An antioxidant plant extract composition

Abstract

The present application belongs to the technical field of plant extracts, and particularly relates to an antioxidant plant extract composition, which is prepared by mixing the following raw materials in parts by mass: Passiflora edulis fruit extract 30-40 parts, blackcurrant fruit extract 25-35 parts, and Ampelopsis megalophylla leaf extract 20-30 parts. The present application first compounds three plant extracts of Passiflora edulis fruit, blackcurrant fruit and Ampelopsis megalophylla leaf, which can enrich the types of related products. Moreover, Passiflora edulis fruit is rich in flavones such as apigenin and luteolin, blackcurrant fruit is rich in anthocyanins such as cyanidin and delphinidin, and Ampelopsis megalophylla leaf is rich in flavonols such as dihydromyricetin. The synergistic effect of the three extracts can scavenge mitochondrial ROS, protect mitochondrial membrane potential, and improve the activity of respiratory chain complex enzymes, and has good mitochondrial protection and antioxidant damage repair effects, and has important application value for the protection of mitochondrial oxidative damage in the middle-aged and elderly population.

Description

Technical Field

[0001] This invention belongs to the field of plant extract technology, and specifically relates to an antioxidant plant extract composition. Background Technology

[0002] Mitochondria are the main source of reactive oxygen species (ROS) in cells. Excessive ROS can attack mitochondrial DNA, membrane lipids, and respiratory chain proteins, causing mitochondrial dysfunction and leading to apoptosis and organ dysfunction. To address this issue, researchers extracted antioxidant active ingredients such as polyphenols, flavonoids, and anthocyanins from various plants and prepared antioxidant compositions through compounding.

[0003] Currently, the commonly used method involves extracting antioxidant components from traditional medicinal and edible plants such as rosemary, grape seed, and green tea through water extraction, alcohol extraction, or ultrasound-assisted extraction to obtain active substances, often in combination with two or more plant extracts. However, existing plant raw materials are mostly widely used plants like rosemary, grape seed, and green tea, resulting in high repetition in combinations and a lack of novel products. Moreover, the extraction process relies on traditional water or alcohol extraction, which has limited damage to plant cell walls, leading to low extraction rates of active ingredients such as flavonoids and anthocyanins. Furthermore, the products are not highly targeted at mitochondrial oxidative damage in middle-aged and elderly individuals, and their effectiveness needs further improvement.

[0004] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0005] The purpose of this invention is to provide an antioxidant plant extract composition to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: An antioxidant plant extract composition, comprising, by weight, 30-40 parts passionflower fruit extract, 25-35 parts black chokeberry fruit extract, and 20-30 parts amaryllis leaf extract.

[0007] Furthermore, the preparation method of the passion fruit extract is as follows: after cleaning fresh passion fruit, remove the shell and extract the pulp, then pulp it. Add deionized water at a material-to-liquid ratio of 1:8-12 and adjust the pH value to 4.5-5.5. Then, add a complex enzyme composed of cellulase and pectinase for ultrasonic-assisted enzymatic hydrolysis. Then, inoculate with Lactobacillus plantarum for fermentation. After fermentation, centrifuge, concentrate, and dry to obtain the passion fruit extract.

[0008] Furthermore, the mass ratio of cellulase to pectinase is 1:1, the amount of the compound enzyme added is 0.3-0.5% of the pulp mass, and the enzymatic hydrolysis is performed at a temperature of 45-55℃ and a power of 200-300W with ultrasound assistance for 40-60 minutes.

[0009] Furthermore, the inoculum amount of *Lactobacillus plantarum* is 2-4% of the volume of the enzymatic hydrolysate, and fermentation is carried out at a temperature of 35-37°C for 24-36 hours.

[0010] Furthermore, the preparation method of the black chokeberry fruit extract is as follows: the black chokeberry fruit is cleaned, dried, and crushed. A 50-60% ethanol solution is added at a material-to-liquid ratio of 1:10-15 for ultrasonic-assisted extraction. Then, the extract is centrifuged, concentrated, and dried to obtain the black chokeberry fruit extract.

[0011] Furthermore, ultrasonic extraction was performed at a temperature of 50-60℃ and a power of 250-350W for 30-45 minutes.

