Biological preparation based on reduced coenzyme Q10 as well as preparation method and application of biological preparation

Abstract

The invention discloses a biological preparation based on reduced coenzyme Q10 as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The biological preparation is prepared from reduced coenzyme Q10, juniperus formosana extract, rubus idaeus extract and pharmaceutically acceptable auxiliary materials through a nano-emulsification process. Wherein the mass ratio of the reduced coenzyme Q10 to the juniperus formosana extract to the rubus idaeus extract is (2-10): (0.2-1): (0.2-1). A non-alcoholic fatty liver disease rat experiment proves that the biological agent can effectively regulate dyslipidemia, inhibit inflammation, relieve oxidative stress injury and improve liver mitochondrial dysfunction through a multi-target synergistic effect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to biological agents based on reduced coenzyme Q10, their preparation methods, and applications. Background Technology

[0002] Non-alcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases worldwide. Its spectrum encompasses simple hepatic steatosis (NAFL) and non-alcoholic steatohepatitis (NASH), and can further progress to cirrhosis and even hepatocellular carcinoma, making it a significant public health concern. The pathogenesis of this disease is complex, involving multiple core components such as insulin resistance, lipid metabolism disorders, and chronic inflammation. Studies have shown that mitochondrial dysfunction and its mediated oxidative stress play a crucial role in connecting these pathological processes and driving the occurrence and progression of NAFLD.

[0003] In NAFLD, excessive lipid accumulation in the liver promotes enhanced fatty acid β-oxidation, leading to increased electron flow in the mitochondrial electron transport chain (ETC) and elevated reactive oxygen species (ROS) production, potentially exceeding the clearance capacity of the endogenous antioxidant system. Excessive ROS can damage mitochondrial components and mitochondrial DNA, affecting ETC function and creating a vicious cycle of ROS accumulation and mitochondrial damage. This further activates inflammatory pathways such as the NLRP3 inflammasome and NF-κB, inducing hepatocyte apoptosis, pyroptosis, and ferroptosis, resulting in liver inflammation, fibrosis, and functional impairment. Therefore, targeted intervention of mitochondrial oxidative stress has become a promising strategy for the prevention and treatment of NAFLD.

[0004] At present, intervention strategies for mitochondrial oxidative stress are mainly divided into four categories, but each has certain limitations: (1) Conventional antioxidants (such as vitamin E, N-acetylcysteine, etc.) can directly remove ROS, but they have problems such as low bioavailability, insufficient mitochondrial targeting, and short duration of action, and their efficacy is limited in long-term use; (2) Mitochondrial-targeting drugs, such as mitochondrial-targeting peptide SS-31, mitochondrial-targeting antioxidant MitoQ, and coenzyme Q10 (CoQ10) with mitochondrial function, can specifically act on mitochondria and reduce oxidative stress by stabilizing ETC or removing mitochondrial ROS. For example, reduced coenzyme Q10 (panthenol), as an electron carrier and endogenous antioxidant, can improve hepatic steatosis by activating the AMPK pathway, but its effect on regulating systemic metabolic disorders and inhibiting multiple pathological processes such as inflammation and fibrosis is relatively limited when used alone; (3) Natural active ingredients (such as resveratrol, quercetin, curcumin, etc.) usually have multi-target effects and can synergistically exert antioxidant, anti-inflammatory and metabolic regulation effects by activating signaling pathways such as SIRT1 / PGC-1α and Nrf2 / Keap1, but their components are complex, their standardization is not uniform, and their mechanisms are not yet fully understood; (4) Signaling pathway regulators (such as AMPK agonists, Nrf2 activators, etc.) can regulate metabolism and antioxidant defense from upstream, but their clinical translation is not yet mature, and their specificity and safety need further verification. Therefore, their application still faces challenges.

[0005] In summary, existing intervention strategies are either insufficiently targeted, have limited effects, or lack clear mechanisms, making it difficult to comprehensively address the complex pathological network of NAFLD. Therefore, developing a synergistic compound preparation with multiple mechanisms, including source-targeted repair, systemic metabolic regulation, and anti-inflammatory and anti-fibrotic effects, has become an important research direction for overcoming current treatment bottlenecks.

[0006] Based on this, the present invention uses reduced coenzyme Q10 as a base and combines it with juniper extract and pine berry extract to provide a comprehensive, targeted and relatively clear treatment plan for NAFLD, thereby enriching the drug treatment options for this disease. Summary of the Invention

[0007] Therefore, this invention provides a biological agent based on reduced coenzyme Q10, its preparation method, and its application, in order to overcome the shortcomings of the prior art.

[0008] To achieve the above objectives, the present invention provides the following technical solution: According to a first aspect of the invention, a biological agent based on reduced coenzyme Q10 is provided, comprising reduced coenzyme Q10, juniper extract and pine berry extract.

[0009] Furthermore, the mass ratio of the reduced coenzyme Q10, juniper extract and pine berry extract is (2-10):(0.2-1):(0.2-1).

[0010] Furthermore, the mass ratio of the reduced coenzyme Q10, juniper extract, and pine berry extract is (4-10):(0.2-0.8):(0.2-0.6).

[0011] Furthermore, it also includes pharmaceutically acceptable excipients.

