on day 7 after DSS administration. The colon was resected and opened longitudinally. Macroscopic lesions were measured by a blinded observer using the following scoring system: 0: normal; 1: congestion, edema, no ulcers; 2: congestion, edema, small linear ulcers or petechiae; 3: congestion, edema, wide ulcers, necrosis or adhesions; 4: congestion, edema, megacolon, stricture or perforation.

[0151] The weight loss, diarrhea, blood in stool and macroscopic scores of mice with DSS-induced colitis were shown in Figure 2. Figure 1-4 As shown. Figure 1 It can be seen that the weight of the mice in the experimental group decreased significantly compared with the mice in the control group. Figure 2 and Figure 3 It can be seen that the diarrhea and bloody stool scores of the experimental group mice were higher than those of the control group. Figure 4 It can be seen that the macroscopic scores of the mice in the experimental group were significantly increased compared with those in the control group. These results indicate that the DSS-induced colitis mouse model in the experimental group was successfully established.

[0152] 3. Experimental study confirms that IBD mice are more susceptible to thrombosis

[0153] The superior mesenteric artery, which has a diameter of approximately 100 μm, was used to investigate whether IBD mice are more susceptible to thrombosis. To this end, a rhodamine 6G solution was injected into the tail vein and leukocytes and platelets in the peripheral blood of mice were labeled. The mesenteric arterioles injured by FeCl3 were monitored by intravital microscopy, and the time to complete blood flow occlusion was recorded. In addition, thrombosis was observed using the inferior vena cava stenosis model, a widely used model for inducing deep vein thrombosis in mice, and thrombi were removed 48 h after surgery for measurement. The results are shown in Figure 2. Figures 5 to 11 shown.

[0154] from Figure 5 It can be seen that the vascular occlusion time of the experimental group mice was shorter than that of the control mice, and there was a significant difference between the two groups (P < 0.01). In addition, peripheral blood cell analysis was performed. From the test results ( Figure 6 and Figure 7 ) It can be seen that the neutrophil count of the experimental group mice increased significantly compared with the normal control group mice (P < 0.01), while there was no statistical difference in platelet count between the two groups (P > 0.05). Figure 8 It can be seen that compared with the control group, the thrombus weight of the mice in the test group increased significantly, and the two showed a significant difference (P < 0.01). Fig. 9It can be seen that compared with the control group mice, the thrombus length of the experimental group mice increased significantly, and there was a certain difference between the two (P < 0.05). In addition, the tail bleeding time and hemoglobin level of the experimental group mice were significantly lower than those of the normal group mice (P < 0.01) ( Fig.10 and Fig.11 ). In summary, it can be seen that the DSS-induced colitis mice in the experimental group are more likely to develop thrombosis than the healthy mice in the control group.

[0155] 4. Effects of DSS colitis on light / dye-induced thrombosis in the arterioles and venules of the cremaster muscle in mice Fig.12 and Fig.13 As shown, it can be seen that the time required for thrombosis to begin and the time to complete flow arrest are much longer in arterioles than in venules. Although there was no difference in the time to onset or flow arrest between venules of control mice and experimental mice, both variables were significantly reduced in the cremaster arterioles of experimental mice compared with control mice. Since DSS-induced enhanced extraintestinal thrombosis was not observed in mouse veins, all subsequent experiments focused on thrombosis studies in arterioles.

[0156] 5. Study on the effect of lutein formula on the changes of inflammatory factors

[0157] Proinflammatory cytokines are considered to be an important link between inflammation and the hypercoagulable, prothrombotic state observed in certain pathological conditions. Therefore, we examined the changes in several inflammatory factors (TNF-α / IL-6 / IL-1β) in the colon and skeletal muscle of mice with colitis and thrombotic symptoms. Figure 14-16 shown.

[0158] from Figure 14-16 The results showed that the changes of TNF-α, IL-1β and IL-6 in the colon of the experimental group of mice were more obvious than those in skeletal muscle. Therefore, the changes of inflammatory factors in the colon were selected in subsequent studies.

[0159] 6. The effect of lutein combination on thrombosis in cremaster arterioles was investigated. Fig.17 shown.

