Composition for improving oral bioavailability of ursolic acid and preparation method thereof

Abstract

The invention discloses a composition for improving oral bioavailability of ursolic acid and a preparation method thereof, and the method comprises the following steps: mixing a hydrogen acceptor and a hydrogen donor to obtain a eutectic solvent; the hydrogen acceptor is menthol; the hydrogen donor is butyric acid, hexanoic acid, n-caprylic acid, capric acid or oleic acid; mixing the eutectic solvent with ursolic acid, and swirling at 60 DEG C; the molar ratio of the hydrogen acceptor to the hydrogen donor is 2: 1, 1: 1 or 1: 2. The invention solves the problem of low bioavailability of terpenoids in the existing apple peel extract. When the amount-of-substance ratio of menthol to capric acid is 1: 2, the bioavailability of ursolic acid in a cell experiment is up to 18.6%, the total cell absorption rate is up to 82%, the cell uptake rate is up to 19.3%, and the cell secretion rate at 4 hours is up to 61.99%; the bioaccessibility of ursolic acid in an in-vitro experiment reaches up to 23%.

Description

Technical Field

[0001] The invention relates to a method for improving the bioavailability of terpenoid active ingredients, and in particular to a method for improving the bioavailability of terpenoid active ingredients in apple peel extracts. Background Art

[0002] Triterpenoids are a class of natural organic compounds widely found in nature. Their main sources include plants (such as ginseng, bupleurum, etc.), fungi (such as ganoderma lucidum) and marine organisms (such as sponges). They have multiple biological activities such as anti-inflammatory, antioxidant, anti-tumor, antibacterial and immune system regulation. Due to their wide application in the pharmaceutical, health care and cosmetic industries and their significant biological activities, triterpenoids have huge market value and broad development prospects. In particular, the growing demand for natural health products and the global aging population trend provide new growth opportunities for the triterpenoid market. With the in-depth study of their pharmacological effects and the innovation of extraction technology, the application scope of triterpenoids is expected to expand further, especially in the fields of anti-inflammatory, antioxidant, anti-tumor and immune regulation. However, improving their water solubility and bioavailability is still an important challenge in the application of triterpenoids. Ursolic acid, as a natural triterpenoid with a wide range of biological activities, is widely used in food and medicine. In the pharmaceutical field, ursolic acid has been widely studied for its anti-inflammatory, antioxidant, anti-tumor and liver-protective effects, especially showing potential in the development of anti-cancer drugs; in the food industry, ursolic acid is used as a functional food ingredient to develop foods and beverages with health benefits. However, the application of ursolic acid still faces challenges such as low bioavailability, poor stability and high extraction cost.

[0003] At present, the technologies for improving the bioavailability of triterpenoids such as ursolic acid include nanotechnology, solid dispersion technology, chemical modification, liposomes, cyclodextrin inclusion, microemulsion, self-microemulsion, etc. These methods effectively improve the absorption and bioavailability of triterpenoids in the body by improving their water solubility, stability and protecting them from degradation in the body. Although some progress has been made in the methods for improving the bioavailability of triterpenoids, there are still some defects, including the possible toxicity and stability problems of nanocarriers, the changes in biological activity after chemical modification, the high production cost of liposomes and nanoparticles, and the technical challenges of large-scale production. In addition, the solubility and compatibility problems that may exist in cyclodextrin inclusion and microemulsion systems, as well as the limited effect of improving the bioavailability of oral and transdermal delivery systems, have limited the widespread promotion of these technologies in actual production and clinical applications.

[0004] Natural Deep Eutectic Solvents (NADESs) are a new type of green solvent formed by mixing two or more natural compounds in a certain proportion. They have the characteristics of low melting point, good biodegradability, low toxicity and renewability. In the food field, NADESs are used to extract natural active ingredients such as polyphenols, pigments and alkaloids due to their excellent solubility and biocompatibility. They also show application potential in food analysis, food processing and the improvement of food packaging materials, such as as a carrier of food additives or for the development of new food preservation technologies. With the increasing demand for environmentally friendly solvents, the application prospects of NADESs in the food industry are promising. In recent years, NADESs have shown significant application potential in improving the bioavailability of bioactive substances. With their unique solubility characteristics and biocompatibility, they are used to effectively extract active ingredients in natural products, such as polyphenols, flavonoids and vitamins, and enhance their bioavailability by improving the solubility and stability of these ingredients. However, for many hydrophobic active ingredients, NADESs formed by hydrophilic components are difficult to effectively improve their solubility and bioavailability. Finding more suitable solvent components and component ratios is one of the ways to solve the ideal delivery carriers of hydrophobic bioactive substances such as triterpenoids and improve their bioavailability.