[0012] Furthermore, the preparation method of the *Ampelopsis grossedentata* leaf extract is as follows: clean, dry, and crush the *Ampelopsis grossedentata* leaves, add deionized water at a material-to-liquid ratio of 1:6-10, add cellulase for enzymatic hydrolysis, extract in a boiling water bath for 30-40 minutes, filter, concentrate, and dry to obtain the *Ampelopsis grossedentata* leaf extract.

[0013] Furthermore, the amount of cellulase added is 0.2-0.4% of the leaf powder of *Ampelopsis grossedentata*, and enzymatic hydrolysis is performed at 50-55°C for 30-45 minutes.

[0014] Compared with the prior art, the present invention has the following beneficial effects: This invention is the first to combine extracts from three plants: passion fruit, black chokeberry fruit, and Ampelopsis grossedentata leaf. This combination enriches the variety of related products. Passion fruit is rich in flavonoids such as apigenin and luteolin, black chokeberry fruit is rich in anthocyanins such as cyanidin and delphinidin, and Ampelopsis grossedentata leaf is rich in flavonols such as dihydromyricetin. The three extracts work synergistically to scavenge mitochondrial ROS, protect mitochondrial membrane potential, and enhance the activity of respiratory chain complex enzymes. They exhibit good mitochondrial protection and antioxidant damage repair effects, making them of significant application value in protecting mitochondrial oxidative damage in middle-aged and elderly individuals. Detailed Implementation

[0015] The technical solution of this invention patent will be clearly and completely described below. Obviously, the described embodiments are some embodiments of this invention, but not all embodiments.

[0016] 1. Materials and Methods 1.1 Passion fruit extract Take 10 kg of fresh passion fruit, wash it thoroughly, remove the shells, extract the pulp, and pulp it. Add 100 L of deionized water and adjust the pH to 5.0. Then add a compound enzyme consisting of 20 g of cellulase (enzyme activity 20000 U / g) and 20 g of pectinase (enzyme activity 30000 U / g), and sonicate at 50℃ (power 250W) for 50 min to assist enzymatic hydrolysis. Then inoculate with a bacterial concentration of 10... 8 3 L of *Lactobacillus plantarum* (BNCC192575) culture (cfu / mL) was fermented at 36℃ for 30 h. After fermentation, the fermentation broth was centrifuged at 4000 rpm for 15 min, and the supernatant was concentrated under vacuum to a solids content of 30%. The resulting product was then spray-dried (inlet air temperature 180℃, outlet air temperature 85℃) to obtain passion fruit extract powder. Analysis showed that 1.2 kg of passion fruit extract powder contained 28.5 wt% total flavonoids.

[0017] 1.2 Black-fruited chokeberry fruit extract Eight kilograms of black chokeberry fruit were taken, washed clean, and then freeze-dried under vacuum at -40°C for 24 hours. The fruit was then pulverized and passed through a 40-mesh sieve. 100 L of 55% ethanol solution was added, and extraction was performed at 55°C with ultrasonic extraction (300W power) for 40 minutes. After centrifugation at 4000 rpm for 15 minutes, the supernatant was collected and concentrated under reduced pressure until no ethanol odor remained. The extract was then freeze-dried under vacuum at -50°C for 48 hours to obtain the black chokeberry fruit extract powder. The proanthocyanidin content of 1.1 kg of the black chokeberry fruit extract powder was measured to be 32.3 wt%.

[0018] 1.3 Extract of *Vitis pachydon* leaf Take 6 kg of Ampelopsis grossedentata leaves, wash and dry them, pulverize them through a 30-mesh sieve, add 50 L of deionized water, add 24 g of cellulase (enzyme activity 20000 U / g), enzymatically hydrolyze at 52℃ for 40 min, then transfer to a boiling water bath for extraction for 35 min, filter and collect the supernatant, vacuum concentrate to a solid content of 25%, and spray dry (inlet air temperature 180℃, outlet air temperature 85℃) to obtain the Ampelopsis grossedentata leaf extract dry powder. The mass of the Ampelopsis grossedentata leaf extract dry powder was measured to be 0.9 kg, and the dihydromyricetin content was 42.1 wt%.