[0012] According to a second aspect of the present invention, a method for preparing a biological agent based on reduced coenzyme Q10 is provided, comprising the following steps: (1) Preheat the lipid matrix at 55-65℃ and stir continuously, then add reduced coenzyme Q10, juniper extract and pine pine extract in sequence until the system is mixed evenly to form a homogeneous oil phase. (2) Mix the additive with part of the solvent and stir continuously at 55-65℃ for 12-20 min until the system is uniformly dispersed and a transparent and homogeneous aqueous phase is formed; (3) Under high-speed shearing, the oil phase in step (1) is slowly added to the aqueous phase in step (2) to obtain a crude emulsion; (4) The crude emulsion in step (3) is subjected to homogenization treatment at a pressure of 40-60 MPa for 2-3 cycles. After homogenization, it is diluted with the remaining solvent to the full volume of the prescription and filtered through a 0.45 μm microporous membrane to obtain the biological agent.

[0013] Furthermore, the preparation method of the juniper extract mentioned in step (1) is as follows: Pulverize dried Siberian juniper branches and leaves, pass through an 80-mesh sieve, add 50% ethanol aqueous solution at a material-to-liquid ratio of 1:25 (g / mL), and extract by reflux at 55-65℃ for 1-3 hours; filter, and repeat the extraction once with the same method on the residue; combine the two filtrates, concentrate under reduced pressure to 1 / 10 of the original volume, disperse with hot water at 55-65℃ to obtain a suspension, defatted with petroleum ether 1-3 times; extract the defatted aqueous phase with n-butanol 2-4 times, combine the n-butanol phases, concentrate under reduced pressure to a relative density of 1.1-1.2, and vacuum dry at 45-55℃ to obtain juniper extract.

[0014] Furthermore, the volume ratio of petroleum ether to suspension is (1-2):1, and the volume ratio of n-butanol to aqueous phase is (1-2):1.

[0015] Furthermore, the volume ratio of petroleum ether to suspension is 1:1; the volume ratio of n-butanol to aqueous phase is 1:1; the heating reflux temperature is 60°C for 2 hours; and the vacuum drying temperature is 50°C.

[0016] Furthermore, the preparation method of the pine-red plum extract described in step (2) is as follows: Pulverized dried pine needle plum leaves were crushed and passed through a 60-mesh sieve. A 70% acetone aqueous solution was added at a material-to-liquid ratio of 1:20 (g / mL). Extraction was performed with ultrasonic assistance, followed by filtration. The residue was extracted once more under the same conditions. The two filtrates were combined and concentrated under reduced pressure to 1 / 10 of the original volume to obtain an aqueous concentrate. This concentrate was extracted 2-4 times with ethyl acetate. All ethyl acetate phases were combined and, while maintaining a constant temperature, concentrated under reduced pressure to a relative density of 1.1-1.2. The concentrate was then vacuum dried at 40-50℃ to obtain the pine needle plum extract.

[0017] Furthermore, the volume ratio of ethyl acetate to the aqueous concentrate is (1-2):1.

[0018] Furthermore, the volume ratio of the ethyl acetate to the aqueous concentrate is 1:1; the vacuum drying temperature is 45°C.

[0019] Furthermore, the conditions for ultrasound-assisted extraction were: ultrasound power 300W, temperature 40℃, and time 30min.

[0020] Furthermore, the parameters for high-speed shearing in step (3) are: rotation speed 10000-15000 r / min, time 3-5 min.

[0021] Further, the adjuvants are emulsifiers and stabilizers, and the mass ratio of the emulsifiers and stabilizers is 8:1 to 12:1; the lipid matrix includes, but is not limited to, ethyl oleate; the emulsifier includes, but is not limited to, soybean lecithin; the stabilizer includes, but is not limited to, poloxamer 188; and the solvent includes, but is not limited to, deionized water.

[0022] Furthermore, the mass ratio of the emulsifier to the stabilizer is 10:1.

[0023] According to a third aspect of the invention, the use of the biological agent prepared by the method described above in the preparation of medicaments for the treatment and / or prevention of non-alcoholic fatty liver disease is provided.

[0024] Compared with the prior art, the present invention has the following advantages: (1) Multi-target synergistic intervention This invention's biological agent combines reduced coenzyme Q10, juniper extract, and pine berry extract to target key stages in the development of NAFLD: reduced coenzyme Q10 directly targets liver cell mitochondria, enhancing fatty acid oxidation efficiency and neutralizing oxidative stress; juniper extract regulates lipid metabolism and inhibits inflammatory responses by modulating metabolic and inflammatory pathways such as AMPK / PPAR-α; and pine berry extract exerts anti-inflammatory and antioxidant effects through its active components (such as β-caryophyllene and triketones and other terpenoids), and may inhibit the activation potential of hepatic stellate cells by targeting the initiation stage of liver fibrosis, thus potentially blocking the progression of NAFLD to fibrosis and cirrhosis. Animal experiments have confirmed that the combined use of the three components is significantly superior to combinations containing only reduced coenzyme Q10, as well as combinations of reduced coenzyme Q10 with either of the two plant extracts, demonstrating synergistic therapeutic advantages in regulating blood lipids, inhibiting inflammation, reducing oxidative stress damage, and improving liver mitochondrial dysfunction.

[0025] (2) Good safety Thanks to the fact that all active ingredients are of natural origin and exhibit synergistic effects, this biopharmaceutical demonstrates good biocompatibility. Preliminary cytotoxicity experiments showed that the biopharmaceutical achieved a cell viability rate of over 85% in HaCaT cells.

[0026] (3) The preparation process is reasonable. This biopharmaceutical employs a nanoemulsification process to integrate hydrophobic reduced coenzyme Q10 with two plant extracts into a homogeneous and stable emulsion system. This process not only solves the problems of dissolution and dispersion of the components, but also creates a nano / submicron-scale drug delivery system that helps improve bioavailability, ensuring that the three active ingredients can be synergistically delivered to the liver target site, thereby effectively exerting a multi-target synergistic effect in vivo.

