Method for integrally extracting active ingredients in seedless roxburgh rose pomace and application

Abstract

The invention discloses a method for integrally extracting active ingredients from seedless roxburgh rose (rosa sterilis) pomace and application. According to the method, the contents of multiple water-soluble and fat-soluble components of the seedless roxburgh rose fruit, juice and pomace are measured, the proportion of each component in the fruit is analyzed, and the availability of the seedless roxburgh rose pomace resource is discussed. Further experiments find that fat-soluble components in the seedless roxburgh rose residues can be extracted by a supercritical CO2 extraction technology, and water-soluble components in the seedless roxburgh rose residues can be extracted by a low-temperature high-pressure crushing extraction technology. The formed fat-soluble extract and water-soluble extract are proved to have a variety of biological activities and uses. The two extraction technologies are combined for extraction, and comprehensive utilization of the two components in the seedless roxburgh rose residues can be achieved. According to the method, processing and utilization of the seedless roxburgh rose can be achieved, and reference is provided for quality evaluation and comprehensive development and utilization of the seedless roxburgh rose.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine extraction and preparation and its applications, and particularly relates to a method for the overall extraction of active ingredients from the fruit residue of seedless Rosa roxburghii tratt (golden Rosa roxburghii tratt) and its applications. Background Art

[0002] The seedless Rosa roxburghii tratt is the fruit of Rosa sterilis S.D.Shi, a plant of the genus Rosa in the family Rosaceae, also known as golden Rosa roxburghii tratt. It is a unique species in Guizhou, China, and is distributed in places such as Anshun, Guizhou. It mostly grows at an altitude of 1500 meters, can grow in areas such as rocky desertification areas, has relatively strong adaptability, is relatively light-loving, and the root growth is facilitated when the ground temperature is relatively high. In recent years, the area of artificial cultivation has been expanding year by year. However, the current research on the development and utilization of seedless Rosa roxburghii tratt is not yet mature.

[0003] With the continuous development of the seedless Rosa roxburghii tratt industry, its quality problems have gradually attracted attention. However, the quality evaluation system for this specific variety is not yet perfect. At present, the relevant research mainly focuses on water-soluble components such as vitamin C, polysaccharides, polyphenols, and flavonoids, and the research on the fat-soluble components in seedless Rosa roxburghii tratt is relatively scarce. Moreover, there is no systematic quality evaluation research based on multiple water-soluble and fat-soluble components and their source parts.

[0004] In industrial production, the utilization of seedless Rosa roxburghii tratt is mainly to obtain seedless Rosa roxburghii tratt juice through physical pressing for use in the production of food and health products. However, the seedless Rosa roxburghii tratt residue, which accounts for 60% - 70% of the dry fruit weight obtained during pressing, may retain a large number of active ingredients in seedless Rosa roxburghii tratt but has not been well utilized, resulting in waste of resources and environmental pollution. It is reported that the extraction of active ingredients from seedless Rosa roxburghii tratt residue mainly uses methods such as hot water extraction, ultrasonic-assisted extraction, and supercritical fluid extraction, and mostly focuses on the extraction of a single component such as total polyphenols or total flavonoids. There is still a lack of in-depth and systematic research on the comprehensive utilization of the overall extract and the synergistic effect between multiple components. At present, the extraction of polyphenol and flavonoid components from the fruit residue often uses methanol or ethanol as solvents, but the use of organic solvents may bring residue problems and affect safety; according to the literature and preliminary experiments, the seedless Rosa roxburghii tratt residue contains various unstable active ingredients such as total flavonoids, total polyphenols, and total proanthocyanidins, which are easily degraded under the influence of factors such as air, light, pH, and metal ions. Traditional high-temperature extraction methods are prone to cause changes in components and a decrease in content.

[0005] Alcoholic Liver Injury (ALI) is a relatively common liver disease, and its main inducing factor is frequent excessive drinking. If not controlled, alcoholic liver disease may gradually progress to liver fibrosis, cirrhosis, and even further develop into liver cancer. The pathogenesis of alcoholic liver injury is complex, involving multiple aspects such as the body's alcohol metabolism, hepatic steatosis, oxidative stress, inflammatory response, immune abnormalities, and gut microbiota dysbiosis. Therefore, it is of innovative pharmacological value to carry out systematic research on the potential protective effect of seedless Rosa roxburghii tratt on liver injury. Summary of the Invention

[0006] An object of the present invention is to overcome the deficiencies of the prior art and provide a method for the overall analysis of active ingredients in seedless Rosa roxburghii tratt.

[0007] Another object of the present invention is a method for the overall extraction of active ingredients from the pomace of seedless Rosa roxburghii tratt.

[0008] Another object of the present invention is the application of the above method.

[0009] The object of the present invention is achieved by the following technical solutions:

[0010] A method for the overall analysis of active ingredients in seedless Rosa roxburghii tratt, including the detection of water-soluble components and the detection of fat-soluble components; wherein: the detection of water-soluble components includes the following steps:

[0011] (1) Preparation of the test solution: Accurately weigh the freeze-dried powder of seedless Rosa roxburghii tratt fruit, juice or pomace, add 70% methanol solution, extract by ultrasonic, centrifuge after cooling, retain the supernatant, add 70% methanol solution to the residue for extraction again, centrifuge, combine the supernatants, and make up the volume with 70% methanol solution to obtain the test solution;

[0012] (2) Detection of water-soluble components:

[0013] The contents of total polyphenols, total flavonoids, total proanthocyanidins, and total polysaccharides in the test solution are determined by the UV method - standard curve method; the content of VC in the test solution is determined by the HPLC method - standard curve method, wherein the chromatographic column is an Agilent C18 column; the mobile phase is a 0.1% (volume ratio) phosphoric acid solution: methanol isocratic elution; the flow rate is 1.0 mL / min; the detection wavelength is 242 nm; the column temperature is 30 °C; the injection volume is 10 μL;

[0014] The linear relationships of total polyphenols, total flavonoids, total proanthocyanidins, total polysaccharides, and VC are as follows: y = 4.9503x + 0.0174, R 2 = 0.9990; y = 1.0757x + 0.0007, R 2= 0.9997; y = 1.5120x - 0.0238, R 2 = 0.9997; y = 11.2500x - 0.0454, R 2 = 0.9991; y = 30852x + 173588R 2 = 0.9996;

[0015] The detection of fat-soluble components includes the following steps:

[0016] (1) Preparation of the test solution: Weigh the freeze-dried powder of seedless Rosa roxburghii fruit, juice or residue separately, add a methanol:dichloromethane solution with a volume ratio of 1:1, extract by ultrasonic treatment, centrifuge after cooling, retain the supernatant, and make up the volume to obtain the test solution with a methanol:dichloromethane solution with a volume ratio of 1:1;

[0017] (2) Detection of fat-soluble components:

[0018] The contents of lutein, zeaxanthin, β-carotene, DL-α-tocopherol, β-sitosterol, and squalene in the test solution were determined by HPLC-standard curve method. Among them, the chromatographic conditions for lutein, zeaxanthin, and β-carotene were as follows: Use the chromatographic column Cosmosil PBr; mobile phase A: methanol, mobile phase B: methyl tert-butyl ether, and perform gradient elution: 0 - 30 min, 4% B; 30 - 35 min, 4% - 7% B; 35 - 45 min, 7% B; 45 - 50 min, 7% - 15% B; 50 - 65 min, 15% - 24% B; 65 - 75 min, 24% - 24% B; 75 - 85 min, 24% - 80% B; 85 - 90 min, 80%; flow rate: 1.0 mL / min; detection wavelength 450 nm; column temperature: 25 °C; injection volume 20 μL; The chromatographic conditions for DL-α-tocopherol, β-sitosterol, and squalene were as follows: Chromatographic column: Hypersil ODS C18; isocratic elution with 100% methanol for 60 min; flow rate: 0.7 mL / min; detection wavelength 205 nm; column temperature: 30 °C; injection 20 μL;

[0019] The linear relationships of lutein, zeaxanthin, β-carotene, DL-α-tocopherol, β-sitosterol, and squalene are as follows: Y = 99618X + 31248, R 2 = 0.9997; Y = 168910X + 13839, R 2 = 0.9997; Y = 148575X + 64098, R 2 = 0.9999; Y = 90502X + 58590, R 2 = 0.9993; Y = 10934X - 2259, R 2= 0.9998; Y = 106223X - 10991R 2 = 0.9992.