[0005] Hydrophobic Deep Eutectic Solvents (H-DES) are a class of low polarity and high hydrophobicity deep eutectic solvents. They are usually composed of hydrophobic hydrogen bond donors and hydrogen bond acceptors. This property makes H-DES excellent in dissolving non-polar or hydrophobic compounds. In the field of food and medicine, H-DES has been used as an effective extraction medium to extract hydrophobic active ingredients from natural resources, such as terpenoids, fat-soluble vitamins and polyphenols. Therefore, based on the green, natural and safe characteristics of HDES, its use as an oral delivery carrier for hydrophobic active ingredients is an effective strategy to solve their low bioavailability. Summary of the invention

[0007] The purpose of the present invention is to provide a composition for improving the oral bioavailability of ursolic acid and a preparation method thereof, which solves the problem of low bioavailability of existing hydrophobic bioactive ingredients such as ursolic acid. The present invention not only improves the bioaccessibility of ursolic acid (UA), but also significantly improves the bioavailability, so HDES can be used to deliver hydrophobic active substances.

[0008] In order to achieve the above object, the present invention provides a composition for improving the oral bioavailability of ursolic acid, which is a blended system comprising a low eutectic solvent and ursolic acid, wherein the low eutectic solvent is a mixture of a hydrogen acceptor and a hydrogen donor, wherein the hydrogen acceptor is menthol, and wherein the hydrogen donor is a fatty acid.

[0009] The present invention also provides the above composition and a preparation method thereof, the steps are as follows: (1) mixing a hydrogen acceptor and a hydrogen donor to obtain a low eutectic solvent; wherein the hydrogen acceptor is selected from menthol; and the hydrogen donor is selected from fatty acids; (2) Mix the low eutectic solvent and ursolic acid and vortex them in a magnetic stirrer at 60 °C for 1 h.

[0010] Preferably, the fatty acid in the above preparation method can be selected from butyric acid, caproic acid, caprylic acid, capric acid or oleic acid.

[0011] Preferably, the molar ratio of the hydrogen acceptor to the hydrogen donor in the above preparation method is 2:1, 1:1 or 1:2; more preferably, the molar ratio of the hydrogen acceptor to the hydrogen donor is 1:1 or 1:2; most preferably, the molar ratio of the hydrogen acceptor to the hydrogen donor is 1:2.

[0012] Preferably, the mass volume ratio of ursolic acid in the above preparation method is 1%.

[0013] The method of the present invention for improving the bioavailability of terpenoid active ingredients in apple peel extract solves the problem of low bioavailability of terpenoid substances in existing apple peel extracts and has the following advantages: The present invention uses natural HDES to deliver hydrophobic active substances, wherein the hydrogen donor is any one of fatty acids such as butyric acid, caproic acid, caprylic acid, capric acid or oleic acid, and the hydrogen acceptor is menthol. When the molar ratio of menthol to fatty acid is 1:2, the bioavailability of ursolic acid in cells can be as high as 18.6%, the total cell absorption rate can be as high as 82%, the cell uptake rate can be as high as 19.3%, and the cell secretion rate can be as high as 61.99% at 4 hours; in in vitro experiments, the bioaccessibility of ursolic acid is as high as 23%.

[0014] The invention also provides a method for preparing the composition. The prepared composition can significantly improve the oral bioavailability of ursolic acid and has significant application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The results are the test results of the bioaccessibility of ursolic acid to the blend prepared in the present invention.