[0027] (4) Clear application prospects Based on a clear pathological mechanism and component synergy theory, this biological agent has been demonstrated in animal models to have a comprehensive effect on improving the core pathological links of NAFLD (lipid deposition, inflammation, oxidative stress, and mitochondrial dysfunction), and has the potential to be developed into an innovative drug or functional agent for the prevention and / or treatment of NAFLD. Attached Figure Description

[0028] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0029] The structures, proportions, sizes, etc. illustrated in this specification are only for the purpose of assisting those skilled in the art in understanding and reading the content disclosed herein, and are not intended to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in the proportions, or adjustments to the size, without affecting the effects and objectives that the present invention can produce, should still fall within the scope of the technical content disclosed in the present invention.

[0030] Figure 1 These are the cytotoxicity test results of the biological agents in the embodiments of the present invention; statistical analysis: compared with the blank control group, #### P <0.0001, ## P <0.01; compared with the model group, **** P <0.0001, *** P <0.001, ** P <0.01, * P <0.05; Figure 2 The results of the effects of biological agents on total cholesterol (TC) levels in rats with non-alcoholic fatty liver disease (NAFLD) in the embodiments and comparative examples of this invention; statistical analysis: compared with the blank control group, #### P <0.0001, ## P <0.01; compared with the model group, **** P <0.0001, *** P <0.001, ** P <0.01, * P <0.05; Figure 3 The results of the effects of biological agents on low-density lipoprotein cholesterol (LDL-C) levels in rats with non-alcoholic fatty liver disease (NAFLD) in the embodiments and comparative examples of this invention; statistical analysis: compared with the blank control group, #### P <0.0001, ## P <0.01; compared with the model group, **** P <0.0001, *** P <0.001, ** P<0.01, * P <0.05; Figure 4 The results of the effects of biological agents on high-density lipoprotein cholesterol (HDL-C) levels in rats with non-alcoholic fatty liver disease (NAFLD) in the embodiments and comparative examples of this invention; statistical analysis: compared with the blank control group, #### P <0.0001, ## P <0.01; compared with the model group, **** P <0.0001, *** P <0.001, ** P <0.01, * P <0.05; Figure 5 The results of the effects of biological agents on the levels of tumor necrosis factor-α (TNF-α) in rats with non-alcoholic fatty liver disease (NAFLD) in the embodiments and comparative examples of this invention; statistical analysis: compared with the blank control group, #### P <0.0001, ## P <0.01; compared with the model group, **** P <0.0001, *** P <0.001, ** P <0.01, * P <0.05; Figure 6 The results of the effects of biological agents on the levels of interleukin-1β (IL-1β) in rats with non-alcoholic fatty liver disease (NAFLD) in the embodiments and comparative examples of this invention; statistical analysis: compared with the blank control group, #### P <0.0001, ## P <0.01; compared with the model group, **** P <0.0001, *** P <0.001, ** P <0.01, * P <0.05; Figure 7The results of the effects of biological agents on malondialdehyde (MDA) levels in rats with non-alcoholic fatty liver disease (NAFLD) in the embodiments and comparative examples of this invention; statistical analysis: compared with the blank control group, #### P <0.0001, ## P <0.01; compared with the model group, **** P <0.0001, *** P <0.001, ** P <0.01, * P <0.05; Figure 8 The results of the effects of biological agents on superoxide dismutase (SOD) activity in rats with non-alcoholic fatty liver disease (NAFLD) in the embodiments and comparative examples of this invention; statistical analysis: compared with the blank control group, #### P <0.0001, ## P <0.01; compared with the model group, **** P <0.0001, *** P <0.001, ** P <0.01, * P <0.05; Figure 9 The results of the effects of biological agents on the activity of mitochondrial respiratory chain complex I in rats with non-alcoholic fatty liver disease (NAFLD) in the embodiments and comparative examples of this invention; statistical analysis: compared with the blank control group, #### P <0.0001, ## P <0.01; compared with the model group, **** P <0.0001, *** P <0.001, ** P <0.01, * P <0.05; Figure 10 The results of the effects of biological agents on adenosine triphosphate (ATP) levels in rats with non-alcoholic fatty liver disease (NAFLD) in the embodiments and comparative examples of this invention; statistical analysis: compared with the blank control group, #### P<0.0001, ## P <0.01; compared with the model group, **** P <0.0001, *** P <0.001, ** P <0.01, * P <0.05. Detailed Implementation

[0031] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] According to a first aspect of the invention, a biological agent based on reduced coenzyme Q10 is provided, comprising reduced coenzyme Q10, juniper extract and pine berry extract.

[0033] Furthermore, the mass ratio of reduced coenzyme Q10, juniper extract and pine pine extract is (2-10):(0.2-1):(0.2-1).

[0034] Furthermore, the mass ratio of reduced coenzyme Q10, juniper extract and pine berry extract is (4-10):(0.2-0.8):(0.2-0.6).

[0035] Furthermore, it also includes pharmaceutically acceptable excipients.