[0020] A method for the overall extraction of active ingredients from seedless Rosa roxburghii Tratt pomace, comprising the following steps:

[0021] S1. Weigh fresh seedless Rosa roxburghii Tratt fruits, and obtain seedless Rosa roxburghii Tratt juice and the remaining pomace through pressing, centrifuging, and filtering;

[0022] S2. Take the remaining pomace from step S1, put it into a supercritical CO2 extraction kettle, and the extraction conditions are as follows: extraction time is 3 ± 0.5 h, the entrainer is 95% ethanol, the extraction pressure is 50 ± 10 MPa, the temperature is 42 ± 2 °C, the separation I pressure is 8 ± 1 MPa, the temperature is 60 ± 5 °C, the separation II pressure is 6 ± 1 MPa, the temperature is 45 ± 5 °C, and the CO2 flow rate is 25 ± 5 L·h -1 ; Collect the extract containing the entrainer in the separation kettle II, and concentrate and dry the solvent under reduced pressure to obtain the supercritical CO2 extract and the remaining residue;

[0023] S3. Take the residue remaining in step S2, add water, and stir evenly; use a low-temperature high-pressure crushing and extraction device to reduce the water circulation temperature of the device to 4 °C, pour the solvent into the feed port for drainage, then pour the evenly mixed residue solution into the feed port, extract at a pressure of 100 ± 10 mPa for 20 ± 5 min, centrifuge to remove the precipitate, and take the supernatant for freeze-drying to obtain the freeze-dried powder of the water-soluble extract.

[0024] Further, the extraction conditions in step S2 are as follows: extraction time is 3 h, the entrainer is 95% ethanol, the extraction pressure is 40 MPa, the temperature is 42 °C, the separation I pressure is 8 MPa, the temperature is 60 °C, the separation II pressure is 6 MPa, the temperature is 45 °C, and the CO2 flow rate is 25 L·h -1 .

[0025] Further, the extraction in step S2 is carried out at a pressure of 100 MPa for 20 min.

[0026] An extract of active ingredients from seedless Rosa roxburghii Tratt pomace, comprising the supercritical CO2 extract and / or the water-soluble extract obtained by the above method.

[0027] The application of the above method for the overall extraction of active ingredients from seedless Rosa roxburghii Tratt pomace includes at least one of the following applications:

[0028] (1) Application in the preparation of products for treating liver injury;

[0029] (2) Application in the preparation of products with anti-inflammatory effects;

[0030] (3) Application in the preparation of skin whitening and freckle removing products.

[0031] Furthermore, the product described in (1) is a drug for treating liver injury or a food with an auxiliary protective function against chemical liver injury.

[0032] Furthermore, the product described in (2) is a drug with an anti-inflammatory effect.

[0033] Furthermore, the product described in (3) is a whitening and freckle-removing cosmetic additive.

[0034] Furthermore, Application (1) is the application of the seedless Rosa roxburghii Tratt juice, supercritical CO2 extract and / or water-soluble extract obtained by the method described above in the preparation of a drug for treating liver injury or a food with an auxiliary protective function against chemical liver injury.

[0035] Furthermore, Application (2) is the application of the supercritical CO2 extract obtained by the method described above in the preparation of an anti-inflammatory drug.

[0036] Furthermore, Application (3) is the application of the water-soluble extract obtained by the method described above in the preparation of a whitening and freckle-removing product.

[0037] The present invention has the following advantages and effects compared with the prior art:

[0038] The present invention determines the contents of water-soluble and fat-soluble components in seedless Rosa roxburghii Tratt fruits, juices and residues, and analyzes the proportion of each component in the fruits, so as to explore the availability of seedless Rosa roxburghii Tratt fruit residue resources.

[0039] Further experiments found that supercritical CO2 extraction technology can be used to extract fat-soluble components from seedless Rosa roxburghii Tratt fruit residue, and low-temperature high-pressure crushing extraction technology can be used to extract water-soluble components from seedless Rosa roxburghii Tratt fruit residue. By combining the two extraction technologies, the comprehensive utilization of the two components in seedless Rosa roxburghii Tratt fruit residue can be realized. The present invention can realize the processing and utilization of seedless Rosa roxburghii Tratt, and provide a reference for the quality evaluation and comprehensive development and utilization of seedless Rosa roxburghii Tratt.

[0040] On this basis, through in vitro and in vivo activity analysis, the protective effect of seedless Rosa roxburghii Tratt fruit residue extract on liver injury and the possible mechanism are further studied. Description of the Drawings

[0041] Figure 1 It is a percentage diagram of the contents of various water-soluble components in seedless Rosa roxburghii Tratt juice and fruit residue in the corresponding components of dried seedless Rosa roxburghii Tratt fruits;

[0042] Figure 2 It is a percentage diagram of the contents of various fat-soluble components in seedless Rosa roxburghii Tratt juice and fruit residue in the corresponding components of dried seedless Rosa roxburghii Tratt fruits;

[0043] Figure 3It is the research result diagram of the effect of seedless Rosa roxburghii Tratt juice on H2O2-induced LO2 cell damage; compared with the normal group, P < 0.001; compared with the model group, ***P < 0.001;

[0044] Figure 4 It is the result diagram of the determination of the anti-inflammatory activity of the supercritical CO2 extract of seedless Rosa roxburghii Tratt pomace;

[0045] Figure 5 It is the research result diagram of the effect of RSPLTE on H2O2-induced LO2 cell damage; compared with the normal group, P < 0.001; compared with the model group, ***P < 0.001;

[0046] Figure 6 It is the result diagram of the determination of the effects of RSPLTE on GSH, CAT, SOD and MDA in alcohol-induced liver injury mice, compared with the normal group ### P < 0.001; compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001;

[0047] Figure 7 It is the effect of RSPLTE on IL-6, IL-1β and TNF-α in the liver of alcohol-induced liver injury mice, compared with the normal group # P < 0.05, ## P < 0.01; compared with the model group, *P < 0.05, **P < 0.01;

[0048] Figure 8 It is the research result diagram of the effect of the water-soluble components in the residue extracted by supercritical fluid from seedless Rosa roxburghii Tratt pomace on tyrosinase activity. Specific embodiments

[0049] The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0050] The instruments and materials involved in the following embodiments:

[0051] Instruments: UV-2600 ultraviolet-visible spectrophotometer (Shimadzu Corporation (China)); LC-20AT high performance liquid chromatograph (Shimadzu Corporation (China)); ME204 electronic analytical balance (Mettler Toledo International Co., Ltd.); JP-060S ultrasonic cleaner (Shenzhen Jiemeng Cleaning Equipment Co., Ltd.); XD-2000A rotary evaporator (Shanghai Xiande Experimental Instrument Co., Ltd.); Bios afer-10A vacuum freeze dryer (Saifei (China) Co., Ltd.); YB-150 high-speed multi-functional grinder (Yongkang Sufen Industry and Trade Co., Ltd.); water bath (Feisifu Instruments (Hebei) Co., Ltd.).

[0052] Materials: Seedless Rosa roxburghii Tratt fresh fruits, fruit juice, and fruit pomace (industrially pressed) are products of the seedless Rosa roxburghii Tratt (Golden Rosa roxburghii Tratt) series from Guizhou Anshun, provided by Guizhou Debao Agricultural Science and Technology Co., Ltd. The fresh fruits, fruit juice, and fruit pomace are sealed in fresh-keeping foam boxes and transported back to the laboratory in a 4°C in-vehicle refrigerator. The fresh fruits, fruit juice, and fruit pomace are freeze-dried in a vacuum freeze-dryer, pulverized, and passed through an 80-mesh sieve to obtain freeze-dried powder samples of seedless Rosa roxburghii Tratt fruits, seedless Rosa roxburghii Tratt juice, and seedless Rosa roxburghii Tratt fruit pomace for content determination and ratio analysis; the fruit pomace samples stored in a low-temperature refrigerator for 8 - 9 months are freeze-dried in a vacuum freeze-dryer, pulverized, and passed through an 80-mesh sieve to obtain samples for the preparation and content determination of seedless Rosa roxburghii Tratt fruit pomace extract.

[0053] Gallic acid reference substance (batch number C10067994, purity ≥99%), procyanidin reference substance (batch number C13135785, purity ≥95%), DL-α-tocopherol reference substance (batch number C10124841, purity 96%), Shanghai Macklin Biochemical Co., Ltd.; D-anhydrous glucose (batch number CS5047, purity ≥99.8%), Jinclon (Beijing) Biotechnology Co., Ltd.; Rutin reference substance (batch number D13HB202516, purity ≥98%), β-sitosterol reference substance (batch number M021B205118, purity ≥98%), Shanghai Yuanye Bio-Technology Co., Ltd.; VC (batch number H1723006, purity ≥99%), squalene reference substance (batch number E1731028, purity 98%), Shanghai Aladdin Biochemical Technology Co., Ltd.; Zeaxanthin reference substance (batch number WP23102414, purity ≥98%), β-carotene reference substance (batch number WP23090710, purity ≥98%), Sichuan Weikeqi Biological Technology Co., Ltd.; Lutein reference substance (batch number 24040370, purity 97.6%), Tianmo Quality Inspection Technology Co., Ltd.; Sodium nitrite (batch number L1904014), Sodium hydroxide (batch number G2031120), Shanghai Aladdin Biochemical Technology Co., Ltd.; Aluminum nitrate (batch number C10094520, Shanghai Macklin Biochemical Co., Ltd.; Folin-Ciocalteu reagent (batch number J24GS142729), Shanghai Yuanye Biotechnology Co., Ltd.; Methyl tert-butyl ether and methanol are of chromatographic purity, Shanghai Macklin Biochemical Co., Ltd.; Absolute ethanol, dichloromethane, and n-butanol are of analytical purity, Tianjin Zhiyuan Chemical Reagent Co., Ltd.; Ammonium ferric sulfate (batch number 24240414001), Solarbio Science & Technology Co., Ltd.; Water is secondary water.