[0016] Figure 2 The figure is the uptake result of ursolic acid by the Caco-2 cells of the present invention. DETAILED DESCRIPTION

[0017] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0018] The present invention uses HDES to deliver the natural pentacyclic triterpenoid compound UA, and the delivery system is evaluated for its bioaccessibility and bioavailability using an in vitro simulated digestion model and a Caco-2 cell model. The specific method is as follows: 1. Preparation of UA-HDES blend system of ursolic acid (UA) and different hydrophobic low eutectic solvents (HDES) HDES was composed of different hydrogen acceptors (HBA) and hydrogen donors (HBD), 5 mL of HDES was weighed and mixed with 1% UA in a mass volume ratio (w / v), and vortexed in a magnetic stirrer at 60°C for 1 h to obtain a blend system of UA and HDES, UA-HDES. The selection of HDES is shown in Table 1.

[0019] Table 1 Composition and molar ratio of deep eutectic solvent HDES

[0020] 2. In vitro digestion of UA-HDES 2.1 Oral fluid According to the literature (Brodkorb, A., et al., INFOGEST static in vitro simulation of gastrointestinal food digestion. Nature Protocols, 2019. 14(4): p. 991-1014), the coordinated static in vitro digestion model proposed by INFO-GEST was used to study the bioaccessibility of UA and HDES. A blend of 5 mL HDES and 1% UA or a blend of 5 mL dimethyl sulfoxide (DMSO) and 1% UA was prepared. 0.05 g of UA solid powder was mixed with 4 mL NaCl (2 M) solution, and 30 μL 1.5 mM CaCl was added. 2 , distilled water 970 μL, and then mixed with the two blends respectively to a final volume of 10 mL. After incubation at 37°C for 2 min, the oral phase liquid was obtained.

[0021] 2.2 Preparation of gastric dysentery samples Mix 10 mL of the above oral phase solution with 8 mL of NaCl (2 M) solution and add 5 μL of CaCl2 (0.15 mM), 1.0 M HCL was added to adjust the pH to 3.0 to prepare gastric juice (SGF). Porcine pepsin (2000 U / mL) and gastric lipase (60 U / mL) were added and dispersed in SGF, and distilled water was added to adjust the final volume to 20 mL. The mixture was incubated at 37°C with constant temperature stirring (200 rpm) for 2 h to obtain gastric necrosis samples.

[0022] 2.3. Preparation of the intestinal phase Take 20 mL of the above gastric chyme sample in a clean beaker and incubate it in a water bath (37°C) for 10 min. Preheat NaCl (2 M) in a 37°C water bath, add 8 mL of NaCl solution, and then adjust the pH to 7 with NaOH solution (0.05-1 M). Take 3 mL of bile solution (10 mM) and trypsin (100 U / mL), stir and add to 20 mL of gastric chyme sample, and adjust the mixture to pH = 7; then add 40 μL of CaCl 2 Solution (0.3 M) was added and adjusted to pH = 7.0; the resulting mixture was incubated at 37 °C and 200 rpm for 2 h; during the incubation period, 0.1 M sodium hydroxide standard solution was added dropwise to maintain the pH at 7.0, and the volume of sodium hydroxide consumed at different digestion times was recorded.

[0023] 2.4. Determination of bioaccessibility of UA-HDES The bioaccessibility of UA in the intestinal phase after digestion was determined. The specific steps were as follows: after digestion, the intestinal phase was centrifuged at 8000 rpm for 10 min. After centrifugation, the intermediate micelles were collected with a syringe. 2 mL of the intermediate micelles were vortexed with 6 mL of anhydrous ethanol at room temperature for 30 min and then 1 mL of the supernatant was passed through a membrane (0.22 μm). The UA content in the micelles was determined by high-performance liquid chromatography. The HPLC determination was performed using an Agilent 1260infinity liquid chromatography system equipped with a binary pump and a four-channel vacuum online degasser, an autosampler and a diode array detector (DAD). The detection wavelength was 210 nm. The separation was performed using a YMC-Pack ODS-A (250×4.60 mm) chromatographic column. The column box and autosampler were maintained at 30°C. The mobile phase was chromatographic grade methanol and 0.1% phosphoric acid (volume ratio of 92.5%:7.5%) at a flow rate of 1 mL / min. Each sample was analyzed at least three times. A series of ursolic acid standards with concentrations of 2, 10, 50, 250 and 1250 μg / mL were prepared, and the standard curve of ursolic acid was plotted with the concentration of ursolic acid as the horizontal axis and the peak area as the vertical axis. The linear regression equation was Y=0.822X-22.073 (R 2 The bioaccessibility formula of UA is as follows (1):

[0024] Bioaccessibility % = C Micelle ×C Digesta ×100% (1) In formula (1), C Micelle is the concentration of UA in the micelle fraction, C Digesta is the concentration of UA in the whole sample after pH-stat experiment.

