Rose extract as well as preparation method and application thereof
By using a ternary synergistic solvent to extract, separate, and identify compounds such as ellagic acid, urolithin M5, and apigenin from rose petals, the problem of insufficient inhibitory ability of rose petal extract on pancreatic lipase was solved, achieving effective inhibition of human pancreatic lipase and preventing obesity.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZUNYI MEDICAL UNIVERSITY
- Filing Date
- 2026-02-11
- Publication Date
- 2026-05-19
AI Technical Summary
Currently, rose extract has not been found to inhibit pancreatic lipase, and therefore cannot effectively reduce the absorption of dietary fat to prevent obesity.
A ternary synergistic solvent composed of water, anhydrous ethanol, and a natural eutectic solvent was used to extract rose powder by ultrasonic treatment and stirring. The supernatant was collected by centrifugation and freeze-dried into a solid powder. Compounds such as ellagic acid, urolithin M5, and apigenin were isolated and identified, which can be used to inhibit the activity of human pancreatic lipase.
A rose extract that inhibits human pancreatic lipase was successfully prepared, significantly reducing the absorption of fatty acids and monoacylglycerols and effectively preventing obesity.
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Figure CN122056946A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of plant extraction technology, specifically to a rose extract, its preparation method, and its application. Background Technology
[0002] Obesity not only affects people's quality of life but is also a major risk factor for a range of serious chronic diseases, including type 2 diabetes, cardiovascular diseases (such as hypertension and coronary heart disease), hyperlipidemia, non-alcoholic fatty liver disease, certain types of cancer, and osteoarthritis. The root cause of obesity is a long-term imbalance between energy intake and energy expenditure. Dietary fat is the nutrient with the highest energy density, and its digestion and absorption are key steps in controlling energy intake.
[0003] Pancreatic lipase, a key digestive enzyme secreted by the pancreas, plays a crucial role in the hydrolysis and absorption of dietary fat. It is responsible for hydrolyzing 50%-70% of total dietary fat in the proximal small intestine and is an important target for the prevention and treatment of obesity. Our daily diet consists of 90% mixed triglycerides. Exogenous fats cannot be directly utilized by the body and must be hydrolyzed for absorption. Pancreatic lipase hydrolyzes long-chain triglycerides (TAGs) in the diet into free fatty acids and monoacylglycerols, thus playing a key role in dietary fat absorption. Therefore, inhibiting pancreatic lipase activity can effectively reduce the hydrolysis of dietary triglycerides, thereby reducing the absorption of free fatty acids and monoacylglycerols, which is of great significance for controlling fat intake and preventing obesity.
[0004] Currently, there are over 100 varieties of roses worldwide. Among them, some roses, specially cultivated and meeting food safety standards, are edible and can be consumed directly or through processing. These are not only beautiful ornamental flowers but also valuable "food ingredients" and "medicinal herbs," boasting a long history of application and broad development prospects in my country. Common edible rose varieties in my country include double-petaled roses, dark red roses, golden-edged roses, and bitter water roses. Double-petaled roses are a traditional and widely used edible variety, with a rich and pure aroma, often used to make rose jam, rose sugar, and rose wine. Dark red roses are commonly used to make flower tea, flower cakes, and for extracting pigments. Golden-edged roses are one of the representative varieties of Yunnan's specialty flower teas. Bitter water roses are an important raw material for extracting rose essential oil, making rose tea, and pastry fillings.
[0005] Furthermore, patent application publication number CN 110638704 A discloses a method for extracting rose extract, the extracted rose extract, its application, and cosmetics. The rose extract obtained by this method has a total polyphenol content as high as 56.3%. Antioxidant assays, such as DPPH free radical and ABTS scavenging, potassium ferricyanide reducing power, and FRAP value determination, all show that this rose extract exhibits strong antioxidant capacity. Patent authorization publication number CN 116712484 B discloses a rose extract, its preparation method, and its application in the preparation of drugs with neuraminidase inhibitory activity. The rose extract obtained by this method has excellent neuraminidase inhibitory activity; its inhibitory effect on neuraminidase is significantly higher than that of oseltamivir carboxylic acid.