[0054] Example 1: Establish an analytical method for determining the contents of water-soluble components and fat-soluble components in seedless Rosa roxburghii Tratt

[0055] 1. Detection of water-soluble components

[0056] Preparation of test solution: Accurately weigh 0.5 g of freeze-dried powder of seedless Rosa roxburghii fruit, juice, and residue respectively and put them into 50 mL centrifuge tubes. Add 30 mL of 70% methanol solution, extract by ultrasonic wave (power 180 W, frequency 40 kHz) for 30 min, cool and centrifuge at 5000 r / min for 15 min. Filter the supernatant into a 50 mL volumetric flask; add 10 mL of 70% methanol solution to the residue and extract again for 30 min, centrifuge at 5000 r / min for 15 min; combine the supernatants and make up the volume to the mark with 70% methanol solution to obtain the test solution.

[0057] (1) Detection of total polyphenols: Precisely pipette 0.5 mL of gallic acid reference solution with a concentration range of 0.025 - 0.175 mg / mL into 10 mL graduated test tubes respectively. Add 2.5 mL of Folin-Ciocalteu reagent diluted tenfold, react for 5 min, then add 2.0 mL of 10% Na2CO3 solution, make up the volume to the mark with water, mix well, react in the dark at room temperature for 1 h, and measure the absorbance at a wavelength of 735 nm by ultraviolet-visible spectrophotometry. Conduct parallel tests three times. Use the concentration of the gallic acid reference solution as the abscissa and the absorbance as the ordinate to plot the standard curve, and obtain the regression equation as y = 4.9503x + 0.0174, R 2 = 0.9990. Accurately pipette 0.5 mL of the test solution and dilute it by a certain multiple. Follow the above steps to calculate the content of total polyphenols in the sample.

[0058] (2) Detection of total flavonoids: Precisely pipette 1.0 mL of rutin reference solution with a concentration range of 0.100 - 0.700 mg / mL into a 10 mL graduated test tube, add 0.3 mL of 5% sodium nitrite solution, shake well, let stand for 6 min, then add 0.3 mL of 10% aluminum nitrate solution, shake well, let stand for 6 min, and finally add 4.0 mL of 4% sodium hydroxide solution, dilute to the mark with 70% ethanol, let stand for 20 min, measure the absorbance at a wavelength of 499 nm by ultraviolet-visible spectrophotometry. Conduct parallel tests three times. Use the concentration of the rutin reference solution as the abscissa and the absorbance as the ordinate to plot the standard curve, and obtain the regression equation as y = 1.0757x + 0.0007, R 2 = 0.9997. Accurately pipette 1.0 mL of the test solution and dilute it by a certain multiple. Follow the above steps to calculate the content of total flavonoids in the sample.

[0059] (3) Detection of total proanthocyanidins: Precisely pipette 1.0 mL of the proanthocyanidin reference solution with a concentration range of 0.20 - 0.60 mg / mL into a 10 mL volumetric flask, add 6 mL of n-butanol - hydrochloric acid solution (95:5) and 0.2 mL of ammonium ferric sulfate solution (0.1 g of ammonium ferric sulfate dissolved in 2 mol / L hydrochloric acid, prepared freshly before use), shake well, heat in a boiling water bath for 40 min, take out, immediately cool in a cold water bath for 15 min, and measure the absorbance at a wavelength of 550 nm using ultraviolet - visible spectrophotometry. Conduct parallel tests three times. With the concentration of the proanthocyanidin reference solution as the abscissa and the absorbance as the ordinate, plot the standard curve, and obtain the regression equation as y = 1.5120x - 0.0238, R 2 = 0.9997. Accurately pipette 1.0 mL of the test solution, dilute it by a certain multiple, and follow the above steps to calculate the content of total proanthocyanidins in the sample.

[0060] (4) Detection of total polysaccharides: Precisely pipette 1.0 mL of the anhydrous glucose reference solution with a concentration range of 0.020 - 0.100 mg / mL into a 10 mL graduated test tube, add 1 mL of 5% phenol solution, shake well, add 5 mL of concentrated sulfuric acid solution in an ice - water bath, mix well, let it cool, then boil in a boiling water bath for 20 min, take out, immediately cool in an ice - water bath for 10 min, and take the corresponding reagent as the blank. Measure the absorbance at a wavelength of 486 nm using ultraviolet - visible spectrophotometry. Conduct parallel tests three times. With the concentration of the anhydrous glucose reference solution as the abscissa and the absorbance as the ordinate, plot the standard curve, and obtain the regression equation as y = 11.2500x - 0.0454, R 2 = 0.9991.

[0061] Accurately weigh 1.000 g of the freeze - dried powder of seedless Rosa roxburghii fruit, juice, and residue respectively, place them in a 100 mL round - bottom flask, precisely add 50 mL of water, reflux at 99 °C for 2 h, filter, retain the filter residue, and repeat the extraction 2 times. Combine the filtrates after the two filtrations in a 100 mL volumetric flask, add water to the scale line, and shake well. Then transfer 1.0 mL of the obtained solution to a 100 mL volumetric flask, add water to the scale line to make the volume constant, and shake well to obtain the sample extraction solution. Pipette 1.0 mL of the sample extraction solution, follow the above steps to calculate the content of total polysaccharides in the sample.

[0062] (5) Detection of VC: Accurately weigh 1.000 g of the freeze - dried powder of seedless Rosa roxburghii fruit, juice, and residue respectively into a 50 mL beaker, add 20 mL of 0.1% phosphoric acid aqueous solution, stir evenly, extract by ultrasonic wave (power 180 W, frequency 40 kHz) for 30 min, take the upper clear liquid into a 50 mL volumetric flask, add 0.1% phosphoric acid water to make the volume constant, and shake well. Dilute it by a certain multiple, filter through a 0.45 μm microporous filter membrane, and perform HPLC detection. Each sample is repeated 3 times.

[0063] Accurately weigh 20.00 mg of each VC reference substance, dissolve it with methanol and dilute it to 10.00 mL to prepare a series of reference substance solutions with concentrations of 0.001 - 1.000 mg / mL. Filter through a 0.45 μm microporous membrane and perform HPLC detection.

[0064] The chromatographic column is an Agilent C18 column (250 mm × 4.6 mm, 5 μm); the mobile phase is 0.1% phosphoric acid solution: methanol (2:98) for isocratic elution; the flow rate is 1.0 mL / min; the detection wavelength is 242 nm; the column temperature is 30 °C; the injection volume is 10 μL. Using the concentration of the VC reference substance solution as the abscissa and the peak area as the ordinate, plot the standard curve to obtain the regression equation y = 30852x + 173588R 2 = 0.9996, and calculate the content of VC in the sample.

[0065] 2. Detection of fat-soluble components

[0066] Preparation of reference substance solution: Accurately weigh appropriate amounts of lutein, zeaxanthin, and β-carotene reference substances, add methanol:dichloromethane (V:V = 1:1) to a 50 mL brown volumetric flask to prepare a mixed reference substance solution with mass concentrations of 0.244 μg / mL, 0.280 μg / mL, and 0.628 μg / mL respectively. After shaking well, filter through a 0.45 μm microporous membrane and set aside; accurately weigh appropriate amounts of DL-α-tocopherol, β-sitosterol, and squalene reference substances, add methanol:dichloromethane (V:V = 1:1) to a 50 mL brown volumetric flask to prepare a mixed reference substance solution with mass concentrations of 1.275 mg / mL, 1.033 mg / mL, and 1.025 mg / mL respectively. After shaking well, filter through a 0.45 μm microporous membrane and set aside.

[0067] Preparation of test solution: Accurately weigh 10.0 g of the freeze-dried powder of seedless Rosa roxburghii fruit, juice, and residue respectively, put them into 50 mL stoppered conical flasks, add 80 mL of methanol:dichloromethane (V:V = 1:1) solution, extract by ultrasound (power 180 W, frequency 40 kHz) for 30 min, cool and centrifuge at 5000 r / min for 15 min. Filter the supernatant into a 100 mL volumetric flask and dilute to volume with methanol:dichloromethane (V:V = 1:1) solution to obtain the test solution. Filter through a 0.45 μm microporous membrane and perform HPLC detection, with 3 replicates for each sample.