[0036] According to a second aspect of the present invention, a method for preparing a biological agent based on reduced coenzyme Q10 is provided, comprising the following steps: (1) Preheat the lipid matrix at 55-65℃ and stir continuously, then add reduced coenzyme Q10, juniper extract and pine pine extract in sequence until the system is mixed evenly to form a homogeneous oil phase. (2) Mix the additive with part of the solvent and stir continuously at 55-65℃ for 12-20 min until the system is uniformly dispersed and a transparent and homogeneous aqueous phase is formed; (3) Under high-speed shearing, the oil phase in step (1) is slowly added to the aqueous phase in step (2) to obtain a crude emulsion; (4) The crude emulsion in step (3) is subjected to homogenization treatment at a pressure of 40-60 MPa for 2-3 cycles. After homogenization, it is diluted with the remaining solvent to the full volume of the prescription and filtered through a 0.45 μm microporous membrane to obtain the biological agent.

[0037] Furthermore, the preparation method of the juniper extract in step (1) is as follows: Pulverize dried Siberian juniper branches and leaves, pass through an 80-mesh sieve, add 50% ethanol aqueous solution at a material-to-liquid ratio of 1:25 (g / mL), and extract by reflux at 55-65℃ for 1-3 hours; filter, and repeat the extraction once with the same method on the residue; combine the two filtrates, concentrate under reduced pressure to 1 / 10 of the original volume, disperse with hot water at 55-65℃ to obtain a suspension, defatted with petroleum ether 1-3 times; extract the defatted aqueous phase with n-butanol 2-4 times, combine the n-butanol phases, concentrate under reduced pressure to a relative density of 1.1-1.2, and vacuum dry at 45-55℃ to obtain juniper extract.

[0038] Furthermore, the volume ratio of petroleum ether to suspension is (1-2):1, and the volume ratio of n-butanol to aqueous phase is (1-2):1.

[0039] Furthermore, the volume ratio of petroleum ether to suspension is 1:1; the volume ratio of n-butanol to aqueous phase is 1:1; the heating reflux temperature is 60℃ for 2 hours; and the vacuum drying temperature is 50℃.

[0040] Furthermore, the preparation method of the pine-red plum extract in step (2) is as follows: Pulverized dried pine needle plum leaves were crushed and passed through a 60-mesh sieve. A 70% acetone aqueous solution was added at a material-to-liquid ratio of 1:20 (g / mL). Extraction was performed with ultrasonic assistance, followed by filtration. The residue was extracted once more under the same conditions. The two filtrates were combined and concentrated under reduced pressure to 1 / 10 of the original volume to obtain an aqueous concentrate. This concentrate was extracted 2-4 times with ethyl acetate. All ethyl acetate phases were combined and, while maintaining a constant temperature, concentrated under reduced pressure to a relative density of 1.1-1.2. The concentrate was then vacuum dried at 40-50℃ to obtain the pine needle plum extract.

[0041] Furthermore, the volume ratio of ethyl acetate to the aqueous concentrate is (1-2):1.

[0042] Furthermore, the volume ratio of ethyl acetate to the aqueous concentrate was 1:1; the vacuum drying temperature was 45°C.

[0043] Furthermore, the conditions for ultrasound-assisted extraction were: ultrasound power 300W, temperature 40℃, and time 30min.

[0044] Furthermore, the parameters for high-speed shearing in step (3) are: rotation speed 10000-15000 r / min, time 3-5 min.

[0045] Furthermore, the adjuvants are emulsifiers and stabilizers, with a mass ratio of emulsifier to stabilizer of 8:1 to 12:1; the lipid matrix includes, but is not limited to, ethyl oleate; the emulsifier includes, but is not limited to, soybean lecithin; the stabilizer includes, but is not limited to, poloxamer 188; and the solvent includes, but is not limited to, deionized water.

[0046] Furthermore, the mass ratio of emulsifier to stabilizer is 10:1.

[0047] According to a third aspect of the invention, the use of the biological agent prepared by the method is provided in the preparation of medicaments for the treatment and / or prevention of non-alcoholic fatty liver disease.

[0048] The reagents used in this experiment were obtained from the following sources: Human liver cancer cell line (HepG2 cells) was purchased from Beijing Eta Biotechnology Co., Ltd. 0.5% sodium carboxymethyl cellulose aqueous solution, purchased from Beijing Solarbio Science & Technology Co., Ltd. 60% high-fat feed, purchased from Beijing Fubo Biotechnology Co., Ltd.; Pioglitazone hydrochloride (CAS: 112529-15-4) and ethyl oleate (CAS: 111-62-6) were both purchased from Shanghai Sangon Biotech Co., Ltd. Reduced coenzyme Q10 (CAS: 992-78-9), soybean lecithin (CAS: 8002-43-5), and poloxamer 188 (CAS: 691397-13-4) were all purchased from Sichuan Weikeqi Biotechnology Co., Ltd.

[0049] Preparation Example 1 Preparation of Juniperus chinensis extract Dry Siberian juniper branches and leaves were pulverized and passed through an 80-mesh sieve. 10g of juniper powder was added to 250mL of 50% ethanol aqueous solution and extracted by reflux at 60℃ for 2h. The mixture was filtered, and the residue was extracted once more using the same method. The two filtrates were combined and concentrated under reduced pressure to 1 / 10 of the original volume. The mixture was dispersed by stirring with 60℃ hot water and defatted twice with an equal volume of petroleum ether. The petroleum ether phase was discarded. The defatted aqueous phase was extracted three times with an equal volume of n-butanol. All n-butanol phases were combined and concentrated under reduced pressure to a relative density of 1.1-1.2. The mixture was then vacuum dried at 50℃ to obtain the juniper extract.