[0006] However, there are currently no reports of rose extract having the ability to inhibit pancreatic lipase. Summary of the Invention
[0007] The present invention aims to provide a rose extract that has the ability to inhibit human pancreatic lipase.
[0008] A rose extract comprising at least one of the monomers ellagic acid, urolithin M5, and apigenin.
[0009] Preferably, the rose extract contains at least one of the following: ellagic acid monomer, urolithin M5, and apigenin, with ellagic acid monomer content of 50–150 mg / g, urolithin M5 content of 0.5–1.5 mg / g, and apigenin content of 0.5–1.5 mg / g.
[0010] A method for preparing rose extract includes the following steps: mixing rose powder with a ternary synergistic solvent, ultrasonicating at 150-250W for 10-60 min, stirring at 20-50℃ for 1-5 h, and centrifuging to collect the supernatant to obtain rose extract.
[0011] Preferably, after centrifuging to collect the supernatant, the supernatant is concentrated under reduced pressure at 30~60℃ to a paste state, and then the paste is freeze-dried into a solid powder to obtain rose extract.
[0012] Preferably, the rose is at least one of dark red rose, gold-edged rose, bitter rose, and double-petaled red rose. Double-petaled red rose is preferred.
[0013] Preferably, the rose is a dried, unopened rosebud.
[0014] Preferably, the ternary synergistic solvent is composed of water, anhydrous ethanol, and a natural eutectic solvent (NADES). The mass ratio of water, anhydrous ethanol, and NADES is 20-30% : 30-40% : 30-50%.
[0015] Preferably, the natural eutectic solvent is composed of betaine and citric acid mixed in a molar ratio of 1:1 to 1:2.
[0016] Preferably, the ratio of rose powder to ternary synergistic solvent is 1:10-30 (W:V). W:V is g:ml.
[0017] Preferably, when collecting the supernatant by centrifugation, the centrifugation process is carried out at 8000-15000 rpm for 5-20 min.
[0018] The inventors have discovered that extracts of double-petaled red roses, golden-edged roses, dark red roses, and bitter roses obtained using the preparation method of this invention have an inhibitory effect on pancreatic lipase. Therefore, this application also claims protection for the use of rose extracts in the non-disease diagnostic and therapeutic purposes of inhibiting human pancreatic lipase, particularly in the preparation of drugs or health foods for the prevention of obesity or drugs with pancreatic lipase inhibitory effects.
[0019] To further verify the key substances in rose extract that exert the inhibitory effect on human pancreatic lipase, the inventors focused on double-petaled red roses and successfully separated and identified multiple highly active monomers using advanced separation and identification techniques (HPLC / Q-TOF-MS); thus clarifying the key substances in double-petaled red rose extract that exert the inhibitory effect on human pancreatic lipase.
[0020] The highly active monomers include, but are not limited to, ellagic acid, apigenin, and urolithin M5. This invention reveals their specific use in inhibiting human pancreatic lipase. This discovery pinpoints the key substances in rose extract that exert their inhibitory effect on human pancreatic lipase.
[0021] This invention uses a chromatographic column to separate compounds with human pancreatic lipase inhibitory activity from the water extract of double-petaled red rose, obtaining a separated solution. The liquid chromatography conditions are as follows: column: Zafex Pre JX – C18; mobile phase A: 0.1% formic acid water, mobile phase B: methanol; gradient elution conditions: 0-5 min, 10%-50% B, 50-70 min, 50% B, 70-75 min, 50%-10% B; flow rate: 1 mL / min.
[0022] Furthermore, this invention uses human pancreatic lipase for in vitro activity verification, which differs from the porcine pancreatic lipase commonly used in existing technologies. This eliminates species differences and enhances the application value.