[0068] Chromatographic conditions: Chromatographic conditions for lutein, zeaxanthin, and β-carotene: Use a Cosmosil PBr column (250 mm × 4.6 mm, 5 μm); mobile phase A: methanol, mobile phase B: methyl tert-butyl ether, and perform gradient elution: 0 - 30 min, 4% B; 30 - 35 min, 4% - 7% B; 35 - 45 min, 7% B; 45 - 50 min, 7% - 15% B; 50 - 65 min, 15% - 24% B; 65 - 75 min, 24% - 24% B; 75 - 85 min, 24% - 80% B; 85 - 90 min, 80%; flow rate: 1.0 mL / min; detection wavelength 450 nm; column temperature: 25 °C; injection volume 20 μL. Prior to the investigation of the chromatographic conditions for lutein, zeaxanthin, and β-carotene, through the investigation of different chromatographic columns (Hypersil ODS C18, Cosmosil PBr, and Phenomenex Luna C18), it was found that the Cosmosil PBr chromatographic column achieved a better separation effect for the chromatographic peaks in the experimental samples, and the peak shapes of each component were better. At the same time, the wavelengths (450 nm, 500 nm) were investigated, and it was found that at 450 nm, the peak shapes and resolution of each component were better, the baseline was flat, and more component peaks in the sample could be detected. In addition, the mobile phases (methanol - isopropanol - methanol, methyl tert-butyl ether - methanol) were investigated, and the result showed that the methyl tert-butyl ether - methanol system was better. The separation effect of the chromatographic peaks in the experimental samples was relatively ideal, and the peak shapes were good.

[0069] Chromatographic conditions for DL-α-tocopherol, β-sitosterol, and squalene: Chromatographic column: Hypersil ODS C18 (250 mm × 4.6 mm, 5 μm); isocratic elution with 100% methanol for 60 min; flow rate: 0.7 mL / min; detection wavelength 205 nm; column temperature: 30 °C; injection 20 μL.

[0070] Investigation of linear relationship: Accurately pipette appropriate amounts of the mixed reference solution of lutein, zeaxanthin, and β-carotene and the reference solutions of DL-α-tocopherol, β-sitosterol, and squalene, dilute with methanol:dichloromethane (V:V = 1:1) to different mass concentrations, and determine according to the above chromatographic conditions. Using the mass concentration of each component as the abscissa (X) and the peak area as the ordinate (Y), draw a standard curve, and the results are shown in Table 1.

[0071] Table 1 Results of the investigation of the linear relationship, precision, repeatability, and stability of fat-soluble active components

[0072]

[0073] Investigation of linear relationship: Accurately pipette the above-mentioned mixed reference solution, and inject samples continuously for 6 times under the above chromatographic conditions. The RSD of the peak areas of the chromatographic peaks of 6 fat-soluble components is calculated to be 0.71% - 1.23%, indicating good precision of the instrument; accurately weigh the samples related to Rosa sterilis S. D. Shi, prepare the test solution according to the above method, inject and analyze under the above chromatographic conditions, and the RSD of the contents of 6 fat-soluble components in the test solution is calculated to be 1.89% - 2.98%, indicating good repeatability of this method; accurately weigh the samples related to Rosa sterilis S. D. Shi, prepare the test solution according to the above method, place it in the dark at room temperature for 0, 2, 4, 6, 8, 12, 24 h, inject and analyze under the above chromatographic conditions, and the RSD of the contents of 6 fat-soluble components in the test solution is calculated to be 1.25% - 1.93%, indicating good stability of the test solution within 24 h; accurately weigh 6 portions of the same batch of samples with known content, accurately add the mixed reference solution with known concentration, prepare the test solution according to the above method, inject and determine under the above chromatographic conditions, and the average recovery rates of 6 components are calculated to be 99.78% - 100.68%, and the RSD is 0.63% - 0.82%.

[0074] Example 2: Extraction, determination and analysis of active components in Rosa sterilis S. D. Shi

[0075] 1. Accurately weigh 500.0 g of fresh Rosa sterilis S. D. Shi fruits, press, centrifuge and filter them with a juicer to obtain Rosa sterilis S. D. Shi fruit juice and the remaining fruit residues. The Rosa sterilis S. D. Shi fruit juice and residues are freeze-dried under vacuum to obtain 24.05 g of freeze-dried Rosa sterilis S. D. Shi juice powder and 52.20 g of freeze-dried Rosa sterilis S. D. Shi residue powder. The relevant calculation formulas are as follows:

[0076] Taking total polyphenols as an example for the calculation formula, other components (including fat-soluble components) are calculated according to this:

[0077] Mass of total polyphenols in juice (residue) g = Mass of juice (residue) g × Total polyphenol content (mg / g) / 1000

[0078] Mass of total polyphenols in dried fruit g = Mass of total polyphenols in juice g + Mass of total polyphenols in residue g

[0079] Percentage of total polyphenols in juice (residue) in total polyphenols in dried fruit % = Mass of total polyphenols in juice (residue) g / Mass of total polyphenols in dried fruit g * 100%

[0080] Based on the method constructed in Example 1, determine the contents of water-soluble components in Rosa sterilis S. D. Shi fruits, juice and residues: total polyphenols, total flavonoids, total proanthocyanidins, total polysaccharides, and VC.

[0081] Based on the method constructed in Example 1, determine the contents of fat-soluble components in Rosa sterilis S. D. Shi fruits, juice, residues and residues: lutein, zeaxanthin, β-carotene, DL-α-tocopherol, β-sitosterol, and squalene. Specifically as follows:

[0082] The results are shown in Table 2 and Table 3. As can be seen from the table, the contents of total polyphenols, total flavonoids, total proanthocyanidins, total polysaccharides, and VC in the water-soluble components of the dried fruits of Rosa sterilis S. D. Shi are 100.35 mg / g, 111.31 mg / g, 85.32 mg / g, 466.01 mg / g, and 43.76 mg / g, respectively; the contents of the fat-soluble components β-carotene, DL-α-tocopherol, β-sitosterol, and squalene in the dried fruits are 13.62 mg / 100 g, 18.71 mg / 100 g, 69.02 mg / 100 g, and 1.80 mg / 100 g, respectively. Lutein and zeaxanthin were not detected. The contents of total polyphenols, total flavonoids, total proanthocyanidins, total polysaccharides, and VC in the water-soluble components of the dry powder of the pressed juice of Rosa sterilis S. D. Shi are 165.32 mg / g, 117.01 mg / g, 76.70 mg / g, 655.35 mg / g, and 67.20 mg / g, respectively; lutein, zeaxanthin, and squalene in the fat-soluble components of the pressed juice dry powder were not detected, and only a small amount of β-carotene, DL-α-tocopherol, and β-sitosterol were detected, and the contents of the latter three were 0.06 mg / 100 g, 0.71 mg / 100 g, and 4.65 mg / 100 g, respectively; the contents of total polyphenols, total flavonoids, total proanthocyanidins, and total polysaccharides in the water-soluble components of the fruit residue powder of Rosa sterilis S. D. Shi are 80.37 mg / g, 145.00 mg / g, 87.50 mg / g, and 277.16 mg / g, respectively, but VC was not detected; the contents of the fat-soluble components lutein, zeaxanthin, β-carotene, DL-α-tocopherol, β-sitosterol, and squalene in the fruit residue powder are 1.02 mg / 100 g, 0.15 mg / 100 g, 13.98 mg / 100 g, 35.70 mg / 100 g, 150.22 mg / 100 g, and 7.17 mg / 100 g, respectively.

[0083] From the above direct content data, it can be seen that in addition to rich water-soluble active components, the fruits of Rosa sterilis S. D. Shi also contain a variety of fat-soluble active components; the pressed fruit juice and fruit residue contain most of the water-soluble components. Among them, the contents of total polyphenols and total polysaccharides in the fruit juice are higher than those in the fruit residue, while the contents of total flavonoids and total proanthocyanidins in the fruit residue are higher than those in the fruit juice, and VC was not detected in the residue; the fat-soluble components mainly exist in the fruit residue and are less in the fruit juice. Among them, lutein, zeaxanthin, and squalene were only detected in the fruit residue, while the contents of β-carotene, DL-α-tocopherol, and β-sitosterol are all higher in the fruit residue than in the fruit juice.