[0050] Preparation Example 2 Preparation of Pine-leaf Plum Extract Take dried pine needle plum leaves, pulverize them, pass them through a 60-mesh sieve, weigh 10g of pine needle plum powder and add it to 200mL of 70% acetone aqueous solution. Sonicate at 300W power and 40℃ for 30min, filter, and repeat the extraction once with the residue under the same conditions. Combine the two filtrates and concentrate them under reduced pressure at 40℃ to 1 / 10 of the original volume. Extract three times with an equal volume of ethyl acetate. Combine all ethyl acetate phases and concentrate them under reduced pressure to a relative density of 1.1-1.2. Dry them under vacuum at 45℃ to obtain the pine needle plum extract.

[0051] Example 1 5.4 mg of ethyl oleate was preheated at 50 °C and stirred continuously. 4 mg of reduced coenzyme Q10, 0.2 mg of juniper extract, and 0.2 mg of pine berry extract were added sequentially until the system was homogeneous and formed a uniform oil phase. 15 mg of soybean lecithin, 1.5 mg of poloxamer 188, and 2 mL of deionized water were mixed and stirred continuously at 65 °C for 15 min until the system was uniformly dispersed and formed a transparent and homogeneous aqueous phase. The oil phase was slowly added to the aqueous phase, and the mixture was sheared at 10000 r / min for 5 min to obtain an emulsion. The crude emulsion was homogenized three times under a pressure of 50 MPa. After homogenization, the emulsion was diluted with deionized water to a final volume of 5 mL. Finally, the emulsion was filtered through a 0.45 μm microporous membrane to obtain the biological agent.

[0052] Example 2 8.4 mg of ethyl oleate was preheated at 50 °C and stirred continuously. 6 mg of reduced coenzyme Q10, 0.5 mg of juniper extract, and 0.4 mg of pine berry extract were added sequentially until the system was homogeneous and formed a uniform oil phase. 17 mg of soybean lecithin, 1.7 mg of poloxamer 188, and 2 mL of deionized water were mixed and stirred continuously at 65 °C for 15 min until the system was uniformly dispersed and formed a transparent and homogeneous aqueous phase. The oil phase was slowly added to the aqueous phase, and the mixture was sheared at 10000 r / min for 5 min to obtain an emulsion. The crude emulsion was homogenized three times under a pressure of 50 MPa. After homogenization, the emulsion was diluted with deionized water to a final volume of 5 mL. Finally, the emulsion was filtered through a 0.45 μm microporous membrane to obtain the biological agent.

[0053] Example 3 13.9 mg of ethyl oleate was preheated at 50 °C and stirred continuously. 10 mg of reduced coenzyme Q10, 0.8 mg of juniper extract, and 0.6 mg of pine berry extract were added sequentially until the system was homogeneous and formed a uniform oil phase. 27 mg of soybean lecithin, 2.7 mg of poloxamer 188, and 2 mL of deionized water were mixed and stirred continuously at 65 °C for 15 min until the system was uniformly dispersed and formed a transparent and homogeneous aqueous phase. The oil phase was slowly added to the aqueous phase, and the mixture was sheared at 10000 r / min for 5 min to obtain an emulsion. The crude emulsion was homogenized three times under a pressure of 50 MPa. After homogenization, the emulsion was diluted with deionized water to a final volume of 5 mL. Finally, the emulsion was filtered through a 0.45 μm microporous membrane to obtain the biological agent.

[0054] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that juniper extract is missing in step (1).

[0055] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that the pine pine extract is missing in step (1).

[0056] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that step (1) lacks juniper extract and pine pine extract.

[0057] Test Example 1 HepG2 cells were cultured in complete medium (DMEM containing 10% fetal bovine serum) and routinely passaged in an incubator at 37°C and 5% CO2 to form a single-cell suspension. The cultured cells were then seeded into 96-well plates at a density of 5 × 10⁶ cells per well. 3 After the cells adhered to the culture medium, the cells were replaced and the following grouping and treatment were performed: Experimental groups: Example 1 group: replaced with a complete culture medium containing the biological agent prepared in Example 1; Example 2 group: replaced with a complete culture medium containing the biological agent prepared in Example 2; Example 3 group: replaced with a complete culture medium containing the biological agent prepared in Example 3. Negative control group: replaced with an equal volume of complete culture medium without biological agents; Positive control group: replaced with complete culture medium containing 10 µmol / L doxorubicin; Blank control group: Contains no cells, only an equal volume of complete culture medium; Each group has 8 duplicate holes.

[0058] Incubate at 37℃ and 5% CO2 for 72 h, then add 10 μL of MTT solution (5 mg / mL) to each well and incubate for another 4 h. Carefully aspirate all supernatant from the wells, then add 100 µL of dimethyl sulfoxide (DMSO) to each well. Place the culture plate on a shaker and shake at low speed for 10 min. Measure the absorbance (OD value) of the culture at 490 nm using a microplate reader and calculate the cell viability. The specific calculation formula is as follows: ; Depend on Figure 1 It can be seen that the cell survival rates of groups 1, 2 and 3 in Examples are (96.05±1.15)%, (93.47±1.73)% and (86.83±2.61)%, respectively, indicating that the biological agent of the present invention has good biological safety.

[0059] Test Example 2 1. Establishment of non-alcoholic fatty liver disease (NAFLD) rats Healthy SD rats weighing 200-230g were used and acclimatized for one week under standard experimental conditions. They were then weighed and randomly divided into two groups: the normal control group was fed a normal maintenance diet throughout the entire period; the model group was fed a 60% high-fat diet for 84 consecutive days to induce the NAFLD model.