[0023] The qualitative analysis method for compounds exhibiting human pancreatic lipase inhibitory activity in rose extract, as described in this invention, includes the following steps: qualitative analysis of compounds with human pancreatic lipase inhibitory effects in rose extract using HPLC / Q-TOF-MS. High performance liquid chromatography (HPLC) conditions: ShimNex HE C18-AQ column, 2.1 × 150 mm, 5 μm; mobile phase A: acetonitrile; mobile phase B: 0.1% (v / v) formic acid aqueous solution; gradient elution conditions: 0.01–2 min, 95% B; 2–10 min, 95%–5% B; 10–12 min, 5% B; 12–12.5 min, 5%–95% B; 12.5–15 min, 95% B; flow rate: 0.4 mL / min; Mass spectrometry conditions: electron spray ionization source, positive and negative ion mode, capillary voltage 4500 V, evaporation temperature 450 ℃, collision energy 35 eV, scan range 100–2000 amu. Attached Figure Description
[0024] Figure 1 A comparison of fingerprint spectra between the double-petaled red rose extract prepared by the method of this patent and commercially available double-petaled red rose extract.
[0025] Figure 2 This is a liquid phase separation diagram for the preparation of extract from double-petaled red roses.
[0026] Figure 3 This is a graph showing the inhibitory effect of the extract of double-petaled red rose on human pancreatic lipase.
[0027] Figure 4 IC50 of apigenin, ellagic acid, and urolithin M5 on human pancreatic lipase 50 Measurement results. Detailed Implementation
[0028] The identification and application of compounds with human pancreatic lipase inhibitory activity in rose extract provided by this invention will be described clearly and completely below.
[0029] Example 1: Preparation of Rose Extract (1) Processing of medicinal materials: crush the dried rose buds into powder, pass them through a 30-mesh sieve to obtain rose bud powder; (2) Preparation of ternary synergistic solvent: The ternary synergistic solvent is composed of water, ethanol and natural eutectic solvent (NADES), wherein the mass fraction of water is 20%, the mass fraction of ethanol is 30%, and the remainder is NADES, which is prepared by betaine and citric acid in a molar ratio of 1:1.
[0030] Preparation method of ternary synergistic solvent: Weigh 19.0 g of betaine and 31.0 g of citric acid and mix them in a round-bottom flask. Add 20 g of water and stir at 80 ℃ for 3 h to obtain a clear and transparent liquid. After cooling, add 30 g of anhydrous ethanol and mix evenly to obtain the target ternary synergistic solvent.
[0031] (3) Extraction: Rose bud powder was added to the above ternary synergistic solvent at a ratio of 1 g: 20 mL. The mixture was ultrasonicated at 200 W for 30 minutes at room temperature, then stirred and extracted at room temperature for 3 h. After centrifugation at 12000 rpm for 10 min, the mixture was filtered to obtain rose extract.
[0032] (4) Drying: The rose extract is concentrated under reduced pressure at 30~60℃ to a paste state, and the paste is freeze-dried into a solid powder to obtain the product.
[0033] Example 2: Preparation of Rose Extract (1) Processing of medicinal materials: Same as in Example 1.
[0034] (2) Preparation of ternary synergistic solvent: The ternary synergistic solvent is composed of water, ethanol and natural eutectic solvent (NADES), wherein the mass fraction of water is 30%, the mass fraction of ethanol is 30%, and the remainder is NADES, which is prepared by betaine and citric acid in a molar ratio of 1:1.
[0035] Preparation method of ternary synergistic solvent: Weigh 15.1 g of betaine and 24.9 g of citric acid and mix them in a round-bottom flask. Add 30 g of water and stir at 80 ℃ for 3 h to obtain a clear and transparent liquid. After cooling, add 30 g of anhydrous ethanol and mix evenly to obtain the target ternary synergistic solvent.
[0036] (3) Extraction: Same as in Example 1.
[0037] (4) Drying: Same as in Example 1.
[0038] Example 3: Preparation of Rose Extract (1) Processing of medicinal materials: Same as in Example 1.
[0039] (2) Preparation of ternary synergistic solvent: The ternary synergistic solvent is composed of water, ethanol and natural eutectic solvent (NADES), wherein the mass fraction of water is 20%, the mass fraction of ethanol is 40%, and the remainder is NADES, which is prepared by betaine and citric acid in a molar ratio of 1:1.