[0025] Bioaccessibility is defined as the fraction released from the food matrix and incorporated into mixed micelles in the gastrointestinal tract. The effects of different types of HDES and HDES with different molar ratios on the bioaccessibility of UA were investigated. The effects of different types of HDES-UA on the bioaccessibility of UA were investigated, with UA alone as a control. The effects of HDES composed of different molar ratios of HBA and HBD on the bioaccessibility of UA were further investigated.

[0026] like Figure 1 The results of the determination of the bioaccessibility of the blends provided by the present invention are shown, wherein A is the bioaccessibility of different blends of HDES and UA at a molar ratio of HBA to HBD of 1:1, and B is the bioaccessibility of menthol (Men) and oleic acid and menthol (Men) and capric acid at a molar ratio of HBA to HBD of 2:1, 1:1 and 1:2. Figure 1 From A, we can see that the bioavailability of UA alone is only 0.3452%. The low bioaccessibility of UA alone may be due to the poor water solubility of UA itself, which leads to its low solubility in digestive fluid. The low solubility of UA in digestive micelles leads to low bioaccessibility. The bioaccessibility of the HDES-UA system is significantly improved compared with that of UA alone. The results showed that it was also significantly improved compared with the DMSO-UA system (DMSO-UA 2.77%). From the perspective of HDES type, there is no obvious regularity with the increase of fatty acid chain length. After digestion, the bioaccessibility of UA in Men-Cia 1∶1-UA is significantly higher than that in other groups. In the presence of digestible lipids, small lipid molecules are mixed with phospholipids, bile salts and cholesterol to form a complex mixture of colloidal nanostructures (mixed micelles, vesicles, liquid crystals), which can encapsulate non-polar bioactive substances. This may cause Men-Cia 1∶1-UA to be encapsulated by HDES during digestion and transported to the intestinal stage, releasing UA and improving the bioaccessibility of UA. Figure 1It can be seen from B that in the HDES group of Men-Oa, as the molar ratio of oleic acid increases, its bioaccessibility decreases. Free linoleic acid and oleic acid have been shown to have inhibitory effects on pancreatic lipase in vitro. Therefore, as Oa increases, the inhibitory effect of Men-Oa-UA on pancreatic lipase increases, reducing the micellar phase produced during in vitro digestion. Therefore, as Oa increases, its bioaccessibility gradually decreases. On the contrary, the bioaccessibility of UA in the HDES blend composed of Men-Cia increases with the increase in the molar ratio of capric acid, and its bioaccessibility is as high as 23%. This shows that HDES composed of Men-Cia at a molar ratio of 1:2 can serve as a good carrier to deliver UA, which helps to improve the bioaccessibility of UA.

[0027] 3. Construction of Caco-2 cell model 3.1 Caco-2 cell culture Caco-2 cells were grown in 4 mL MEM (containing 20% ​​FBS) with a final pH of 7.2-7.4 at 37°C and 5% CO. 2 Incubate at 95% air and maintain humidity. Change the culture medium every day until the cells reach 80-90% confluence, then digest with trypsin for 2.5 min before subculturing.