[0084] Table 2 Determination results of water-soluble component contents in fruits, juices, and residues of Rosa sterilis S. D. Shi (n = 3)

[0085]

[0086] Note: All the samples to be tested are dried products, and ND indicates that the content is lower than the detection limit

[0087] Table 3 Determination results of the contents of fat-soluble active components in seedless Rosa roxburghii Tratt fruits, juices and residues (n = 3)

[0088]

[0089] Note: All the samples to be tested are dried products. ND indicates that the content is lower than the detection limit

[0090] 2. Proportion analysis of water-soluble and fat-soluble components in seedless Rosa roxburghii Tratt fruits, juices and residues

[0091] After the seedless Rosa roxburghii Tratt fruits are pressed, calculated according to the freeze-dried powder, the weight proportion analysis of the dry weights of its fruit juice and residue in the dried seedless Rosa roxburghii Tratt fruits is as follows: the dry powder of fruit juice accounts for 32% and the dry powder of fruit residue accounts for 68%. According to the proportion of the dry products of fruit juice and fruit residue and the aforementioned content results, the mass distribution of each water-soluble component in the fruit juice and fruit residue of seedless Rosa roxburghii Tratt is shown in Table 4, and the distribution of each water-soluble component in the fruit juice and fruit residue in the dried seedless Rosa roxburghii Tratt fruits can be further calculated. The results are as Figure 1 shown

[0092] The results show that after pressing and processing, VC mainly exists in the seedless Rosa roxburghii Tratt juice; 72.90% of the total flavonoids, 71.23% of the total proanthocyanidins, 51.34% of the total polyphenols, and 47.86% of the total polysaccharides still remain in the fruit residue

[0093] Table 4 Mass distribution of each water-soluble component in the seedless Rosa roxburghii Tratt samples

[0094]

[0095] Note: All the samples to be tested are dried products

[0096] Similarly, the mass distribution results of each fat-soluble component in the fruit juice and fruit residue of seedless Rosa roxburghii Tratt are shown in Table 5, and the distribution results of each fat-soluble component in the fruit juice and fruit residue are further calculated as Figure 2 shown

[0097] The results show that more than 97% of the fat-soluble active components in the seedless Rosa roxburghii Tratt fruits exist in the fruit residue of seedless Rosa roxburghii Tratt, while the proportion of fat-soluble active components in its fruit juice is very small

[0098] Table 5 Mass distribution of each fat-soluble component in the seedless Rosa roxburghii Tratt samples

[0099]

[0100] Note: All the samples to be tested are dried products

[0101] According to the research results, the seedless Rosa roxburghii Tratt pomace contains almost all the fat-soluble components and most of the water-soluble components in the seedless Rosa roxburghii Tratt. These components have good pharmacological activities and health-care functions, and directly discarding them is a huge waste of resources. Therefore, it is of great significance to efficiently and greenly extract and analyze the content of a large number of bioactive components remaining in the seedless Rosa roxburghii Tratt pomace for developing health products for preventing related diseases from waste and realizing the high-value comprehensive utilization of the seedless Rosa roxburghii Tratt.

[0102] Example 3: Overall extraction of active components from seedless Rosa roxburghii Tratt

[0103] 1. Preparation of seedless Rosa roxburghii Tratt juice

[0104] Accurately weigh 500.0 g of fresh seedless Rosa roxburghii Tratt fruits, press, centrifuge, and filter them with a juicer to obtain seedless Rosa roxburghii Tratt fruit juice and the remaining pomace. The seedless Rosa roxburghii Tratt fruit juice and pomace are freeze-dried under vacuum to obtain freeze-dried powder of seedless Rosa roxburghii Tratt juice and freeze-dried powder of seedless Rosa roxburghii Tratt pomace.

[0105] 2. Extraction of fat-soluble components from seedless Rosa roxburghii Tratt pomace

[0106] Supercritical CO2 extraction: Weigh an appropriate amount of seedless Rosa roxburghii Tratt pomace powder and put it into a supercritical CO2 extraction kettle. The extraction conditions are as follows: extraction time is 3 h, the entrainer is 95% ethanol, the extraction pressure is 40 MPa, the temperature is 42 °C, the separation I pressure is 8 MPa, the temperature is 60 °C, the separation II pressure is 6 MPa, the temperature is 45 °C, and the CO2 flow rate is 25 L·h -1 . Collect the extract containing the entrainer in the separation kettle II, concentrate and dry the solvent under reduced pressure to obtain the supercritical CO2 extract for standby.

[0107] Ethanol reflux extraction (traditional method): Accurately weigh the seedless Rosa roxburghii Tratt pomace powder, add absolute ethanol according to the solid-liquid ratio of 1:20 (g:mL), place it in a water bath at 80 °C and reflux for 1.5 h, filter, take the filtrate and concentrate and dry it under reduced pressure to obtain the ethanol reflux extract for standby.

[0108] Ultrasonic ethanol-assisted extraction (traditional method): Accurately weigh the seedless Rosa roxburghii Tratt pomace powder and place it in a 50 mL stoppered glass conical flask. Add absolute ethanol according to the solid-liquid ratio of 1:20 (g:mL), weigh it, ultrasonically treat it (power 180 W, frequency 40 kHz) for 30 min, cool, make up the lost mass with absolute ethanol, shake well, filter, take the filtrate and concentrate and dry it under reduced pressure to obtain the ultrasonic ethanol-assisted extract for standby.

[0109] Determine the contents of lutein, zeaxanthin, β-sitosterol, DL-α-tocopherol, β-sitosterol, and squalene in three different extracts, namely, the ethanol reflux extract, the ultrasonic ethanol-assisted extract, and the supercritical CO2 extract of the seedless Rosa roxburghii Tratt pomace. The results are shown in Table 6.

[0110] The results showed that among the extracts obtained by different extraction methods, except for zeaxanthin, there were significant differences in the contents of each component (P < 0.05). There was no significant difference in the lutein content between the ultrasonic ethanol-assisted extract and the ethanol reflux extract or the supercritical CO2 extract (P > 0.05). The contents of each component in the supercritical CO2 extract were significantly higher than those in the ultrasonic ethanol-assisted extract and the ethanol reflux extract.

[0111] Table 6 Yield and content of fat-soluble extracts from seedless Rosa roxburghii Tratt pomace (n = 3)

[0112]

[0113] Note: Different lowercase letters on the right shoulder of the same column indicate significant differences between the extracts (P < 0.05).

[0114] 3. Extraction of water-soluble components from the residue of supercritical extraction of seedless Rosa roxburghii Tratt pomace

[0115] Low-temperature high-pressure crushing water extraction: Accurately weigh the residue powder obtained by supercritical extraction and place it in a 250 mL stoppered glass conical flask. Add secondary water according to the solid-liquid ratio of 1:20 (g:mL), stir evenly, use a low-temperature high-pressure crushing extraction device to reduce the water circulation temperature of the device to 4 °C, pour 5 mL of solvent into the feed inlet for drainage, and then pour the evenly mixed seedless Rosa roxburghii Tratt pomace solution into the feed inlet in small amounts and multiple times. Extract at a pressure of 100 mPa for 20 min, centrifuge to remove the precipitate, and take the supernatant. The extract freeze-dried powder is obtained by vacuum freeze-drying method. Store it in a -20 °C refrigerator for later use.

[0116] Water stirring extraction (traditional method): Accurately weigh the residue powder obtained by supercritical extraction and place it in a 50 mL stoppered glass conical flask. Add secondary water according to the solid-liquid ratio of 1:20 (g:mL) and mix well. Add a magnetic stir bar to the conical flask and stir with a magnetic stirrer at 300 rpm and 25 °C for 60 min. Filter, take the filtrate, and then obtain the extract freeze-dried powder by vacuum freeze-drying method. Store it in a -20 °C refrigerator for later use.

[0117] Ultrasonic water-assisted extraction (traditional method): Accurately weigh the residue powder obtained by supercritical extraction and place it in a 50 mL stoppered glass conical flask. Add secondary water according to the solid-liquid ratio of 1:20 (g:mL) and mix well. Weigh and determine the mass, ultrasonically treat it (power 180 W, frequency 40 kHz) for 40 min, cool, make up the reduced mass with secondary water, shake well, filter, take the filtrate, and then obtain the extract freeze-dried powder by vacuum freeze-drying method. Store it in a -20 °C refrigerator for later use.

[0118] The contents of water-soluble components in three different extracts, namely the ethanol reflux extract, ultrasonic ethanol-assisted extract, and supercritical CO2 extract of the residue obtained from the supercritical extraction of seedless Rosa roxburghii Tratt pomace, were determined, and the results are shown in Table 7.

[0119] The results showed that the three extraction methods could all extract the total polyphenols, total flavonoids, total proanthocyanidins, and total polysaccharides, which are water-soluble components. There were significant differences in the contents of total polyphenols, total flavonoids, total proanthocyanidins, and total polysaccharides in the extracts obtained by different extractions. Among them, the contents of total polyphenols, total flavonoids, total proanthocyanidins, and total polysaccharides in the low-temperature high-pressure crushing water extract were all higher than those in the water stirring extract and ultrasonic water-assisted extract, reaching 68.5 mg / g, 77.25 mg / g, 47.74 mg / g, and 285.61 mg / g. It can be seen that the extraction, development, and utilization of the water-soluble components of seedless Rosa roxburghii Tratt pomace are suitable for using the low-temperature high-pressure crushing extraction technology.

[0120] Table 7 Yields and contents of water-soluble extracts from different seedless Rosa roxburghii Tratt pomaces (n = 3)

[0121]

[0122] Note: Different lowercase letters indicate significant differences among the extracts (P < 0.05).

[0123] In summary, the supercritical CO2 extraction technology can be selected to extract the fat-soluble components from seedless Rosa roxburghii Tratt pomace, and the low-temperature high-pressure crushing extraction technology can be selected to extract the water-soluble components from seedless Rosa roxburghii Tratt pomace. By combining the two extraction technologies, the comprehensive utilization of the two components in seedless Rosa roxburghii Tratt pomace can be realized. The present invention can realize the processing and utilization of seedless Rosa roxburghii Tratt, and provide a reference for the quality evaluation and comprehensive development and utilization of seedless Rosa roxburghii Tratt.