[0040] Preparation method of ternary synergistic solvent: Weigh 15.2 g of betaine and 24.8 g of citric acid and mix them in a round-bottom flask. Add 20 g of water and stir at 80 ℃ for 3 h to obtain a clear and transparent liquid. After cooling, add 40 g of anhydrous ethanol and mix evenly to obtain the target ternary synergistic solvent.
[0041] (3) Extraction: Same as in Example 1.
[0042] (4) Drying: Same as in Example 1.
[0043] Example 4: Preparation of Rose Extract (1) Processing of medicinal materials: Same as in Example 1.
[0044] (2) Preparation of ternary synergistic solvent: The ternary synergistic solvent is composed of water, ethanol and natural eutectic solvent (NADES), wherein the mass fraction of water is 30%, the mass fraction of ethanol is 40%, and the remainder is NADES, which is prepared by betaine and citric acid in a molar ratio of 1:1.
[0045] Preparation method of ternary synergistic solvent: Weigh 11.4 g of betaine and 18.6 g of citric acid and mix them in a round-bottom flask. Add 30 g of water and stir at 80 ℃ for 3 h to obtain a clear and transparent liquid. After cooling, add 40 g of anhydrous ethanol and mix evenly to obtain the target ternary synergistic solvent.
[0046] (3) Extraction: Same as in Example 1.
[0047] (4) Drying: Same as in Example 1.
[0048] Example 5: Preparation of Rose Extract (1) Processing of medicinal materials: Same as in Example 1.
[0049] (2) Preparation of ternary synergistic solvent: The ternary synergistic solvent is composed of water, ethanol and natural eutectic solvent (NADES), wherein the mass fraction of water is 20%, the mass fraction of ethanol is 30%, and the remainder is NADES, which is prepared by betaine and citric acid in a molar ratio of 1:2.
[0050] Preparation method of ternary synergistic solvent: Weigh 11.7 g of betaine and 38.3 g of citric acid and mix them in a round-bottom flask. Add 20 g of water and stir at 80 ℃ for 3 h to obtain a clear and transparent liquid. After cooling, add 30 g of anhydrous ethanol and mix evenly to obtain the target ternary synergistic solvent.
[0051] (3) Extraction: Same as in Example 1.
[0052] (4) Drying: Same as in Example 1.
[0053] Example 6: Preparation of Rose Extract (1) Processing of medicinal materials: Same as in Example 1.
[0054] (2) Preparation of ternary synergistic solvent: The ternary synergistic solvent is composed of water, ethanol and natural eutectic solvent (NADES), wherein the mass fraction of water is 20%, the mass fraction of ethanol is 40%, and the remainder is NADES, which is prepared by betaine and citric acid in a molar ratio of 1:2.
[0055] Preparation method of ternary synergistic solvent: Weigh 9.3 g of betaine and 30.7 g of citric acid and mix them in a round-bottom flask. Add 20 g of water and stir at 80 ℃ for 3 h to obtain a clear and transparent liquid. After cooling, add 40 g of anhydrous ethanol and mix evenly to obtain the target ternary synergistic solvent.
[0056] (3) Extraction: Same as in Example 1.
[0057] (4) Drying: Same as in Example 1.
[0058] Example 7: Preparation of Rose Extract (1) Processing of medicinal materials: Same as in Example 1.
[0059] (2) Preparation of ternary synergistic solvent: The ternary synergistic solvent is composed of water, ethanol and natural eutectic solvent (NADES), wherein the mass fraction of water is 30%, the mass fraction of ethanol is 30%, and the remainder is NADES, which is prepared by betaine and citric acid in a molar ratio of 1:2.
[0060] Preparation method of ternary synergistic solvent: Weigh 9.3 g of betaine and 30.7 g of citric acid and mix them in a round-bottom flask. Add 30 g of water and stir at 80 ℃ for 3 h to obtain a clear and transparent liquid. After cooling, add 30 g of anhydrous ethanol and mix evenly to obtain the target ternary synergistic solvent.