[0160] from Fig.17It can be seen that the lutein composition obtained in Examples 1-9 (Y1-Y9) has a certain effect on the start time and stop time of thrombosis, but the effect on the stop time is more obvious. In addition, it can be seen from the figure that the extraintestinal thrombosis stop time of the DSS model group mice is significantly lower than that of the control group mice, and there is a significant difference between the two (P < 0.01), indicating that the mice in the experimental group have obvious extraintestinal thrombosis. When the lutein composition obtained in Examples 1-9 was used to intervene in the treatment of colitis mice, the extraintestinal thrombosis phenomenon of colitis mice was alleviated to varying degrees. Fig.17 We can further find that after using the lutein composition obtained in Example 4 to intervene in mice with colitis and extraintestinal thrombosis, the improvement effect of extraintestinal thrombosis is most obvious. The time for extraintestinal thrombosis to stop flowing is significantly higher than that of the model group mice (DSS group), and there is a significant difference between the two (P < 0.01); secondly, the lutein composition obtained in Example 2 also has a good effect on improving extraintestinal thrombosis in mice. After intervention with the lutein composition obtained in Example 2, it can be seen that the time for extraintestinal thrombosis to stop flowing in mice with colitis is significantly higher than that of the model group mice (DSS group) (P < 0.01). Again, from Fig.17 It can be seen that after the intervention of the lutein composition obtained in Example 7 on the mice with colitis and extraintestinal thrombosis, its improvement effect on extraintestinal thrombosis is worse than that of Example 2 and Example 4, but it still shows a difference compared with the model group mice (DSS group) (P < 0.05), indicating that the lutein composition obtained in Example 7 also has a certain improvement effect on the symptoms of mice with colitis and extraintestinal thrombosis, and the lutein composition obtained in Example 5 also has a similar effect as Example 7. In addition, it can be found that the improvement effects of the remaining lutein prescriptions (Examples 1, 3, 6, 8, and 9) on extraintestinal thrombosis in colitis mice are almost the same, but the improvement effect is far less than that of Examples 2, 4, and 7, and there is no difference compared with the model group mice (DSS group), but Examples 3, 6, and 9 have a certain difference compared with the normal control group mice (P < 0.05). This change shows that the lutein composition obtained in Examples 3, 6, and 9 has little effect on the improvement of the symptoms of extraintestinal thrombosis in colitis mice. Therefore, in subsequent studies, it is not necessary to continue to study these three prescriptions.

[0161] 7. Effects of lutein formula on changes of three inflammatory factors in the colon of colitis mice

[0162] In order to further verify the conclusions and confirm the effect of the lutein compositions obtained in Examples 1, 3, 6 and 9 on improving inflammation and extraintestinal thrombosis in colitis mice, the 9 lutein formulations were further used to carry out experiments on the changes in inflammatory factors in colitis mice to further confirm whether the lutein formulation of the present invention regulates colitis-related thrombosis by affecting the changes in these three inflammatory factors in the colon. The results are as follows: Figure 18-20 shown.

[0163] 1) From Fig.18 It can be seen that after the lutein composition obtained in Examples 2, 4 and 7 was used to intervene in colitis mice, the level of the pro-inflammatory factor TNF-α in the mouse colon decreased significantly, showing a significant difference compared with the model group mice (DSS group) (P < 0.01). Of course, there were also certain differences between the mice in Examples 1, 5 and 8 and the model group (DSS group) (P < 0.05), but the intervention effect on colitis mice was still slightly inferior to that of Examples 2, 4 and 7. However, the inflammation improvement effect of Examples 3, 6 and 9 on colitis mice was relatively weak, and its effect on the inflammatory factor TNF-α was almost similar to that of the model group mice (DSS group), indicating that the intervention effect of Examples 3, 6 and 9 on inflammatory factors was not ideal.

[0164] 2) From Fig.19 It can be seen from the improvement effects of each formula on the proinflammatory factor IL-1β that the intervention effect of the lutein composition obtained in Examples 2, 4 and 7 is still the most ideal, which is significantly different from the model group (P < 0.01). The intervention effects of Examples 5 and 8 are second, which are somewhat different from the model group (P < 0.05). However, Examples 3, 6 and 9 have relatively poor improvement effects on the proinflammatory factor IL-1β in the colon of colitis mice, with an improvement effect of almost 0, and the change of IL-1β is similar to that of the model group. This also shows that the lutein composition obtained in Formula Examples 3, 6 and 9 has a very poor effect on improving inflammation in colitis mice.