[0124] Example 4: Activity of seedless Rosa roxburghii Tratt extract

[0125] Cell culture: LO2 cells were cultured under the conditions of 37 °C and 5% CO2, using DMEM high-glucose medium containing 10% fetal bovine serum and 1% double antibody. When the cell density reached 75% - 80%, subculture operations were carried out, and the 4th to 10th generation cells were selected for subsequent experiments.

[0126] Establishment of H2O2-induced LO2 cell injury model: Preparation of H2O2 mother liquor: Take 57 μL of 3% H2O2 solution, add 4943 μL of distilled water, and dilute to 1 mol / L to obtain the H2O2 mother liquor. Filter the solution using a 0.22 μm filter membrane and transfer it to an EP tube for standby. Cell treatment and grouping: In a 96-well plate, LO2 cells were seeded at 5 × 10 3Inoculate at a density of cells per well, and replace the culture medium with fresh medium after 24 hours of culture. The cells are divided into two groups: a control group and an H2O2 treatment group (concentration 200 - 1000 μM). The control group uses 200 μL of serum-free DMEM medium, and the H2O2 treatment group adds 10 mM H2O2 solution at different concentrations. Each group has 6 replicate wells and is incubated at 37°C and 5% CO2 for 1 hour. After incubation, replace the medium and continue to culture for 24 hours. Finally, use a CCK-8 kit to detect the cell viability of each group.

[0127] (1) Activity of seedless Rosa roxburghii juice

[0128] 1) Effect of seedless Rosa roxburghii juice on the proliferation of LO2 cells

[0129] In a 96-well plate, inoculate logarithmic-phase LO2 cells at 5×10 3 cells per well, add complete medium, and culture at 37°C and 5% CO2 for 24 hours, then replace with fresh medium. The cells are divided into two groups: a normal group and a treatment group with seedless Rosa roxburghii juice solutions at different concentrations (18.75, 37.5, 75, 150, 300, 600 μg / mL). The working solutions of each concentration treatment group are prepared with complete medium. The normal group adds 100 μL of complete medium as a control, and the treatment groups add the medium containing the extract according to the concentration. Each experimental group has 3 replicate wells and continues to culture for 24 hours. Use the CCK-8 method to detect the cell viability of each group and evaluate the effect of the extract on the proliferation of LO2 cells. Cell viability = OD value of the test well / average OD value of the normal group.

[0130] 2) Effect of seedless Rosa roxburghii juice on the growth of H2O2-induced damaged LO2 cells

[0131] For the extract, in a 96-well plate, inoculate logarithmic-phase LO2 cells at 5×10 3 cells per well, add complete medium to each well, and culture in an incubator at 37°C and 5% CO2 for 24 hours, then discard the original medium. The cells are assigned to different groups: a normal group, a model group, a positive control group, and a drug treatment group. The positive drug is N-acetylcysteine (NAC), 2 mM, and the concentrations of the drug treatment group are 75, 150, 300 μg / mL. The normal group adds serum-free DMEM medium, and the model group and the treatment groups add 600 μM H2O2 solution. Each group has 6 replicate wells. After incubating for 1 hour, discard the medium, then the treatment groups are replaced with seedless Rosa roxburghii juice solutions at different concentrations, while the control group keeps the medium unchanged and continues to culture for 24 hours. After incubation, wash the cells twice with PBS buffer, add CCK-8 reagent and continue to incubate for 4 hours, and finally determine the cell viability by measuring the absorbance value. Cell viability = OD value of the test well / average OD value of the normal group.

[0132] 3) Effects of Seedless Rosa roxburghii Juice on Liver Function and Oxidation Indexes of H2O2-Induced Damaged L02 Cells

[0133] In a 6-well plate, 4×10 5 logarithmic phase LO2 cells were inoculated, and complete medium was added to each well. They were cultured for 24 hours in an environment of 37°C and 5% CO2. After the culture ended, the old medium was discarded. The cells were assigned to different groups: normal group, model group, positive control group, and drug treatment group. The positive drug was NAC at 2 mM, and the concentrations of the drug treatment group were 150, 300, and 600 μg / mL respectively. Each group had 3 replicate wells. The normal group was added with an equal amount of serum-free medium. The model group and each treatment group were added with 600 μM H2O2 solution according to the pre-experiment protocol. Each group was incubated for 1 hour at 37°C and 5% CO2, and then the medium was discarded. After that, the treatment group was changed to complete medium containing the extract, and the normal group and the model group were added with the same volume of complete medium and continued to be incubated for 24 hours. After the incubation ended, the cells were washed twice with PBS buffer, and indexes such as ALT, AST, MDA, SOD, and GSH were detected according to the kit instructions.

[0134] As Figure 3 shown, (a) in the figure is the effect of seedless Rosa roxburghii juice on the proliferation of normal LO2 cells. As shown in the figure, within the concentration range of 18.75 - 600 μg / mL of Rosa roxburghii juice, there was no obvious cytotoxicity to LO2 cells, indicating that within the experimental concentration range, each group did not show obvious cytotoxicity. Therefore, the subsequent experiments can be carried out within the above concentration range. (b) in the figure is the effect of seedless Rosa roxburghii juice on the proliferation of normal LO2 cells. As shown in the figure, the cell viability of the model group was significantly lower than that of the normal group (P < 0.0001), indicating that the liver injury model was successfully established. Compared with the model group, the cell viabilities of the 150, 300, and 600 μg / mL concentration groups of seedless Rosa roxburghii juice were all significantly increased (P < 0.001), indicating that seedless Rosa roxburghii juice has the effect of reducing the damage of H2O2-induced LO2 cells. (2) Activity of Supercritical CO2 Extracts from Seedless Rosa roxburghii Pomace

[0135] 1) Anti-inflammatory Activity of Supercritical CO2 Extracts from Seedless Rosa roxburghii Pomace

[0136] Experimental method: RAW264.7 cells were cultured in DMEM high-glucose medium containing 10% FBS (containing 1% penicillin and streptomycin) in an incubator at 37°C and 5% CO2. RAW264.7 cells were not passaged using trypsin. After discarding the old medium, 3 mL of fresh complete medium was added, and the cells at the bottom of the culture dish were gently blown into a cell suspension, and then they could be passaged at a ratio of 1:2 - 1:3.

[0137] RAW264.7 cells in the logarithmic growth phase were taken, counted, and made into a cell suspension. Then, 100 μL of the cell suspension was aspirated and inoculated into a 96-well cell culture plate at a density of 3×10 4 cells per well, and incubated in an incubator for 24 h until the cells adhered to the wall. The old culture medium was discarded, and 100 μL of the drug-containing culture medium was added according to the negative control group, LPS group, and drug group (drug + LPS). Three replicate wells were set in each group, and the cells were intervened with the drug for 24 h. After the intervention, 50 μL of the supernatant of the cells was aspirated into a new 96-well plate, and 50 μL of Griess I reagent and 50 μL of Griess II reagent were added to the test wells in sequence. After reacting in the dark for 5 min, the absorbance of each well at 540 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader. The NO release was calculated by substituting into the standard curve.

[0138] The measurement results are shown in Figure 4 (a) and (b). Compared with the negative control, the NO release in the LPS group increased significantly (p < 0.001), while the supercritical CO2 extract of the seedless Rosa roxburghii Tratt pomace had an inhibitory effect on NO release in LPS-induced RAW264.7 cells (p < 0.001 or p < 0.05), and the lowest effective concentration for anti-inflammatory was 37.5 μg / ml.

[0139] (3) Activity of water-soluble components (RSPLTE) in the residue extracted by supercritical CO2 from seedless Rosa roxburghii Tratt pomace

[0140] A. Effect of RSPLTE on H2O2-induced liver injury

[0141] Refer to the method in (1) to analyze the effects of RSPLTE on the proliferation of LO2 cells, the growth of L02 damaged cells induced by H2O2, and the liver function and oxidation indexes of L02 damaged cells induced by H2O2.

[0142] As shown in Figure 5 (a), in the range of 18.75 - 600 μg / mL of RSPLTE concentration, there was no obvious cytotoxicity to LO2 cells and it had a certain effect of promoting proliferation, but there was no significant difference compared with the normal group (P > 0.05). The concentrations of 75, 150, and 300 μg / mL were selected for preliminary screening in the subsequent pharmacodynamic experiments.

[0143] As shown in Figure 5 (b), the cell viability of the model group was significantly lower than that of the normal group (P < 0.001), indicating that the liver injury model was successfully established. Compared with the model group, the cell viability of the positive drug NAC group increased significantly (P < 0.001). Compared with the model group, the cell viabilities of the 75, 150, and 300 μg / mL concentration groups of RSPLTE all increased significantly (P < 0.001), indicating that RSPLTE had the effect of reducing the damage of LO2 cells induced by H2O2.