[0061] (3) Extraction: Same as in Example 1.
[0062] (4) Drying: Same as in Example 1.
[0063] Example 8: Preparation of Rose Extract (1) Processing of medicinal materials: Same as in Example 1.
[0064] (2) Preparation of ternary synergistic solvent: The ternary synergistic solvent is composed of water, ethanol and natural eutectic solvent (NADES), wherein the mass fraction of water is 30%, the mass fraction of ethanol is 40%, and the remainder is NADES, which is prepared by betaine and citric acid in a molar ratio of 1:2.
[0065] Preparation method of ternary synergistic solvent: Weigh 7.0 g of betaine and 23.0 g of citric acid and mix them in a round-bottom flask. Add 30 g of water and stir at 80 ℃ for 3 h to obtain a clear and transparent liquid. After cooling, add 40 g of anhydrous ethanol and mix evenly to obtain the target ternary synergistic solvent.
[0066] (3) Extraction: Same as in Example 1.
[0067] (4) Drying: Same as in Example 1.
[0068] Example 9: In vitro IC50 assay of rose extracts in different proportions of ternary synergistic solvents on human pancreatic lipase 50 Measurement Weigh appropriate amounts of rose extract powder according to the extraction rate and dissolve them in ultrapure water to prepare a stock solution equivalent to 100 mg / mL of raw material. The stock solution was diluted to a series of concentration gradients (final concentrations of 12.5, 10, 7.5, 5, 2.5, 1, 0.5, 0.25, and 0 μM), with a total reaction volume of 200 μL, including buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, and 1 mM CaCl2), bile salts (0.1 mg / mL), and human pancreatic lipase (0.2 μg / mL, final concentration). After reacting each concentration of rose extract with human pancreatic lipase at 37 °C for 3 min, the substrate DDAO-ol (10 μM, final concentration) was added to initiate the reaction. After reacting at 37 °C for 20 min, the fluorescence signal of the hydrolysis products was detected using a fluorescence microplate reader at an excitation wavelength of 600 nm and an emission wavelength of 660 nm (gain=500). Orlistat was selected as a positive inhibitor.
[0069] IC of different mass fractions 50 As shown in Table 1, the IC50 values of each rose extract against hPL were obtained when the ternary synergistic solvents had water and ethanol mass fractions of 30% and the molar ratio of betaine to citric acid was 1:2. 50 The values were the lowest, with double-petaled red rose showing the strongest inhibitory effect on hPL, IC50. 50 Only 0.63 μg / mL.
[0070] Table 1. IC50 of each rose extract in Examples 1-8 against hPL 50 value
[0071] Example 10: Comparison of double-petaled red rose extract prepared by the method of this patent with commercially available double-petaled red rose extract. The double-petaled red rose extract prepared according to the method described in Example 8 was diluted with ultrapure water to a concentration equivalent to 1 mg / mL of the raw rose. The double-petaled red rose extract purchased from Fufeng Sinote Biotechnology Co., Ltd. was diluted with ultrapure water to concentrations of 1 mg / mL and 10 mg / mL, respectively. The fingerprint chromatograms were plotted using high performance liquid chromatography.
[0072] The high-performance liquid chromatography (HPLC) conditions were as follows: column: Zafex Pre JX – C18, 250 × 10 mm, 5 μm; column temperature: 30 ℃; mobile phase A: 0.1% formic acid in water, mobile phase B: methanol; gradient elution conditions: 0-5 min, 10%-50% B, 50-70 min, 50% B, 70-80 min, 50%-10% B; flow rate: 1 mL / min.
[0073] The results are as follows Figure 1 As shown, the content of active ingredients in the double-petal red rose extract prepared using the preparation method described in this patent is 1 mg / mL, which is comparable to 10 mg / mL in commercially available products. It can be seen that the content of active ingredients in the double-petal red rose extract extracted using this patent is ten times higher than that in commercially available products, which greatly improves the extraction rate of active ingredients.