[0060] 2. Experimental grouping and dosing regimen Rats that successfully developed the model were randomly divided into 8 groups of 8 rats each, with a blank control group (normally fed rats), for a total of 9 groups. The specific grouping and drug administration regimens are as follows: Blank control group: an equal volume of 0.5% sodium carboxymethyl cellulose aqueous solution; Model control group: an equal volume of 0.5% sodium carboxymethyl cellulose aqueous solution; Positive control group: pioglitazone hydrochloride; Low-dose group: the biological agent in Example 1; Medium-dose group: Biologic agent from Example 2; High-dose group: Biologics from Example 3; Comparative Example 1: The biological agent in Comparative Example 1; Comparative Example 2: Biological agent in Comparative Example 2; Comparative Example 3: Biological agents in Comparative Example 3; Administer once daily by gavage at a dose of 20 mg / kg for 8 consecutive weeks.

[0061] 3. Sample Collection and Testing 3.1 Serum and Tissue Collection Twelve hours after the last administration, rats were anesthetized by intraperitoneal injection of 20% urethane (0.5 mL / 100 g). Blood was collected via the abdominal aorta, allowed to stand at room temperature for 0.5 hours, and then centrifuged at 3000 r / min for 15 minutes at 4°C to separate the serum. The serum was then aliquoted and stored at -80°C for later use. The rats were subsequently sacrificed, and liver tissue from the same lobe was divided into two parts: one part was rinsed with physiological saline pre-cooled at 4°C, flash-frozen in liquid nitrogen, and stored at -80°C for preparing liver tissue homogenate; the other part was placed in mitochondrial separation medium pre-cooled at 4°C for preparing mitochondrial suspension.

[0062] 3.2 Blood lipid level testing Using a fully automated biochemical analyzer and its matching reagent kits, and strictly following the instructions, the serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) of each group of rats were measured.

[0063] 3.3 Serum inflammatory factor detection The levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in the serum of rats in each group were measured using an enzyme-linked immunosorbent assay (ELISA) kit.

[0064] 3.4 Detection of liver oxidative stress indicators A 10% tissue homogenate was prepared from liver tissue stored at -80℃. The homogenate was centrifuged at 12000 r / min for 10 min at 4℃. The supernatant was collected and the malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were determined using a standardized kit.

[0065] 3.5 Liver mitochondrial function testing Fresh liver tissue was collected and rapidly minced under ice bath conditions. An appropriate amount of pre-cooled mitochondrial separation medium was added, and the mixture was homogenized. Mitochondria were then separated using differential centrifugation (4℃, 600 r / min for 10 min, supernatant collected; subsequently, 4℃, 12000 r / min for 15 min, precipitate collected as mitochondria). The mitochondrial suspension was resuspended in mitochondrial preservation solution. The protein concentration of the mitochondrial suspension was determined using the BCA method for standardization, and the activity of mitochondrial respiratory chain complex I (expressed as NADH dehydrogenase activity) and adenosine triphosphate (ATP) content were measured using a dedicated kit.

[0066] 4. Statistical Analysis GraphPad Prism 9 was used for data analysis, and the quantitative data are expressed as mean ± standard deviation (mean ± SD). p A value <0.05 is considered statistically significant.

[0067] 5 Results and Analysis 5.1 Blood lipid level test results Depend on Figure 2-4 As shown in the figure, the changes in serum levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) in rats of each group after treatment can be summarized as follows: Compared with the blank control group, the levels of TC and LDL-C in the model group rats were significantly increased ( p＜ 0.0001), HDL-C levels decreased significantly ( p＜ The result of 0.01 indicates that the NAFLD model was successfully established.

[0068] (1) TC level: Compared with the model control group, the TC levels of rats in each treatment group decreased to some extent. Among them, the reduction effect of pioglitazone hydrochloride (positive drug control group) and low, medium and high dose biological agents (containing reduced coenzyme Q10, juniper extract and pine berry extract) was extremely significant. p <0.0001); the reduction effect of biological agents (containing reduced coenzyme Q10 and juniper extract) in comparative group 2 was extremely significant ( p <0.001). The reduction effect of the biological agent (containing reduced coenzyme Q10 and pine berry extract) in Comparative Example 1 and Comparative Example 3 (containing reduced coenzyme Q10) was not statistically significant.

[0069] (2) LDL-C level: Compared with the model control group, the LDL-C levels of rats in each treatment group decreased to some extent. Among them, the reduction effect of pioglitazone hydrochloride (positive drug control group) and the high-dose biological agent group (containing reduced coenzyme Q10, juniper extract and pine berry extract) was extremely significant. p <0.0001); the medium-dose biological agent group (containing reduced coenzyme Q10, juniper extract and pine needle extract) showed a significant reduction in efficacy ( p <0.01); while the reduction effect of the low-dose biological agent group (containing reduced coenzyme Q10, juniper extract and pine pine extract), comparative group 1 (containing reduced coenzyme Q10 and pine pine extract) and comparative group 2, as well as the biological agent (containing reduced coenzyme Q10) in comparative group 3, was not statistically significant.

[0070] (3) HDL-C level: Compared with the model control group, the HDL-C levels of rats in each treatment group were increased to some extent. Among them, the increase was significant in pioglitazone hydrochloride (positive drug control group) and the high-dose biological agent group (containing reduced coenzyme Q10, juniper extract and pine berry extract). p<0.05); while the increase in the effects of biological agents (containing reduced coenzyme Q10, juniper extract and pine pine extract) in the low- and medium-dose biological agent groups, in comparative group 1 (containing reduced coenzyme Q10 and pine pine extract) and comparative group 2, and in comparative group 3 (containing reduced coenzyme Q10) was not statistically significant.

