CN116637106A

CN116637106A - Health care product or medicine with antioxidation effect

Info

Publication number: CN116637106A
Application number: CN202210137346.0A
Authority: CN
Inventors: 张仕勇; 廖春燕; 谭毅峰; 雷雨; 吴潇; 赖黄晋; 卢晓鸾
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2022-02-15
Filing date: 2022-02-15
Publication date: 2023-08-25

Abstract

The invention discloses a health product or medicine with an antioxidant effect, and belongs to the technical field of biological medicines. According to the health-care product or medicine, vitamin C (VC) and Lipoic Acid (LA) are combined, so that the VC and the LA can be regenerated in an intracellular circulation mode, and are prevented from being rapidly metabolized, and the bioavailability of the VC and the LA is remarkably improved. The combination of the two has a definite combined antioxidation mechanism, so that not only is an excellent synergistic antioxidation effect realized, but also the combined antioxidation effect of various antioxidants is achieved. The health product or medicine is safe and nontoxic, and can be used for a long time.

Description

Health care product or medicine with antioxidation effect

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a health product or medicine with an antioxidant effect.

Technical Field

Over the course of age, air pollution, ultraviolet radiation (UV), smoking, drinking, viral infection, and the like, cause the body to produce ROS at levels exceeding normal. ROS can act with proteins, DNA and unsaturated fatty acids to cause protein crosslinking, DNA cleavage, lipid peroxidation, etc., and finally cause aging of human body, cardiovascular diseases, senile dementia, atherosclerosis, malignant tumor, etc. The organism is supplemented with antioxidant, which not only eliminates redundant ROS in the body, but also improves the life quality of people. At present, because antioxidants are easy to rapidly clear after entering the body, the clinical antioxidant effect is very limited, and people can only use a combination strategy of increasing dosage or multiple antioxidants. However, large-dose applications tend to increase the risk of biosafety, and there are also significant limitations to the various antioxidant combination strategies available. The combination of antioxidant products on the market at present has an undefined combination mechanism of antioxidants and lacks scientificity. Although some combination schemes do have a certain synergistic antioxidant effect, the improvement of the effect is still very limited, and the improvement has no practical significance for promoting clinical application. Moreover, these combination strategies have not yet completely overcome the drawback of the antioxidants being easily and rapidly cleared after entry into the body, and such combination antioxidant products are wasteful and increase the risk of cancer and cardiovascular disease.

Disclosure of Invention

Aiming at the problems, the invention provides a health care product or medicine with an antioxidation effect, vitamin C (VC) and Lipoic Acid (LA) are scientifically combined, so that the VC and the LA can be regenerated in an intracellular cycle, the VC and the LA are prevented from being rapidly metabolized, the bioavailability of the VC and the LA is obviously improved, and the vitamin C and the LA are combined to have a definite combined antioxidation mechanism, so that an excellent synergistic antioxidation effect is realized, and the combined antioxidation effect of various antioxidants is further achieved. The product should improve organism by resisting oxidation to prevent various diseases.

The invention comprises the following technical scheme:

a health product or medicine with antioxidant effect comprises VC and LA as main active ingredients. The inventor scientifically and reasonably combines VC and LA according to scientific theory, repeated experiments and proportion screening, can remarkably improve the bioavailability of VC and LA, has a clear synergistic antioxidant mechanism, generates a synergistic antioxidant effect, and can realize the combined antioxidant effect of various antioxidants.

The lipoic acid is a broad lipoic acid concept in the present invention, and may be lipoic acid molecules, lipoic acid derivatives or a mixture of the lipoic acid derivatives, wherein the lipoic acid derivatives comprise lipoic acid salts or pharmaceutically acceptable modifications (including but not limited to grafting functional groups on lipoic acid molecules) obtained by non-substantial modification of lipoic acid without affecting the core function of lipoic acid.

The vitamin C is a broad vitamin C concept, and can be a vitamin C molecule, a vitamin C derivative or a mixture of the vitamin C molecule and the vitamin C derivative is a pharmaceutically acceptable modified substance (including but not limited to grafting functional groups on the vitamin C molecule) obtained by non-substantial modification of the vitamin C without affecting the core function of the vitamin C.