[0074] Example 11 Isolation of double-petaled red rose extract The extract of double-petaled red rose was dissolved in ultrapure water to a concentration of 5 mg / mL. After filtration through a 0.22 μm filter membrane, the components in the extract were separated and collected using a preparative phase. The specific chromatographic conditions were as follows: column: Zafex Pre JX – C18, 250 × 10 mm, 5 μm; column temperature: 30 ℃; mobile phase A: 0.1% formic acid water, mobile phase B: methanol; gradient elution conditions: 0–5 min, 10%–50% B, 50–70 min, 50% B, 70–80 min, 50%–10% B; flow rate: 1 mL / min.
[0075] Sixteen fractions were collected from the extract of double-petaled red rose using preparative liquid chromatography. The specific results are as follows: Figure 2 As shown.
[0076] Example 12: Screening of the optimal active extraction fraction using in vitro human pancreatic lipase inhibition experiments The solvent in the 16 separation solutions obtained from the preparative liquid chromatography was removed using a refrigerated centrifuge, and the solutions were redissolved in 200 μL of DMSO, resulting in a total reaction volume of 200 μL. This included buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM CaCl2), bile salts (0.1 mg / mL), and human pancreatic lipase (0.2 μg / mL, final concentration). After reacting the 16 separation solutions with human pancreatic lipase at 37 °C for 3 min, the substrate DDAO-ol (10 μM, final concentration) was added to initiate the reaction. The reaction was then carried out at 37 °C for 20 min. The fluorescence signal of the hydrolysis products was detected using a fluorescence microplate reader at an excitation wavelength of 600 nm and an emission wavelength of 660 nm (gain=500). The results are as follows: Figure 3 As shown, separation solutions 4-10 have a strong inhibitory effect on human pancreatic lipase.
[0077] Example 13: Qualitative analysis of active compounds in double-petaled red rose extract that inhibit human pancreatic lipase. 1. Qualitative: After drying the 4-10 separation solutions dissolved in DMSO using a refrigerated centrifuge, they were redissolved in 200 μL of 50% acetonitrile aqueous solution, centrifuged at 18000 rpm for 20 min, and the supernatant was collected and analyzed by HPLC / Q-TOF-MS.
[0078] High performance liquid chromatography (HPLC) conditions: ShimNex HE C18-AQ column, 2.1 × 150 mm, 5 μm; mobile phase A: acetonitrile; mobile phase B: 0.1% (v / v) formic acid aqueous solution; gradient elution conditions: 0.01–2 min, 95% B; 2–10 min, 95%–5% B; 10–12 min, 5% B; 12–12.5 min, 5%–95% B; 12.5–15 min, 95% B; flow rate 0.4 mL / min.
[0079] Mass spectrometry conditions: electron spray ionization source, positive and negative ion mode, capillary voltage 4500 V, evaporation temperature 450 ℃, collision energy 35 eV, scan range 100 – 2000 amu.
[0080] The identified components are shown in Table 2.