[0165] 3) From Fig. 20 The intervention effects of each formula on the inflammatory factor IL-6 can be seen to have a similar trend to the intervention effects of the previous two inflammatory factors, that is, Example 2 and Example 4 have the most ideal improvement effects on IL-6, although their improvement effects are slightly inferior to those of the previous two factors. This may be because in the colon of colitis mice, the degree to which the inflammatory factor IL-6 participates in the inflammatory process or the corresponding signal pathway is weaker than that of the previous two inflammatory factors. Therefore, the degree of change in its detection process is weaker. However, even if the degree of IL-6 participation in inflammation is weaker, from Fig.13 It can still be seen from the results that the lutein compositions obtained in Examples 3, 6 and 9 have the worst intervention effects, and the effects after intervention are almost not improved compared with the model group, indicating that the prescription effects of these three groups are the worst.

[0166] 4) Therefore, through the changes in the three inflammatory factors, we further verified our conclusion that the lutein compositions obtained in Examples 3, 6 and 9 had the worst effect on improving colon inflammation and the accompanying extraintestinal thrombosis in colitis mice, and we will not explore these three compositions in the future.

[0167] 8. Effects of the three best prescriptions on mesenteric artery thrombosis

[0168] The three lutein compositions with better intervention effects (lutein compositions obtained in Examples 2, 4 and 7) were tested again to explore their effects on mesenteric artery thrombosis. The flow time of arterial thrombus, thrombus weight, thrombus length, number of neutrophils and tail bleeding time were tested respectively. The test results are as follows Figure 21 to Figure 25 .

[0169] 1) From Fig.21 It can be seen that: compared with the model group mice, the arterial thrombosis flow time of the DSS colitis mice showed a significant decrease (P < 0.01), while the arterial thrombosis flow time of the mice intervened by the three lutein formulas increased to varying degrees, among which Example 4 had the most ideal intervention effect. Compared with the model group mice (DSS group), the arterial thrombosis flow time after intervention showed a significant increase, and there was a significant difference between the two (P < 0.01). Of course, Example 2 also had a good intervention effect. Compared with the model group, the arterial thrombosis flow time after intervention recovered to a certain extent, and there was a certain difference between the two (P < 0.05). However, the improvement effect of Example 7 on the change of arterial thrombosis flow time after intervention was not obvious, and there was no difference compared with the model group.

[0170] 2) From Fig. 22 It can be seen that the number of neutrophils in DSS colitis mice was significantly higher than that in the normal group, but after intervention with the three lutein prescriptions, the number of neutrophils decreased to a certain extent compared with the model group mice (DSS group), but there was no difference, and the difference between the three prescriptions was not obvious. This may be because the effects of the three lutein prescriptions on the number of neutrophils were not very different. We can continue to study other indicators to distinguish the differences in the intervention effects of the three prescriptions.

[0171] 3) From Fig.23 It can be seen that compared with the model group mice, the thrombus weight was significantly reduced after the intervention of the lutein formula, among which the intervention effects of Example 2 and Example 4 were the most ideal, and there were significant differences between them compared with the model group mice (P < 0.01). Of course, the intervention effect of Example 7 was also good, and it showed a certain difference compared with the model group (P < 0.05), but compared with Example 2 and Example 4, its intervention effect was second.

[0172] 4) From Fig.24 It can be seen that in terms of the effect on thrombus length, after Example 4 intervened in colitis mice, the degree of reduction in thrombus length was significantly different from that of the model group (P < 0.01); the degree of reduction in thrombus length after intervention in Example 2 and Example 7 was also different from that in the model group (P < 0.05), but the improvement effect was not as good as that in Example 4.

[0173] 5) From Fig.25 It can be seen that Example 4 is still better in terms of the effect on blood flow time. The blood flow time of mice in the model group increased significantly, and there was a significant difference between the two groups (P < 0.01). After intervening in colitis mice, although the blood flow time of Example 2 and Example 7 increased compared with the model group, the difference was not significant (P < 0.05), indicating that the improvement effect of Example 2 and Example 7 on the symptoms of enteritis with thrombosis is still weaker than that of Example 4.

[0174] From the above-mentioned examples, it can be seen that no matter the changes in the start and stop time of thrombosis in the cremaster arterioles or the changes in the three inflammatory factors, a consistent pattern can be seen, that is, three lutein compositions (Examples 2, 4 and 7) have ideal intervention effects on extraintestinal thrombosis and colon inflammation. Further comparison shows that among the three better prescriptions (Examples 2, 4 and 7), the improvement effects of Examples 2 and 4 on intestinal inflammation and thrombosis changes are more significant than those of Example 7. Therefore, the effects of the two best prescriptions (Examples 2 and 4) on intestinal inflammation and corresponding thrombosis are further studied.