[0028] 3.2 Caco-2 cell toxicity assay The cell viability after intestinal digestion was studied by MTT test, and the cytotoxicity of the samples was determined by MTT cell proliferation and cytotoxicity assay kit (Beijing Solarbio Science & Technology Co., Ltd). The experiment set up blank control group, positive control group and experimental group. The blank control group only added complete culture medium; the positive control group only added cell suspension without adding blend; the experimental group added cell suspension and blend. The specific operation was to inoculate 100 μL Caco-2 cells in a 96-well plate and keep the density at 1×10 5 cell / mL, incubated at 37 °C for 24 h to allow cells to attach; then the complete medium was removed, and 100 μL of a series of digests (filtered through a 0.22 μm membrane) diluted with medium were added to the experimental group, the positive control group was added with medium, and the blank control group continued to use only complete medium. After culturing for 24 h, 10 μL of MTT solution was placed in CO 2Continue to culture in the incubator for 4 hours. Add 100 μL of Formazan solution to each well and mix well. Usually, incubate at 37°C for about 3 to 4 hours until all the purple crystals are dissolved. Then use an ELISA reader to measure the absorbance of each well at 570 nm and calculate the cell viability (Viability %) of each well according to formula (2).

[0029] (2) OD in formula (2) T is the absorbance value of the experimental group; OD K is the absorbance value of the blank group; OD C is the absorbance value of the positive control group.

[0030] 3.3 Establishment of Caco-2 cell monolayer The Caco-2 cells that were growing well in the logarithmic phase were washed twice with PBS, digested with 1 mL of trypsin, and then digested with 4 mL of MEM medium. The cells were pipetted repeatedly to ensure that the cells were in a single state. The cells were counted using a hemocytometer under an inverted microscope at 100 times magnification. A certain volume of MEM medium was added to prepare a cell concentration of 1×10 5 cells / mL of cell suspension was inoculated into a pore size of 0.4 μm and 1.12 cm 2 Diameter collagen-coated polytetrafluoroethylene membrane (Corning Inc., MA, USA), i.e., 12-well Transwell plate. 0.5 mL of cell suspension was added to the apical side (AP), and 1.5 mL of culture medium was added to the basal side (BL). The medium was changed the next day. The medium was changed every other day for the next week, and every day after the second week. The cells were completely differentiated after about 21 days of culture.

[0031] 3.4. Measurement of transmembrane electrical resistance (TEER) of Caco-2 cells The transmembrane resistance of cultured Caco-2 cells was measured (0-21 days). Before the measurement, the electrodes were first immersed in 75% alcohol for 15 min for sterilization, taken out and air-dried, and then rinsed with HBSS. The culture medium on the AP and BL sides of the Tanswell plate was aspirated, and the cell layer was washed 1-2 times with HBSS. Then 0.5 mL of HBSS was added to the AP side and 1.5 mL of HBSS was added to the BL side. When measuring the resistance value, the short end of the electrode was vertically inserted into the AP side of the culture chamber (do not touch the cell membrane on the AP side), and the long end was immersed in the BL side of the culture chamber. After the reading stabilized, the resistance value was recorded. The measurement was performed every three days during the culture period. The TEER was calculated as follows (3):

[0032] (3) In formula (3), TEER is the transmembrane resistance of Caco-2 cells, in Ω·cm 2 ; Ω is the transmembrane resistance of the cell membrane in the experimental group, that is, the transmembrane resistance of the growing cells, in Ω; Ω 0 is the resistance of the blank group, that is, the resistance of the blank group without growing cells, Ω; 1.12 is the effective membrane area of ​​the 12-well Transwell plate culture chamber, unit: cm 2 .

[0033] Usually those with resistance values ​​higher than 500 Ω·cm 2 The integrity of the cell monolayer was evaluated using TEER. The Caco-2 cell monolayer constructed in the present invention was cultured for 21 d The TEER value was 510.87 ± 28.00 Ω·cm 2 It can be used for uptake and transport experiments of several subsequent UA digestion systems.

[0034] 3.5. Uptake by Caco-2 cells Caco-2 cells were seeded in 12-well plates at a density of 1×10 5 cells / well. The culture medium was changed every 2 days in the first week and once a day in the second week. The complete culture medium was removed after about 14 days of culture. The Caco-2 cells were rinsed with PBS three times and placed in the incubator for 30 min for the last time, and the buffer was discarded. The cell uptake of UA was tested by adding several UA digestion micelles (600 μL) diluted with PBS to the cells and incubating for 4 h. After incubation, the cell monolayer was washed with PBS three times to remove free samples. Then 1 mL PBS was added, and the cells were placed in an ultra-low temperature refrigerator at -80 °C and repeatedly frozen and thawed four times to disrupt the cells. The cell disruption solution was collected and ultrasonically treated (50% power, 5 min) and centrifuged at 14500 rpm for 10 min to obtain the supernatant. UA in the supernatant was extracted with anhydrous ethanol, and the UA content was determined by high performance liquid chromatography. The uptake rate of UA by Caco-2 cells was calculated according to formula (4).