[0081] Table 2. Detection and identification of active chemical components in double-petaled red rose extract by HPLC / Q-TOF-MS Peak Retention time / min compound Chemical formula Ion mode Theoretical value / (m / z) Measured value / (m / z) Error (ppm) Fragments (m / z) Classification 1 1.52 gallic acid <![CDATA[C7H6O5]]> <![CDATA[[M+H] + ]]> 171.0288 171.0282 -0.9 107.0122，109.0271，125.0233，127.0395，135.0065，153.0182，171.0273 Phenols 2 4.18 Corilagin <![CDATA[C 27 H 22 O 18 ]]> <![CDATA[[M+NH4] + ]]> 652.11444 652.11515 1.1 259.0236，277.0343，278.0380，303.0136，465.0667 Phenols 3 4.388 5-Hydroxyisovanillic acid <![CDATA[C8H8O5]]> <![CDATA[[M+H] + ]]> 185.04445 185.04463 1.0 107.0132，125.0251，138.9554，153.0207，153.9690 Phenols 4 4.757 Urolithin M5 <![CDATA[C 13 H8O7]]> <![CDATA[[M+H] + ]]> 277.03428 277.03453 0.9 157.0299，213.0201，231.0273，277.0377，276.8944 Phenols 5 4.758 Ellagic acid <![CDATA[C 14 H6O8]]> <![CDATA[[M+H] + ]]> 303.01354 303.01466 3.7 275.0145，284.9993，303.0145，303.0347 Phenols 6 4.76 L-Epicatechin <![CDATA[C 15 H 14 O6]]> <![CDATA[[M+H] + ]]> 291.07904 291.0867 1.5 123.0442，139.0396，147.0444，161.0591，165.0567，189.0540，207.0676，231.0651，291.0878 Flavonoids 7 4.87 Catechins <![CDATA[C 15 H 14 O6]]> <![CDATA[[M+H] + ]]> 291.08631 291.08612 -0.7 139.0399，151.0383，207.0666，245.0971 Flavonoids 8 7.138 Celery <![CDATA[C 15 H 10 O5]]> <![CDATA[[M+H] + ]]> 271.0601 271.05979 -1.1 119.0466，153.0189，271.0606 Flavonoids 2. Screening of active compounds with potent in vitro inhibitory ability against human pancreatic lipase The inhibitory capacity and inhibition mechanism of the components identified by HPLC / Q-TOF-MS were investigated in vitro. The experimental procedure was as follows: The total reaction volume was 200 μL, including buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM CaCl2), bile salts (0.1 mg / mL), and human pancreatic lipase (0.2 μg / mL, final concentration). After reacting each standard with human pancreatic lipase at 37 ℃ for 3 min, the substrate DDAO-ol (10 μM, final concentration) was added to initiate the reaction. After reacting at 37 ℃ for 20 min, the fluorescence signal of the hydrolysis products was detected using a fluorescence microplate reader at an excitation wavelength of 600 nm and an emission wavelength of 660 nm (gain=500). The experimental results are as follows. Figure 4 As shown, among the eight active compounds identified, apigenin, ellagic acid, and urolithin M5 exhibited potent inhibitory effects on human pancreatic lipase, with an IC50 value of [missing value]. 50 The values were 19.42 μM, 0.2 μM, and 4.77 μM, respectively.
[0082] Example 14: Quantitative analysis of apigenin, ellagic acid, and urolithin M5 in double-petaled red rose extract Appropriate amounts of apigenin and urolithin M5 standards were weighed and diluted with 10% acetonitrile aqueous solution to prepare a series of concentration gradients (0.1, 0.5, 1, 2, 4, 8, 16, 32, 50, 100 ng / mL). Appropriate amounts of ellagic acid standards were weighed and diluted with 10% acetonitrile aqueous solution to prepare a series of concentration gradients (10, 30, 50, 60, 70, 100, 150, 250 ng / mL). The double-petaled red rose extract was diluted with 10% acetonitrile aqueous solution to a concentration equivalent to 16.2 μg / mL of the raw material. A standard curve was plotted on the standards using an AB SCIEX 4500 triple quadrupole liquid chromatography-mass spectrometry (LC-MS) instrument to further quantify the active ingredients in the double-petaled red rose extract.
[0083] High performance liquid chromatography (HPLC) conditions: Poroshell 120 SB-C18 column, 100 × 2.1 mm, 2.7 μm; mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: acetonitrile; gradient elution conditions: 0–1.5 min, 90% A; 1.5–3 min, 90%–10% A; 4.5–6 min, 10% A; flow rate 0.35 mL / min.
[0084] Mass spectrometry conditions: Electron spray ionization source, negative ion mode, ion spray voltage -4500 V, curtain gas (CUR) 35, source temperature (TEM) 550 ℃, multiple reaction detection (MRM) mode, and the ion pair mass spectrometry detection methods for each compound are shown in Table 3.