[0019] 1.4 Antioxidant Activity Test The oxygen free radical absorption capacity (ORAC method) was used.

[0020] 1.4.1 Reagents 75 mM (pH 7.4) phosphate buffer, 81.6 nM sodium fluorescein solution, 153 mM azobisisobutylamidine dihydrochloride (AAPH) solution, Trolox standard solutions (prepared with phosphate buffer to a series of concentrations of 6.25 μM, 12.5 μM, 25 μM and 50 μM) 1.4.2 Test Methods Dissolve and dilute the samples to be tested in Table 1 with phosphate buffer to a concentration of 1 mg / mL, and filter through a 0.45 μm microporous membrane. Add 25 μL of the sample solution (or Trolox standard solution, or blank control phosphate buffer) and 150 μL of sodium fluorescein solution sequentially to a 96-well black fluorescent microplate. After pre-incubating the microplate at 37°C for 15 min, quickly initiate the oxidation reaction with 25 μL of 153 mM AAPH solution. Immediately place the microplate in a multi-functional microplate reader and continuously measure the fluorescence intensity at 37°C (excitation wavelength 485 nm, emission wavelength 520 nm), measuring every 2 min for a total of 120 min. Plot a fluorescence decay curve with the ratio of fluorescence intensity at each time point to the initial fluorescence intensity as the ordinate and time as the abscissa. Calculate the area under the curve (AUC) for each well using the trapezoidal method. Establish a standard curve by performing a linear regression of the net AUC against the net AUC of the Trolox standard series. The Trolox equivalent concentration (μmol TE / mL) of the sample was calculated based on the standard curve and then converted to the ORAC value per unit mass of sample. Five replicates were set for each group, and the results are shown in Table 1.

[0021] Table 1 ORAC determination results of the test samples

[0022] As shown in Table 1, the ORAC values ​​of the three single plant extracts ranged from 856 to 1243 μmol TE / g, with the extract of *Sorbus nigra* fruit exhibiting the highest activity. The ORAC values ​​of the two-ingredient combination ranged from 1652 to 2056 μmol TE / g, and from 856 to 1243 μmol TE / g, all significantly lower than those of the three-ingredient combination, indicating that the absence of any one component would lead to a significant decrease in the activity of the plant extract components in this application. Furthermore, the ORAC value of the three-ingredient combination in an equal mass ratio was 2986 μmol TE / g, lower than the ORAC value of 3875 μmol TE / g of the optimal ratio of 35:30:25, indicating that the 35:30:25 mass ratio of the three extracts produced a synergistic effect in this invention.

[0023] 1.5 Mitochondrial protective effect test The protective effect of mitochondria was evaluated using an H2O2-induced oxidative damage model in PC12 cells.

[0024] Experimental Groups 1.5.1 Cell Culture PC12 cells were cultured in RPMI-1640 containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin in a 37°C, 5% CO2 incubator. The cells were passaged every 2-3 days, and logarithmically growing cells were used for experiments.

[0025] 1.5.2 Experimental Grouping and Treatment See Table 2.

[0026] Table 2 Experimental Grouping and Treatment

[0027] 1.5.3. Indicator Testing PC12 cells in the logarithmic growth phase were prepared into 5×10⁶ cells. 4 Cell suspension at 100 μL / mL was seeded into 96-well plates and cultured at 37°C and 5% CO2 for 24 h until cell adhesion. 100 μL of the grouped sample was added to each well. After H2O2 damage was eliminated, cell viability was determined by the MTT assay, mitochondrial membrane potential was determined by the JC-1 assay, and ATP content was determined by the luciferase assay. Each group had 5 replicates. Results are shown in Table 3.

[0028] In the MTT assay, the absorbance (OD) value of each well was measured at 570 nm using an ELISA reader. With the OD value of the normal control group as 100%, the cell viability of each group was calculated according to the following formula: Cell viability (%) = (OD value of experimental group / OD value of normal control group) × 100%.