The Lipoic Acid (LA) has excellent biocompatibility and is a metabolic antioxidant, and can be converted into dihydrolipoic acid (reduced form of lipoic acid, dihydrolipoic Acid and DHLA) in vivo, and the two components can directly remove free radicals and active oxygen (such as hydroxyl free radicals, hydrogen peroxide, singlet oxygen, nitric oxide, hypochlorous acid and the like) and can chelate metal ions (such as Fe) which are easy to generate ROS in vivo ²⁺ 、Cu ²⁺ 、Zn ²⁺ 、Pb ²⁺ Etc.) and regenerating other antioxidants (glutathione, vitamin C, vitamin E, coenzyme Q, thioredoxin reductase, etc.), thereby indirectly scavenging active oxygen from various sites (aqueous phase and lipid region). However, LA is liposolubleThe sexual and water-soluble amphoteric molecules are easy to be absorbed and metabolized by organisms, and after LA enters cells, DHLA generated by reduction is easy to be rapidly discharged out of the cells, so that the content of LA and DHLA remained in the cells is reduced, and the physiological efficacy of the cells is reduced.

One of the main active ingredients in the health care product or the medicine disclosed by the invention, the alkene diol group in the Vitamin C (VC) molecule can be converted into a diketone group, so that the Vitamin C (VC) molecule has stronger reducibility. It not only can directly remove ROS, but also can regenerate vitamin E and coenzyme Q, so as to prevent lipid peroxidation. However, VC is prone to losing electrons to produce dehydroascorbic acid (Dehydroascorbic Acid, DHA), which is reversible, and DHA can be reduced by a reducing agent to produce VC, thereby again exerting the efficacy of VC; however, when DHA is further oxidized into diketopyrrolopyrronic acid, the reaction is irreversible, and finally the physiological effect is completely lost, so that VC is easily oxidized after entering the body to lose the physiological effect.

The invention combines LA and VC, and after entering cells, the reduction product of the active ingredient LA of the invention Oxidation product of reducible VC->And further regenerate LA and VC, so that the VC and LA can be regenerated in the intracellular circulation, thereby avoiding rapid metabolism and ensuring the continuous exertion of the efficacy of the LA.

Alternatively, the health product is a health food or a medicine.

Alternatively, in the health product or the medicine, the molar ratio of the vitamin C to the lipoic acid is 10:1-1:10. The ratio range can obviously improve the bioavailability of VC and LA.

Alternatively, in the health product or the medicine, the molar ratio of the vitamin C to the lipoic acid is 2:1. The composition has optimal synergistic antioxidation effect.

Alternatively, in the above health product or medicine, the antioxidant effect includes: reduces MDA content and improves GSH-Px, CAT and SOD activity.

Optionally, the health care product or the medicine further comprises auxiliary materials.

Alternatively, in the health care product or the medicine, the dosage form of the health care product or the medicine is powder, tablet, capsule or injection reagent.

Alternatively, in the above health products or medicines, VC and LA are prepared into coated tablets for oral administration.

Optionally, in the health product or the medicine, the auxiliary materials comprise lactose, microcrystalline cellulose, polyvinylpyrrolidone, corn starch, tartaric acid, magnesium stearate and talcum powder.

Alternatively, the health product or the medicine comprises the following components in percentage by weight: 5-50% of vitamin C, 5-50% of alpha-lipoic acid, 5-25% of microcrystalline cellulose, 10-25% of lactose, 0.1-3% of 5% of polyvinylpyrrolidone 30, 3-15% of corn starch, 0.5-1.5% of tartaric acid, 0.1-1% of magnesium stearate and 0.1-3% of talcum powder.

The invention also provides application of the vitamin composition, which is characterized in that the vitamin composition is used for preparing an antioxidant health-care product or medicine, and the vitamin composition comprises vitamin C and lipoic acid.

All of the features disclosed in this specification, or all of the steps in a method or process disclosed, may be combined in any combination, except for mutually exclusive features and/or steps.