[0175] 9. The experiment on the effect of the lutein composition obtained in Examples 2 and 4 on arterial thrombosis and changes in inflammatory factors was carried out. The results are as follows Figure 26-30 shown.

[0176] (1) From Fig.26 It can be seen that the pro-inflammatory factor TNF-α in the colon of DSS colitis mice was significantly increased compared with the normal group mice (P < 0.01), and after the intervention of Example 4, TNF-α was significantly reduced (P < 0.01); the pro-inflammatory factor TNF-α in the colitis mice intervened by Example 2 also decreased to a certain extent, which was different from the model group (P < 0.05), but the improvement effect was not as obvious as that of Example 4.

[0177] (2) From Fig. 27 It can be seen that Examples 2 and 4 have comparable intervention effects on the inflammatory factor IL-1β in colitis mice, and the effects of the two groups after intervention are significantly different from those of the model group of colitis mice (P < 0.01), indicating that Examples 2 and 4 are equivalent in improving the inflammatory factor IL-1β.

[0178] (3) From Fig.28It can be seen that for the intervention effect of IL-6, compared with the model group, Example 2 showed some improvement but no difference (ns), while after the intervention of Example 4, compared with the colitis mice in the model group, the inflammatory factor IL-6 decreased to a certain extent, but the difference between the two groups was not obvious (P < 0.05). This may be because the change of IL-6, an inflammatory factor, in the symptoms of colitis with thrombosis is not obvious, that is, IL-6 may not be a more obvious marker in the relevant signal system of changes in symptoms of colitis with thrombosis, resulting in the response of Examples 2 and 4 to this inflammatory factor being relatively weak compared with other inflammatory factors (TNF-α, Il-β).

[0179] (4) From Fig.29 It can be seen that in terms of the effect on thrombus length, the thrombus length of colitis mice after intervention in Example 4 was significantly reduced, which was significantly different from the model group (P < 0.01); the thrombus length after intervention in Example 2 was reduced to a certain extent compared with the model group, but the difference was not significant (P < 0.05).

[0180] (5) From Fig.30 It can be seen that for the effect on the onset time of thrombosis, the effect of each formula after intervention did not change significantly, which is consistent with the results of the previous study, because the lutein formula needs a certain time to intervene in inflammation and thrombosis, and the onset time of thrombosis may be shorter, and the response change is not obvious; and for the effect on the cessation time of thrombosis, both formulas showed relatively ideal intervention effects, but it can be seen that the intervention effect of Example 4 is still the most ideal, and its effect after intervention is significantly different from that of the model group (P < 0.01), while the effect of Example 2 after intervention is different from that of the model group but not significant (P < 0.05).

[0181] From the above, it can be seen that the lutein composition obtained by the present invention can have a good intervention and improvement effect on colitis with extraintestinal thrombosis. Animal experiments have confirmed that Example 4 has the most ideal intervention effect on colitis with extraintestinal thrombosis, followed by Example 2. The present invention can play a good role in improving and restoring the symptoms of colitis with extraintestinal thrombosis by improving the changes in inflammatory factors TNF-α, Il-1β, IL-6 and the changes in the number of neutrophils related to the inflammation-coagulation pathway and the length and weight of thrombus.

[0182] 10. Preliminary study of pharmacological mechanism

[0183] Through the above experiments, it has been confirmed that Examples 2 and 4 are the two most ideal formulas for intervening in the symptoms of colitis with extraintestinal thrombosis. The possible mechanisms of action of these two lutein formulas in improving the symptoms of colitis with extraintestinal thrombosis were further studied. Two key cytokines of the intestinal inflammation-related microvascular system-thrombomodulin (TM) and endothelial PC receptors were further detected by flow cytometry to study whether the lutein composition can improve colitis with extraintestinal thrombosis through PC meridians.