[0144] As Figure 5 shown in (c) and (d) below, the activities of AST and ALT in the cells of the model group were significantly increased compared with those of the normal group (P < 0.001), indicating that the liver injury cell model was successfully constructed. Compared with the model group, the activities of AST and ALT in the positive drug NAC group were significantly decreased (P < 0.001 or P < 0.01), and the activities of AST and ALT in the cells of each concentration group of RSPLTE decreased to varying degrees, indicating that this extract has a certain liver protection effect; among them, the activity of AST in the liver injury cells of each concentration group of RSPLTE was significantly decreased (P < 0.001), and the activity of ALT in the cells of the 300 μg / mL group was significantly decreased (P < 0.001). The results showed that RSPLTE has a certain repair effect on damaged LO2 human normal liver cells.

[0145] As Figure 5 shown in (e) below, compared with the normal group, the SOD activity of the cells in the model group was significantly decreased (P < 0.001). Compared with the model group, the SOD activities of the NAC positive group and the 75, 150, and 300 μg / mL groups of RSPLTE were significantly increased (P < 0.001). The results showed that RSPLTE can effectively scavenge superoxide anion radicals in cells, thereby improving cell damage.

[0146] As Figure 5 shown in (f) below, compared with the normal group, the MDA content of the cells in the model group was significantly increased (P < 0.001). Compared with the model group, the MDA content in the cells of the NAC positive group and the 150 and 300 μg / mL groups of RSPLTE was significantly decreased (P < 0.001). It shows that RSPLTE can reduce the degree of oxidative damage of H2O2 to LO2 cells and has a protective effect on LO2 cells in the oxidative stress state.

[0147] As Figure 5 shown in (g) below, the GSH content of the cells in the model group was significantly decreased compared with that of the normal group (P < 0.001), indicating that the liver injury model was successfully constructed. Compared with the model group, the GSH contents of the positive NAC group and the 150 and 300 μg / mL groups of RSPLTE were significantly increased (P < 0.05 or P < 0.01), and the most significant GSH content was in the 300 μg / mL group, indicating that RSPLTE has a good cell protection effect.

[0148] B. Effects of RSPLTE on mice with alcohol-induced liver injury

[0149] 1) Experimental grouping and drug administration

[0150] Healthy KM mice were randomly divided into a normal control group, a model group (56° white liquor), a positive control group (silymarin 150 mg / kg / d), and low, medium, and high-dose groups of RSPLTE (150, 300, 600 mg / kg / d), with 10 mice in each group. After one week of adaptive feeding, gavage treatment began. In the morning, the low, medium, and high-dose groups of RSPLTE were gavaged with different doses of RSPLTE, and the mice in the control group and the model group were gavaged with an equal volume of distilled water. In the afternoon, the mice in other groups except the control group were given 56° white liquor (10 mL / kg) to establish an acute alcoholic liver injury model. The experiment lasted for 16 days. For the last administration of medicine and liquor, the mice were fasted but allowed to drink water for 16 h. Subsequently, the mice were anesthetized with 50 mg / kg sodium pentobarbital and sacrificed by orbital blood collection. The blood was centrifuged at 3000 r / min for 10 min at 4 °C, and the serum was separated and stored in a -80 °C refrigerator for subsequent determination of biochemical indexes. Tissue processing: The liver tissues of the mice were quickly dissected aseptically, weighed, and recorded. Part of the liver tissue was fixed in 4% paraformaldehyde to ensure that the fixed tissue positions of each mouse were the same. The remaining liver tissue was stored in a -80 °C environment for subsequent determination of indexes.

[0151] 2) Determination of mouse body weight and liver coefficient

[0152] During the modeling process, the body weights of the mice in each group were measured every two days to preliminarily evaluate the effect of RSPLTE on the basic body weight of the mice. After the last administration, the mice were fasted for 16 h, allowed to drink water freely, and their livers were removed and processed and weighed during the dissection of the mice. The liver coefficient was calculated based on the liver weight to further observe the effect of the water extract of Rosa sterilis S. D. Shi pomace on the liver coefficient of mice with acute alcohol poisoning. The liver coefficient was calculated using the following formula: Liver index % = liver mass g / mouse body mass g × 100%.

[0153] 3) Detection of liver oxidative stress indexes

[0154] Weigh 0.1 g of mouse liver tissue and store it at -80 °C. Add pre-cooled physiological saline according to a volume ratio of 1:9 to prepare a 10% tissue homogenate. Grind the tissue completely under ice-water bath conditions. After grinding, centrifuge the homogenate at 3000 r / min for 15 min at 4 °C, and aspirate the supernatant. Use a BCA protein concentration assay kit to detect the protein concentration of each group of samples. And according to the method in the kit instructions, determine the activities or contents of malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT).

[0155] 4) Detection of liver tissue inflammatory factors

[0156] According to the instructions and operation requirements of the kit, the levels of inflammatory factors IL-6, IL-1β, and TNF-α in the livers of mice were measured.

[0157] 5) Data processing and analysis

[0158] Experimental data were expressed as mean ± standard deviation (Mean ± SD). All data analysis and chart drawing were completed using GraphPad Prism 12.0 software. One-way analysis of variance (one-way ANOVA) was used for comparison among multiple groups, and statistical significance was determined by P < 0.05.

[0159] During the alcohol gavage period, the body weights of mice in each group increased slowly with the prolongation of the gavage time. There were no significant differences in the body weights of mice in each RSPLTE dose group compared with the normal group (P > 0.05). In addition, at the end of the gavage, no deaths occurred in all mice, which preliminarily indicated that gavage with 56° Baijiu and different doses of RSPLTE had no significant effect on the body weights of mice. The liver coefficient of mice in the model group increased by 0.42% compared with the normal group (P < 0.001), indicating liver enlargement due to excessive alcohol consumption. At the same time, compared with the model group, the liver index of mice in the high-dose group decreased by 0.31% (P < 0.01), indicating that RSPLTE could effectively relieve liver enlargement caused by excessive alcohol consumption.

[0160] As Figure 6 shown, the contents of GSH, CAT, and SOD in the liver tissues of mice in the model group were significantly lower than those in the normal group (P < 0.001), while the content of MDA was significantly increased (P < 0.001), indicating that oxidative stress occurred in the mouse liver and hepatocytes were damaged, further demonstrating the successful establishment of the model. Compared with the model group, the content of malondialdehyde (MDA) in the liver tissues of mice in the positive drug group and different RSPLTE dose groups was significantly decreased (P < 0.05 or P < 0.01). The contents of GSH, CAT, and SOD were significantly increased (P < 0.01 or P < 0.05). These results indicated that RSPLTE could effectively reduce the content of harmful substance MDA, and increase the activities or contents of GSH, CAT, and SOD, thereby enhancing the body's ability to scavenge free radicals, reducing the damage caused by oxidative stress, and thus achieving the effect of protecting the liver.

[0161] As Figure 7As shown, in the livers of the mice in the model group, the levels of pro-inflammatory factors were significantly increased compared with the control group (P < 0.05 or P < 0.01). Compared with the model group, both the positive group and the high-dose RSPLTE group could significantly reduce the levels in the livers of mice (P < 0.05 or P < 0.01). By increasing the levels of pro-inflammatory factors in the liver, RSPLTE can inhibit the production of pro-inflammatory factors, reduce the ethanol-induced inflammatory response, and play a hepatoprotective role.

[0162] C. Effect of RSPLTE on tyrosinase activity

[0163] Refer to the method recorded in the group standard T / GDCA 006-2021 of Guangdong Cosmetics Society (Test Method for Inhibitory Effect of Cosmetic Raw Materials on Tyrosinase Activity (In Vitro Method)) to determine the effect of RSPLTE on tyrosinase activity.

[0164] The results are as Figure 8 shown, RSPLTE has an obvious inhibitory effect on tyrosinase activity. By inhibiting tyrosinase activity, melanin production can be reduced, thus achieving the effects of whitening and lightening spots.

[0165] Comparative Example 1:

[0166] Ultrasonic ethanol-assisted extraction: Weigh accurately the residue of seedless Rosa roxburghii fruit or the powder of Rosa roxburghii fruit residue and place it in a 50 mL stoppered glass conical flask. Add anhydrous ethanol according to the solid-liquid ratio of 1:20 (g:mL). Weigh the mass, perform ultrasonic treatment (power 180 W, frequency 40 kHz) for 30 min, cool, make up the lost mass with anhydrous ethanol, shake well, filter, and take the filtrate for concentration and drying under reduced pressure to obtain the ultrasonic ethanol-assisted extracts of the residue of seedless Rosa roxburghii fruit and Rosa roxburghii fruit residue for standby.

[0167] Refer to Example 1 to determine the content of fat-soluble components in the ultrasonic ethanol-assisted extracts of the residue of seedless Rosa roxburghii fruit and Rosa roxburghii fruit residue. The results are shown in Table 8.

[0168] The results show that β-carotene, DL-α-tocopherol, β-sitosterol, and squalene were not detected in the Rosa roxburghii fruit extract, and the contents of lutein and zeaxanthin were also significantly lower than those in the seedless Rosa roxburghii fruit extract.