[0071] The results above show that the biological agent of the present invention is most effective in reducing TC and LDL-C levels and increasing HDL-C levels, and is superior to the comparative group that lacks Juniperus chinensis extract and / or Prunus armeniaca extract.

[0072] 5.2 Serum inflammatory factor detection results Depend on Figure 5-6 As shown in the figure, the serum levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in rats of each group after treatment revealed the following: Compared with the blank control group, the levels of TNF-α and IL-1β in the model group rats were significantly increased ( p＜ The result was 0.0001, indicating that the inflammation model was successfully established.

[0073] (1) TNF-α level: Compared with the model control group, the TNF-α levels of rats in each treatment group decreased to some extent. Among them, the reduction effect of pioglitazone hydrochloride (positive drug control group) and medium and high dose biological agents (containing reduced coenzyme Q10, juniper extract and pine berry extract) was extremely significant. p <0.0001); the low-dose biological agent group (containing reduced coenzyme Q10, juniper extract and pine needle extract) showed a significant reduction in efficacy ( p <0.01); the reduction effect of biological agents (containing reduced coenzyme Q10 and pine needle extract) in both comparative group 1 (containing reduced coenzyme Q10 and juniper extract) and comparative group 2 was significant ( p <0.05). However, the reducing effect of biological agents (containing reduced coenzyme Q10) in the three control groups was not statistically significant.

[0074] (2) IL-1β level: Compared with the model control group, the IL-1β levels of rats in each treatment group decreased to some extent. Among them, the reduction effect of pioglitazone hydrochloride (positive drug control group) and low, medium and high dose biological agents (containing reduced coenzyme Q10, juniper extract and pine berry extract) was extremely significant. p <0.0001); the reduction effect of biological agents (containing reduced coenzyme Q10 and pine needle extract) in both Comparative Example 1 (containing reduced coenzyme Q10 and juniper extract) and Comparative Example 2 was significant ( p<0.01); the reduction effect of biological agents (containing reduced coenzyme Q10) was significant in the three control groups. p <0.05).

[0075] The results above indicate that low, medium, and high doses of biological agents containing reduced coenzyme Q10, juniper extract, and pine needle plum extract all exhibited strong anti-inflammatory effects and significantly reduced TNF-α and IL-1β levels. Furthermore, the combined use of reduced coenzyme Q10 with juniper extract or pine needle plum extract also demonstrated certain anti-inflammatory effects.

[0076] 5.3 Results of Liver Oxidative Stress Indicators Depend on Figure 7-8 As shown in the figure, the results of the malondialdehyde (MDA) content and superoxide dismutase (SOD) activity tests in the liver tissue of rats in each group after drug treatment indicate that: Compared with the blank control group, the MDA content in the model group rats was significantly increased ( p＜ 0.01), SOD activity decreased significantly ( p＜ The result was 0.0001, indicating that the oxidative stress model was successfully established.

[0077] (1) MDA content: Compared with the model control group, the MDA content of rats in each treatment group decreased to a certain extent. Among them, the reduction effect of pioglitazone hydrochloride (positive drug control group) and high-dose biological agent group (containing reduced coenzyme Q10, juniper extract and pine berry extract) was significant. p <0.05); There was no statistically significant reduction in the effects of biological agents (containing reduced coenzyme Q10, juniper extract and pine pine extract) in the low- and medium-dose biological agent groups, comparative group 1 (containing reduced coenzyme Q10 and pine pine extract), comparative group 2, and comparative group 3 (containing reduced coenzyme Q10).

[0078] (2) SOD activity: Compared with the model control group, the SOD activity of rats in each treatment group was increased to a certain extent. Among them, the increase in SOD activity of pioglitazone hydrochloride (positive drug control group), low, medium and high dose biological agent groups (containing reduced coenzyme Q10, juniper extract and pine pine extract), comparative group 1 (containing reduced coenzyme Q10 and pine pine extract) and comparative group 2 (containing reduced coenzyme Q10 and juniper extract), and comparative group 3 (containing reduced coenzyme Q10) was extremely significant. p＜ 0.0001).

[0079] The results above show that the high-dose biological agent of the present invention is comparable to the positive control drug (pioglitazone hydrochloride) in improving liver oxidative stress damage, and all experimental groups containing reduced coenzyme Q10 can significantly enhance SOD activity, but reducing MDA content must rely on the synergistic effect of the three components.

[0080] 5.4 Results of liver mitochondrial function tests Depend on Figure 9-10 As shown in the figure, after drug treatment, the test results of mitochondrial respiratory chain complex I (expressed as NADH dehydrogenase activity) activity and adenosine triphosphate (ATP) content (normalized by mitochondrial protein content) in the liver tissue of rats in each group show that: Compared with the blank control group, the activity of mitochondrial respiratory chain complex I and the ATP content of rats in the model group were significantly reduced. p＜ The result was 0.0001, indicating that the mitochondrial dysfunction model was successfully established.

[0081] (1) Mitochondrial respiratory chain complex I activity: Compared with the model control group, the activity of mitochondrial respiratory chain complex I in rats of each drug administration group was increased to a certain extent. Among them, the increase was significant with pioglitazone hydrochloride (positive drug control group). p <0.01); the high-dose biological agent group (containing reduced coenzyme Q10, juniper extract and pine needle extract) showed a significant increase in efficacy ( p <0.05); There was no statistically significant increase in the effects of biological agents (containing reduced coenzyme Q10, juniper extract and pine pine extract) in the low- and medium-dose biological agent groups, comparative group 1 (containing reduced coenzyme Q10 and pine pine extract), comparative group 2, and comparative group 3 (containing reduced coenzyme Q10).