[0184] Experimental steps:

[0185] To determine the expression of TM and EPCR in HIMECs, endothelial cells were seeded at a density of 5×10 on fibronectin-coated wells of 24-well cluster plates (4 cells / ml per well). Confluent monolayers were cultured in culture medium for 24 hours. At the end of the culture period, HIMECs were washed five times in cold PBS, and single-cell suspensions were obtained using separation buffer (PBS; 20 mM HEPES, pH 7.4; 10 mM EDTA; and 0.5% BSA), followed by vigorous pipetting. After additional washing, HIMECs were incubated with primary anti-TM or anti-EPCR; the above HIMEC stimulation did not alter cell viability as assessed by trypan blue staining (data not shown). Samples were analyzed by quantitative flow cytometry using a Coulter Epics XL flow cytometer (Beckman Coulter). The experimental results are shown in Figure 31-34 :

[0186] from Figure 31-34 It can be seen that the lutein composition obtained by the present invention will affect the expression levels of EPCR and TM in microvascular endothelial cells, especially the mRNA level. Regardless of the EPCR expression level or the TM expression level, after intervention with the lutein composition, the expression levels of the mice with colitis (DSS group) increased significantly, and there were differences between the groups (P < 0.01, P < 0.05). Because EPCR and TM are two important protein molecules in the PC signaling pathway, it is predicted that the lutein composition obtained by the present invention can achieve an ideal intervention effect of improving colitis with extraintestinal thrombosis through PC dredging.

[0187] The present invention has only conducted a preliminary and superficial exploration of the mechanism of the lutein composition in intervening in the symptoms of colitis with extraintestinal thrombosis, and more animal experiments and clinical trials are needed for further verification.

[0188] The present invention obtains natural lutein by low-temperature extraction of marigold flowers under specific temperature, pressure and solvent conditions, and further obtains multiple lutein prescriptions. Animal experimental studies have confirmed that the lutein compositions obtained in Examples 2 and 4 of the present invention have a relatively ideal improvement effect on the symptoms of colitis with extraintestinal thrombosis, and their mechanism of action may involve one or more of the inflammation-coagulation system-related signal pathways, which can regulate the expression levels of inflammatory factors and related proteins or receptors on the related signal pathways, and can provide reference significance for the future clinical development of drugs for the treatment of diseases such as colitis and thrombosis or atherosclerosis. In addition, the lutein in the lutein prescription of the present invention is extracted from marigold flower particles, the raw materials are natural, and the other raw material components are also medicinal and edible components, without side effects, and can prevent and improve inflammatory bowel disease and prevent and improve symptoms such as thrombosis, especially for middle-aged and elderly people who are prone to both diseases at the same time, it is of great significance, and can greatly reduce the treatment pain of the middle-aged and elderly sick people, improve patient compliance, and thus improve the quality of life of the middle-aged and elderly people.

[0189] The above are only preferred specific implementations of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present application should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. A method for preparing lutein, characterized in that: The following steps are involved: The marigold flower particles are extracted with propane and butane as extraction solvents, and then the obtained extract is evaporated twice and then distilled and concentrated, and then the obtained extract is centrifuged after removing the solvent to obtain the lutein.

2. The method for preparing lutein according to claim 1, characterized in that: In the extraction solvent, the volume ratio of propane to butane is 1:(1.1-1.4).

3. The method for preparing lutein according to claim 1, characterized in that: The mass ratio of the extraction solvent to the marigold flower particles is 1:(2-3).

4. The method for preparing lutein according to claim 1, characterized in that: The extraction temperature is 40° C., the pressure is 0.8-0.9 MPa, and the extraction time is 45-60 min.

5. The method for preparing lutein according to claim 1, characterized in that: The evaporation temperature is 36°C ± 2°C and the pressure is 0.04-0.05MPa; The temperature of the distillation concentration is 60° C. and the pressure is 0.04-0.05 MPa.

6. Lutein prepared by the method for preparing lutein according to any one of claims 1 to 5.

7. Use of the lutein according to claim 6 in the preparation of a drug for preventing and improving IBD and its thrombotic complications.

8. A lutein composition, characterized in that: The raw materials include the following mass fractions: The lutein 8-13.5%, zeaxanthin 5-12%, curcumin 8-16%, quercetin 3-21%, fucoidan 10-13%, honeysuckle extract 6-11.3%, casein 0.8-1.5%, beeswax 0.4-1.2%, soybean lecithin 0.4-1%, soybean oil 5.5-24.2%, glycerol 8-19% and hydroxypropyl methylcellulose 5-7.5% as described in claim 6.

9. A lutein composition according to claim 8, characterized in that: The dosage form of the lutein composition includes one or more of powder, emulsion, capsule and soft candy dosage forms.

10. Use of a lutein composition as claimed in claim 8 or 9 in the preparation of a drug for preventing and improving IBD and its thrombotic complications.

Images