[0029] In the JC-1 method, the ratio of red fluorescence intensity (excitation wavelength 525 nm, emission wavelength 590 nm) to green fluorescence intensity (excitation wavelength 490 nm, emission wavelength 530 nm) represents the mitochondrial membrane potential level. The ratio of red fluorescence intensity to green fluorescence intensity in the normal control group (hereinafter referred to as the red-green ratio) is taken as 100%. The relative level of mitochondrial membrane potential in each group is calculated according to the following formula: Mitochondrial membrane potential (%) = (Red-green ratio of experimental group / Red-green ratio of normal control group) × 100%.

[0030] Table 3 Results of mitochondrial protective effect test

[0031] Table 3 shows that H2O2 damage reduced PC12 cell survival rate by 42.3%, mitochondrial membrane potential to 52.3%, and ATP content to 9.8 nmol / mg prot, indicating severe oxidative damage. While the single-component groups provided some protection, the effect was limited, with cell survival rates only reaching 55.8-62.4%. The two-component combination was more effective than the single-component groups, with the combination of passion fruit extract and black chokeberry fruit extract showing relatively better results, but still significantly lower than normal levels. The three-component combination was more effective than both the two-component and single-component groups, with the 35:30:25 ratio showing the best effect, achieving a mitochondrial membrane potential of 87.7%, an ATP content of 15.2 nmol / mg prot, and a cell survival rate of 85.2%, significantly superior to the other groups. This indicates that the three extracts in a 35:30:25 mass ratio produced a synergistic effect, providing strong protection against oxidative damage to mitochondrial function.

[0032] 1.6 Animal Experimental Studies 1.6.1 Experimental Design Eighteen-month-old senescent C57BL / 6 mice were randomly divided into multiple groups of 10 mice each; another 10 six-month-old C57BL / 6 mice were used as the young control group.

[0033] 1.6.2 Experimental Grouping and Treatment See Table 4.

[0034] Table 4 Experimental Grouping and Treatment

[0035] 1.6.3 Indicator Testing Twenty-four hours after the last drug administration, mice were fasted for 12 hours but allowed free access to water, then euthanized by decapitation, and liver tissue was quickly harvested. Approximately 0.5 g of liver tissue was collected, washed with pre-cooled physiological saline, and blotted dry with filter paper. 5 mL of pre-cooled mitochondrial extraction buffer (0.25 M sucrose, 10 mM Tris-HCl, 1 mM EDTA, pH 7.4) was added, and the mixture was homogenized 10 times in a water bath using a glass homogenizer. The mixture was centrifuged at 1000 g for 10 min at 4°C, and the supernatant was collected. The supernatant was then centrifuged at 1200 g for 15 min at 4°C, and the precipitate was the mitochondria. The mitochondrial precipitate was resuspended in mitochondrial suspension (0.25 M sucrose, 10 mM Tris-HCl, pH 7.4), and the protein concentration was adjusted to 5 mg / mL.

[0036] Mitochondrial respiratory control rate (RCR) determination: 0.5 mL mitochondrial suspension (approximately 250 mg protein), 1.5 mL reaction buffer (0.25 M sucrose, 10 mM KH2PO4, 10 mM Tris-HCl, 5 mM MgCl2, pH 7.4); 5 mM sodium succinate was added to record oxygen consumption rate (state 4 respiration); 200 μM ADP was added to record oxygen consumption rate (state 3 respiration), RCR = state 3 respiration rate / state 4 respiration rate.

[0037] Mitochondrial ATPase activity assay: 50 μL mitochondrial suspension, 150 μL reaction solution (5 mM ATP, 5 mM MgCl2, 50 mM Tris-HCl, pH 8.0). Incubate at 37 °C for 15 min, then add 200 μL 10% TCA to terminate the reaction. Centrifuge at 3000 rpm for 10 min at 4 °C and collect the supernatant. Inorganic phosphorus (Pi) was determined using the ammonium molybdate method. ATPase activity was expressed as the number of micromoles of Pi produced per milligram of ATP hydrolyzed per minute.

[0038] Determination of malondialdehyde (MDA) content: Take 100 μL of mitochondrial suspension and add 900 μL of physiological saline and mix well. Add 1 mL of 20% TCA and 1 mL of 0.67% TBA (prepared with 0.05M NaOH), mix well, and incubate at 95℃ for 30 min. Cool under running water, centrifuge at 4℃ and 4000 rpm for 10 min, collect the supernatant, and measure the absorbance at 523 nm. Calculate the MDA content using tetraethoxypropane as a standard.