[0085] Experimental results showed that the standard curves for ellagic acid, urolithin M5, and apigenin were Y = 329.0X – 1469, Y = 2406X – 3300, and Y = 17871X – 956.2, respectively, with R0. 2 All values were greater than 0.99. By detecting the peak areas of the corresponding compounds in the double-petaled red rose extract, the contents of ellagic acid, urolithin M5, and apigenin in the double-petaled red rose extract were calculated to be 105.28 mg / g, 1.22 mg / g, and 1.06 mg / g, respectively.
[0086] Table 3: Detection methods for ellagic acid, urolithin M5, and apigenin under MRM mode compound Detection of ion pairs DP(V) CE(V) Ellagic acid 301.2→229.0 -80 -42 Urolithin M5 275.0→229.0 -80 -30 Celery 269.0→117.0 -40 -41 The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.
Claims
1. A rose extract, characterized in that, It contains at least one of ellagic acid monomer, urolithin M5 and apigenin, and the content of ellagic acid monomer, urolithin M5 and apigenin must meet at least one of the following conditions: ellagic acid monomer 50-150 mg / g, urolithin M5 0.5-1.5 mg / g and apigenin 0.5-1.5 mg / g.
2. The method for preparing the rose extract according to claim 1, characterized in that, Includes the following steps: Rose powder was mixed evenly with a ternary synergistic solvent and ultrasonically treated at 150–250 W for 10–60 min. Then, it was stirred at 20–50 °C for 1–5 h. After centrifugation at 8000–15000 rpm for 5–20 min, the supernatant was collected to obtain rose extract.
3. The preparation method according to claim 2, characterized in that, After centrifugation to collect the supernatant, the supernatant was concentrated under reduced pressure at 30-60°C to a paste state, and then the paste was freeze-dried into a solid powder to obtain rose extract.
4. The preparation method according to claim 3, characterized in that, The rose is at least one of the following: dark red rose, gold-edged rose, bitter water rose, and double-petaled red rose.
5. The preparation method according to claim 4, characterized in that, The ternary synergistic solvent is composed of water, anhydrous ethanol and a natural eutectic solvent; wherein the mass ratio of water, anhydrous ethanol and natural eutectic solvent is 20~30%:30~40%:30~50%.
6. The preparation method according to claim 5, characterized in that, The natural eutectic solvent is composed of betaine and citric acid mixed in a molar ratio of 1:1 to 1:
2.
7. A method for isolating compounds with human pancreatic lipase inhibitory activity from the rose extract of claim 2, characterized in that, Compounds with human pancreatic lipase inhibitory activity in the aqueous extract of double-petaled red rose were separated using a chromatographic column to obtain the separated solution. The liquid chromatography conditions were as follows: column: Zafex Pre JX – C18; mobile phase A: 0.1% formic acid water, mobile phase B: methanol; gradient elution conditions: 0-5 min, 10%-50% B, 50-70 min, 50% B, 70-75 min, 50%-10% B; flow rate: 1 mL / min.
8. The qualitative analysis method for the separated liquid according to claim 7, characterized in that, Qualitative analysis of the extract was performed using HPLC / Q-TOF-MS. High performance liquid chromatography (HPLC) conditions: ShimNex HE C18-AQ column, 2.1 × 150 mm, 5 μm; mobile phase A: acetonitrile; mobile phase B: 0.1% (v / v) formic acid aqueous solution; gradient elution conditions: 0.01–2 min, 95% B; 2–10 min, 95%–5% B; 10–12 min, 5% B; 12–12.5 min, 5%–95% B; 12.5–15 min, 95% B; flow rate: 0.4 mL / min; Mass spectrometry conditions: electron spray ionization source, positive and negative ion mode, capillary voltage 4500 V, evaporation temperature 450 ℃, collision energy 35 eV, scan range 100–2000 amu.
9. The use of the rose extract according to claim 1 for non-disease diagnostic and therapeutic purposes in inhibiting human pancreatic lipase.
10. The application according to claim 9, characterized in that, The application is in the preparation of drugs, health foods, or drugs with pancreatic lipase inhibitory effects for the prevention and / or treatment of obesity.