[0035] (4) In formula (4) M cell is the amount of UA absorbed by the cells, M micelle is the UA content in the added micelle phase.

[0036] The cellular uptake rate, cellular secretion rate, total cellular uptake rate and bioavailability of micellar dilutions obtained after in vitro simulated digestion of solid UA and two systems of UA-HDES were studied. In order to explore the effects of HDES-UA with the highest bioaccessibility of Men-Cia and Men-Oa and different molar ratios of HBA and HBD on their bioavailability, four HDESs were selected for subsequent bioavailability determination experiments.

[0037] like Figure 2 The figure shows the uptake results of Caco-2 cells of the present invention, wherein A is the absorption rate of UA by Caco-2 cells cultured for 4 hours; B is the secretion rate of Caco-2 at different culture times; C is the total absorption rate of UA by Caco-2 cells cultured for 4 hours and D is the bioavailability of UA. Figure 2 It can be seen from A that after 4 h of incubation, the uptake rate of UA alone was only 3.164%, while the cellular uptake rate of different HDES-UAs was significantly improved. The cellular uptake rate of UA in Men-Oa is lower than that in Men-Cia. In addition, for Men-Cia-UA, as the molar ratio of capric acid in HDES increases, the cellular uptake rate of UA also increases. When the molar ratio of menthol and capric acid is 1:2, the cellular uptake rate is as high as 19.3%. The higher uptake of active substances is related to its good solubility in water and smaller mixed micelles. In general, delivery through HDES can increase the cellular uptake of UA, which provides a basis for the study of the bioavailability of bioactive substances.

[0038] 3.6. Transport in Caco-2 cells Caco-2 cells were seeded in 12-well plates at a density of 1×10 5 The medium was changed every 2 days in the first week and every day in the second week (21-25 days). 2 After culturing for 21 days under the conditions of , the cells were washed three times with HBSS and placed in an incubator for 30 min before being aspirated. In the direction from AL to BL, 0.5 mL of several micelle dilutions containing UA were added to the root tip (AL), and 1.5 mL of blank HBSS was added to the basolateral side. At the specified time (0.5, 1, 2, and 4 h), 200 μL of transport medium from the basolateral side (BL) was collected, and 200 μL of new HBSS was added to make up the liquid volume. UA was extracted with anhydrous ethanol, and the UA content was determined by high performance liquid chromatography. The UA secreted by Caco-2 cells was defined as the ratio of the UA content on the receiving side to the original UA content, and the UA secretion rate was calculated according to the following formula (5).

[0039] (5) In formula (5) M Bas is the content of UA in the basolateral solution, M intial It is the content before UA transportation.

[0040] Depend on Figure 2 As shown in B, the secretion of all systems in Caco-2 cells gradually increased with the extension of incubation time, and after 2 h of incubation, the increase in cell secretion rate showed a gradual trend, and the cell secretion rate of UA reached the highest after 4 h of incubation. Among them, Men-Cia 1∶2-UA had the highest cell secretion rate (61.99%) after 4 h of incubation, while the cell secretion rate of UA alone was only 5.55%. Similar to the situation of cell uptake, the cell secretion rate of HDES-UA composed of Men-Cia increased with the increase of the molar proportion of decanoic acid. The cell secretion rate of Men-Cia-UA is generally higher than that of Men-Oa-UA. Studies have shown that HDES may increase oral absorption by reducing the integrity of intestinal tight junctions. Therefore, by forming a blend of UA and HDES, the integrity of the tight junctions of Caco-2 cells may be destroyed to a certain extent, allowing UA to be transported from the upper layer of Caco-2 cells to the lower layer more smoothly in the presence of HDES, so that the cell secretion rate of UA in the blend is higher than that of UA alone.