[0169] Table 8 Contents of fat-soluble components in the ultrasonic ethanol-assisted extracts of seedless Rosa roxburghii fruit and Rosa roxburghii fruit

[0170]

[0171] Note: — means not detected

[0172] Comparative Example 2:

[0173] Preparation of supercritical CO2 extract: Weigh an appropriate amount of seedless Rosa roxburghii Tratt pomace or Rosa roxburghii Tratt residue powder and put it into the supercritical CO2 extraction kettle. The extraction conditions are as follows: extraction time is 3 h, the entrainer is 95% ethanol, the extraction pressure is 40 MPa, the temperature is 42 °C, the separation I pressure is 8 MPa, the temperature is 60 °C, the separation II pressure is 6 MPa, the temperature is 45 °C, and the CO2 flow rate is 25 L·h -1 . Collect the extract containing the entrainer in the separation kettle II, concentrate and dry the solvent under reduced pressure to obtain the supercritical CO2 extracts of seedless Rosa roxburghii Tratt pomace and Rosa roxburghii Tratt residue.

[0174] Refer to Example 2 to determine the activities of the supercritical CO2 extracts of seedless Rosa roxburghii Tratt pomace and Rosa roxburghii Tratt residue on the lipopolysaccharide-induced anti-inflammatory model of RAW cells. The results are as shown in Figure 4 (c) and (d) below. Compared with the negative control, the NO release in the LPS group increased significantly (p < 0.001). The results showed that the supercritical CO2 extract of seedless Rosa roxburghii Tratt pomace had an inhibitory effect on NO release in LPS-induced RAW264.7 cells (p < 0.001 or p < 0.05), and the lowest effective concentration was 37.5 μg / ml. Compared with the negative control, the NO release in the LPS group increased significantly (p < 0.0001). Compared with the LPS group, the supercritical CO2 extracts of Rosa roxburghii Tratt residue at different concentrations had no significant difference in the NO release in LPS-induced RAW264.7 cells (p > 0.05), that is, there was no anti-inflammatory effect.

[0175] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for the overall analysis of active ingredients in seedless Rosa roxburghii tratt, characterized in that: Including the detection of water-soluble components and the detection of fat-soluble components; among which: the detection of water-soluble components includes the following steps: (1) Preparation of the test solution: Accurately weigh the freeze-dried powder of seedless Rosa roxburghii fruit, juice or residue, add 70% methanol solution, extract by ultrasonic, centrifuge after cooling, retain the supernatant, add 70% methanol solution to the residue for extraction again, centrifuge, combine the supernatants, and make up the volume to obtain the test solution with 70% methanol solution; (2) Detection of water-soluble components: The UV method-standard curve method is used to determine the contents of total polyphenols, total flavonoids, total proanthocyanidins, and total polysaccharides in the test solution; the HPLC method-standard curve method is used to determine the content of VC in the test solution. Among them, the chromatographic column is Agilent C18 column; the mobile phase is 0.1% phosphoric acid solution: methanol with a volume ratio of 2:98 for isocratic elution; the flow rate is 1.0 mL / min; the detection wavelength is 242 nm; the column temperature is 30 °C; the injection volume is 10 μL; The linear relationships of total polyphenols, total flavonoids, total proanthocyanidins, total polysaccharides, and VC are as follows: y = 4.9503x + 0.0174, R 2 = 0.9990; y = 1.0757x + 0.0007, R 2 = 0.9997; y = 1.5120x - 0.0238, R 2 = 0.9997; y = 11.2500x - 0.0454, R 2 = 0.9991; y = 30852x + 173588R 2 = 0.9996; The detection of fat-soluble components includes the following steps: (1) Preparation of the test solution: Weigh the freeze-dried powder of seedless Rosa roxburghii fruit, juice or residue respectively, add a methanol:dichloromethane solution with a volume ratio of 1:1, extract by ultrasonic, centrifuge after cooling, retain the supernatant, and make up the volume to obtain the test solution with a methanol:dichloromethane solution with a volume ratio of 1:1; (2) Detection of fat-soluble components: The HPLC method-standard curve method is used to determine the contents of lutein, zeaxanthin, β-carotene, DL-α-tocopherol, β-sitosterol, and squalene in the test solution. Among them, the chromatographic conditions for lutein, zeaxanthin, and β-carotene are as follows: Use the chromatographic column Cosmosil PBr; mobile phase A: methanol, mobile phase B: methyl tert-butyl ether, for gradient elution: 0-30 min, 4% B; 30-35 min, 4%-7% B; 35-45 min, 7% B; 45-50 min, 7%-15% B; 50-65 min, 15%-24% B; 65-75 min, 24%-24% B; 75-85 min, 24%-80% B; 85-90 min, 80%; the flow rate is 1.0 mL / min; the detection wavelength is 450 nm; the column temperature is 25 °C; the injection volume is 20 μL; the chromatographic conditions for DL-α-tocopherol, β-sitosterol, and squalene are as follows: Chromatographic column: Hypersil ODS C18; isocratic elution with 100% methanol for 60 min; the flow rate is 0.7 mL / min; the detection wavelength is 205 nm; the column temperature is 30 °C; injection volume 20 μL; The linear relationships of lutein, zeaxanthin, β-carotene, DL-α-tocopherol, β-sitosterol, and squalene are as follows: Y = 99618X + 31248, R 2 = 0.9997; Y = 168910X + 13839, R 2 = 0.9997; Y = 148575X + 64098, R 2 = 0.9999; Y = 90502X + 58590, R 2 = 0.9993; Y = 10934X - 2259, R 2 = 0.9998; Y = 106223X - 10991R 2 = 0.9992.

2. A method for the overall extraction of active ingredients from the residue of seedless Rosa roxburghii Tratt, characterized in that: Including the following steps: S1. Weigh fresh seedless Rosa roxburghii fruit, press, centrifuge, and filter to obtain seedless Rosa roxburghii juice and the remaining fruit residue; S2. Take the remaining pomace from step S1 and put it into a supercritical CO2 extraction kettle. The extraction conditions are as follows: extraction time is 3 ± 0.5 h, the entrainer is 95% ethanol, the extraction pressure is 50 ± 10 MPa, the temperature is 42 ± 2 °C, the separation I pressure is 8 ± 1 MPa, the temperature is 60 ± 5 °C, the separation II pressure is 6 ± 1 MPa, the temperature is 45 ± 5 °C, and the CO2 flow rate is 25 ± 5 L·h -1 ; Collect the extract containing the entrainer in the separation kettle II, concentrate and dry the solvent under reduced pressure to obtain the supercritical CO2 extract and the remaining residue; S3. Take the residue remaining in step S2, add water, and stir evenly; use a low-temperature high-pressure crushing extraction device to reduce the water circulation temperature of the device to 4 °C, pour the solvent into the feed port for drainage, then pour the evenly mixed residue solution into the feed port, extract at a pressure of 100 ± 10 mPa for 20 ± 5 min, centrifuge to remove the precipitate, and take the supernatant for freeze-drying to obtain the freeze-dried powder of the water-soluble extract.

3. The method according to claim 2, wherein: The extraction conditions described in step S2 are as follows: extraction time is 3 h, the entrainer is 95% ethanol, the extraction pressure is 40 MPa, the temperature is 42 °C, the separation I pressure is 8 MPa, the temperature is 60 °C, the separation II pressure is 6 MPa, the temperature is 45 °C, and the CO2 flow rate is 25 L·h -1 .

4. The method according to claim 2, wherein: The extraction in step S2 is extraction at a pressure of 100 MPa for 20 min.

5. An extract of active ingredients from the residue of seedless Rosa roxburghii Tratt fruits, characterized in that: Comprising the supercritical CO2 extract and / or water-soluble extract obtained by the method according to any one of claims 2 to 4.

6. Use of the method for overall extraction of active ingredients from seedless Rosa roxburghii Tratt pomace according to any one of claims 2-4, characterized in that: Comprising at least one of the following applications: (1) Application in the preparation of a product for treating liver injury; (2) Application in the preparation of a product having an anti-inflammatory effect; (3) Application in the preparation of a whitening and freckle-removing product.

7. The application according to claim 6, wherein: The product in application (1) is a drug for treating liver injury or a food having an auxiliary protective function against chemical liver injury; The product in application (2) is a drug having an anti-inflammatory effect; The product in application (3) is a whitening and freckle-removing cosmetic additive.

8. The application according to claim 6 or 7, wherein: Application (1) is the application of the seedless Rosa roxburghii juice, supercritical CO2 extract and / or water-soluble extract obtained by the method in the preparation of a product for treating liver injury.

9. The application according to claim 6 or 7, wherein: Application (2) is the application of the supercritical CO2 extract obtained by the method in the preparation of an anti-inflammatory drug.

10. The application according to claim 6 or 7, wherein: Application (3) is the application of the water-soluble extract obtained by the method in the preparation of a whitening and freckle-removing product.

Images