[0082] (2) ATP content: Compared with the model control group, the ATP content of rats in each drug-treated group was increased to a certain extent. Among them, the increase of pioglitazone hydrochloride (positive drug control group) was extremely significant. p <0.001); the high-dose biological agent group (containing reduced coenzyme Q10, juniper extract and pine needle extract) showed a significant increase in efficacy ( p <0.01); the increase in the medium-dose biological agent group (containing reduced coenzyme Q10, juniper extract and pine needle extract) was significant ( p <0.05); There was no statistically significant increase in the effect of biological agents (containing reduced coenzyme Q10, juniper extract and pine pine extract) in the low-dose biological agent group, comparative group 1 (containing reduced coenzyme Q10 and pine pine extract), comparative group 2, and comparative group 3 (containing reduced coenzyme Q10).

[0083] The results above show that the biological agent of the present invention can improve the liver mitochondrial energy metabolism function (enhance the activity of complex I and ATP production), but the effect is weaker than that of the positive control drug (pioglitazone hydrochloride). The comparative group lacking Juniperus chinensis extract and / or Prunus armeniaca extract showed limited improvement in this indicator, further confirming the necessity of the three-component combination.

[0084] 6. Conclusion Based on the above experimental results, it can be seen that the biological agent developed by the present invention based on reduced coenzyme Q10, in combination with juniper extract and pine pine extract, has shown significant effects on NAFLD model rats in regulating blood lipids, inhibiting inflammation, reducing oxidative stress and improving liver mitochondrial dysfunction.

[0085] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A biological agent based on reduced coenzyme Q10, characterized in that, It contains reduced coenzyme Q10, juniper extract, and pine berry extract.

2. The biological agent based on reduced coenzyme Q10 as described in claim 1, characterized in that, The mass ratio of the reduced coenzyme Q10, juniper extract and pine pine extract is (2-10):(0.2-1):(0.2-1).

3. The biological agent based on reduced coenzyme Q10 as described in claim 2, characterized in that, The mass ratio of the reduced coenzyme Q10, juniper extract and pine pine extract is (4-10):(0.2-0.8):(0.2-0.6).

4. The biological agent based on reduced coenzyme Q10 as described in any one of claims 1-3, characterized in that, It also contains pharmaceutically acceptable excipients.

5. A method for preparing a biological agent based on reduced coenzyme Q10, characterized in that, Includes the following steps: (1) Preheat the lipid matrix at 55-65℃ and stir continuously, then add reduced coenzyme Q10, juniper extract and pine pine extract in sequence until the system is mixed evenly to form a homogeneous oil phase. (2) Mix the additive with part of the solvent and stir continuously at 55-65℃ for 12-20 min until the system is uniformly dispersed and a transparent and homogeneous aqueous phase is formed; (3) Under high-speed shearing, the oil phase in step (1) is slowly added to the aqueous phase in step (2) to obtain a crude emulsion; (4) The crude emulsion in step (3) is subjected to homogenization treatment at a pressure of 40-60 MPa for 2-3 cycles. After homogenization, it is diluted with the remaining solvent to the full volume of the prescription and filtered through a 0.45 μm microporous membrane to obtain the biological agent.

6. The method for preparing the biological agent based on reduced coenzyme Q10 as described in claim 5, characterized in that, The preparation method of the juniper extract mentioned in step (1) is as follows: Pulverize dried Siberian juniper branches and leaves, pass through an 80-mesh sieve, add 50% ethanol aqueous solution at a material-to-liquid ratio of 1:25, and extract by reflux at 55-65℃ for 1-3 hours; filter, and repeat the extraction once with the same method on the residue; combine the two filtrates, concentrate under reduced pressure to 1 / 10 of the original volume, disperse with hot water at 55-65℃ to obtain a suspension, defatted with petroleum ether 1-3 times; extract the defatted aqueous phase with n-butanol 2-4 times, combine the n-butanol phases, concentrate under reduced pressure to a relative density of 1.1-1.2, and vacuum dry at 45-55℃ to obtain juniper extract.

7. The method for preparing the biological agent based on reduced coenzyme Q10 as described in claim 5, characterized in that, The preparation method of the pine-red plum extract mentioned in step (2) is as follows: Pulverized dried pine needle plum leaves were sieved through a 60-mesh sieve and extracted with 70% acetone aqueous solution at a material-to-liquid ratio of 1:

20. The extraction was performed with ultrasonic assistance and filtered. The residue was extracted once more under the same conditions. The two filtrates were combined and concentrated under reduced pressure to 1 / 10 of the original volume to obtain an aqueous concentrate. This concentrate was extracted with ethyl acetate 2-4 times. All ethyl acetate phases were combined and concentrated under reduced pressure to a relative density of 1.1-1.2 while maintaining a constant temperature. The concentrate was then dried under vacuum at 40-50℃ to obtain the pine needle plum extract.

8. The method for preparing the biological agent based on reduced coenzyme Q10 as described in claim 7, characterized in that, The conditions for ultrasound-assisted extraction were: ultrasound power 300W, temperature 40℃, and time 30min.

9. The method for preparing the biological agent based on reduced coenzyme Q10 as described in claim 5, characterized in that, The parameters for high-speed shearing in step (3) are: rotation speed 10000-15000 r / min, time 3-5 min.

10. The use of the biological agent prepared by the method according to any one of claims 5-9 in the preparation of a drug for the treatment and / or prevention of non-alcoholic fatty liver disease.

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