[0041] 4. Determination of bioavailability Based on the above results of Caco-2 cell uptake and secretion rates of UA digestion system, the total absorption rate of UA in cells can be calculated according to formula (6): Cell absorption %); while the total bioavailability of UA ( Total bioavailability %) can be expressed as the product of total cellular absorption rate and bioaccessibility, see formula (7).

[0042] (6) (7) Depend on Figure 2 From the C, it can be seen that the nature of the carrier has an impact on cell absorption and transport to a certain extent. As expected, only a small amount of UA was absorbed in the single UA, and its total uptake rate was only 3.30%. The reason is that the low cell uptake rate and low cell secretion rate led to a very low total cell uptake rate. HDES-UA can increase the cell uptake rate and cell secretion rate of UA, thereby increasing its total cell uptake rate. The two different HDES showed different total cell uptake rates when delivering UA. Relatively speaking, the total cell uptake rate of Men-Cia-UA was higher than that of Men-Oa-UA.

[0043] The bioavailability of the encapsulated compound can be defined as the fraction absorbed into the systemic circulation, including bioaccessibility, cellular uptake, and transformation. Although UA has multiple bioactive functions, its limited aqueous solubility restricts its absorption in the body, thus limiting its functional activity.

[0044] Depend on Figure 2 It can be seen from the D that the bioavailability of pure UA solid is only 0.03%, which may be due to the poor water solubility and extremely low bioaccessibility of UA (0.3452%). The use of hydrophobic HDES to deliver UA can improve the bioavailability of UA in Caco-2 cells. For two different HDES, the bioavailability of Men-Cia-UA is higher than that of Men-Oa-UA. This shows that the HDES composed of Men-Cia is more suitable for delivering UA, a pentacyclic triterpenoid. For the HDES of Men-Cia, as the molar ratio changes from 2:1, 1:1 to 1:2, its bioavailability increases accordingly, reaching a maximum of 18.6%.

[0045] Based on the above research, the present invention provides a method for improving the oral bioavailability of terpenoid active ingredients in apple peel extract, especially ursolic acid. The specific examples are as follows: (1) A low eutectic solvent (HDES) is composed of a hydrogen acceptor (HBA) and a hydrogen donor (HBD), wherein the hydrogen acceptor is menthol, the hydrogen donor is butyric acid, hexanoic acid, n-octanoic acid, decanoic acid or oleic acid, and decanoic acid is most preferred, and the molar ratio of the hydrogen acceptor to the hydrogen donor is 2:1, 1:1 or 1:2, and 1:2 is most preferred.

[0046] (2) Mix 5 mL of HDES with 1% ursolic acid (UA) and vortex in a magnetic stirrer at 60 °C for 1 h to obtain a blended system UA-HDES.

[0047] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. A composition for improving the oral bioavailability of ursolic acid, characterized in that: The composition is a blended system comprising a low eutectic solvent and ursolic acid, wherein the low eutectic solvent is obtained by mixing a hydrogen acceptor and a hydrogen donor, the hydrogen acceptor is menthol, and the hydrogen donor is fatty acid.

2. A composition as claimed in claim 1 and a method for preparing the same, characterized in that: Here are the steps: (1) mixing a hydrogen acceptor and a hydrogen donor to obtain a low eutectic solvent; wherein the hydrogen acceptor is menthol; and the hydrogen donor is a fatty acid; (2) Mix the deep eutectic solvent and ursolic acid and vortex at 60°C.

3. The preparation method according to claim 2, characterized in that: The fatty acid comprises butyric acid, caproic acid, caprylic acid, capric acid or oleic acid.

4. The preparation method according to claim 2, characterized in that: In step (1), the molar ratio of the hydrogen acceptor to the hydrogen donor is 2:1, 1:1 or 1:

2.

5. The preparation method according to claim 2, characterized in that: The molar ratio of the hydrogen acceptor to the hydrogen donor is 1:1 or 1:

2.

6. The preparation method according to claim 2, characterized in that: The molar ratio of the hydrogen acceptor to the hydrogen donor is 1:

2.

7. The preparation method according to claim 2, characterized in that: The mass volume ratio of the ursolic acid is 1%.

8. The preparation method according to claim 2, characterized in that: The vortexing was performed in a magnetic stirrer for 1 h.

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