[0039] SOD activity was determined using the xanthine oxidase method.

[0040] Table 5 Results of animal experimental studies

[0041] Table 5 shows that the liver mitochondrial function of mice in the aging model group was severely impaired, with the RCR decreasing to 2.34 (only 55.6% of that in the young group), ATPase activity decreasing by 41.5%, MDA content increasing by 198%, and SOD activity decreasing by 36.5%. While the single-component groups showed some improvement, the effect was limited, with an RCR of only 2.68-2.84 and an MDA content still as high as 1.48-1.62 nmol / mg prot. The two-component combination was more effective than the single-component group, but still significantly lower than the young group. The three-component combination in equal proportions was more effective than all two-component combinations and single-component groups, with an RCR of 3.12 and an MDA of 1.35 nmol / mg prot. The three-ingredient combination in a 35:30:25 ratio exhibited the best improvement effect, with an RCR of 3.78 (reaching 89.8% of the young group), ATPase activity of 16.8 U / mg prot (91.8% of the young group), MDA of 0.89 nmol / mg prot (close to the level of the young group), and SOD activity of 41.3 U / mg prot (91.4% of the young group). These data indicate that the 35:30:25 ratio of this invention has good mitochondrial protection and antioxidant damage repair effects, and has important application value for protecting mitochondrial oxidative damage in middle-aged and elderly populations.

[0042] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. An antioxidant plant extract composition, characterized in that, It is composed of the following ingredients in parts by weight: 30-40 parts passion fruit extract, 25-35 parts black chokeberry fruit extract and 20-30 parts amaryllis leaf extract.

2. The antioxidant plant extract composition according to claim 1, characterized in that, The preparation method of the passion fruit extract is as follows: After cleaning fresh passion fruit, remove the shell and extract the pulp, then pulp it. Add deionized water at a material-to-liquid ratio of 1:8-12 and adjust the pH value to 4.5-5.

5. Then add a complex enzyme composed of cellulase and pectinase for ultrasonic-assisted enzymatic hydrolysis. Then inoculate with Lactobacillus plantarum for fermentation. After fermentation, centrifuge, concentrate and dry to obtain the passion fruit extract.

3. The antioxidant plant extract composition according to claim 2, characterized in that, The mass ratio of cellulase to pectinase is 1:1, the amount of the compound enzyme added is 0.3-0.5% of the pulp mass, and the enzymatic hydrolysis is performed at a temperature of 45-55℃ and a power of 200-300W with ultrasound assistance for 40-60 minutes.

4. The antioxidant plant extract composition according to claim 2, characterized in that, The inoculum amount of *Lactobacillus plantarum* is 2-4% of the volume of the enzymatic hydrolysate, and fermentation is carried out at a temperature of 35-37℃ for 24-36 hours.

5. The antioxidant plant extract composition according to claim 1, characterized in that, The preparation method of the black chokeberry fruit extract is as follows: the black chokeberry fruit is cleaned, dried, and crushed. A 50-60% ethanol solution is added at a material-to-liquid ratio of 1:10-15 for ultrasonic-assisted extraction. Then, the extract is centrifuged, concentrated, and dried to obtain the black chokeberry fruit extract.

6. The antioxidant plant extract composition according to claim 5, characterized in that, Ultrasonic extraction at 50-60℃ and 250-350W for 30-45 minutes.

7. The antioxidant plant extract composition according to claim 1, characterized in that, The preparation method of the Amaranthus praecox leaf extract is as follows: clean, dry and crush the Amaranthus praecox leaves, add deionized water at a material-to-liquid ratio of 1:6-10, add cellulase for enzymatic hydrolysis, extract in boiling water bath for 30-40 minutes, filter, concentrate and dry to obtain the Amaranthus praecox leaf extract.

8. The antioxidant plant extract composition according to claim 7, characterized in that, The amount of cellulase added is 0.2-0.4% of the leaf powder of *Ampelopsis grossedentata*, and enzymatic hydrolysis is performed at 50-55℃ for 30-45 minutes.

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