The invention has the beneficial effects that:

1. the invention combines VC and LA, can effectively reduce the oxidative metabolism speed of VC in cells/bodies, further improve the bioavailability of VC and the concentration of VC in blood and cells; at the same time, the concentration of LA in blood and cells can be increased. In addition, VC and LA can be recycled and regenerated with each other, and other antioxidants such as: vitamin E, glutathione and the like finally exert excellent synergistic antioxidation effect, and the combined antioxidation effect can reach or even exceed the antioxidation effect of astaxanthin (the strongest antioxidant); the combined antioxidation effect of the two can achieve the combined effect of various antioxidants.

2. Compared with the combined antioxidant strategy in the prior art, the vitamin composition has the remarkable effect of enhancing the antioxidant effect by 75 times under the condition of using only two common antioxidants, can achieve a better effect at a lower dosage, effectively avoids the risk of diseases easily caused by mixing a plurality of antioxidants in the prior art, and enables the combined antioxidant to be possible in long-term clinical application.

Description of the drawings:

FIG. 1 shows the changes in MDA (a), GSH-Px (b), CAT (c) and SOD (d) levels in the blood of mice after the effects of the materials of example 3.

FIG. 2 shows the changes in MDA (a), GSH-Px (b), CAT (c) and SOD (d) levels and changes in liver function indices ALT (e) and AST (f) in the livers of mice after the action of the materials of example 3.

FIG. 3 shows the changes in MDA (a), GSH-Px (b), CAT (c) and SOD (d) levels in the brain tissue of mice after the effects of each group of materials in example 3.

FIG. 4 shows the changes in MDA (a), GSH-Px (b), CAT (c) and SOD (d) levels in the blood of mice after the effects of the materials of example 4.

FIG. 5 shows the changes in MDA (a), GSH-Px (b), CAT (c) and SOD (d) levels and changes in liver function indices ALT (e) and AST (f) in the livers of mice after the action of the materials of example 4.

FIG. 6 shows the changes in MDA (a), GSH-Px (b), CAT (c) and SOD (d) levels in the brain tissue of mice after the action of each group of materials in example 4.

FIG. 7 shows the changes in MDA (a), GSH-Px (b), CAT (c) and SOD (d) levels in the blood of mice after the effects of the materials of example 5.

FIG. 8 shows the changes in MDA (a), GSH-Px (b), CAT (c) and SOD (d) levels and changes in liver function indices ALT (e) and AST (f) in the livers of mice after the action of each group of materials in example 5.

FIG. 9 shows the changes in MDA (a), GSH-Px (b), CAT (c) and SOD (d) levels in the brain tissue of mice after the action of each group of materials in example 5.

FIG. 10 is an acute toxicity evaluation of [ VC+LA ] in example 6: weight change of mice (a), weight of isolated tissue of mice (b), kidney function index (c) and liver function index (d).

FIG. 11 is an immunogenicity evaluation of [ VC+LA ] in example 6: white blood cell count (a) and cytokine (TNF-. Alpha.and IL-6) detection (b).

FIG. 12 is microkernel test evaluation of [ VC+LA ] in example 7: microkernel ratio (a) and PCE/NCE ratio (b).

FIG. 13 is a chromosomal aberration evaluation of [ VC+LA ] in example 7: chromosomal aberration rate.

FIG. 14 shows the DPPH-scavenging effect of VC (a) and LA (b) in example 8, and DPPH-scavenging efficiency and Combination Index (CI) analysis (c) of different ratios of the [ VC+LA ] mixture.

FIG. 15 shows the effect of VC (a) and LA (b) on OH removal in example 8, and the effect of varying proportions of the [ VC+LA ] mixture on OH removal and Combination Index (CI) analysis (c).

FIG. 16 shows the toxicity of the [ VC+LA ] mixture of example 8 to normal cells.

FIG. 17 is H in example 8 ₂ O ₂ Toxicity to normal cells.

FIG. 18 shows the intracellular ROS scavenging effect of VC (a) and LA (b) of example 8, and the intracellular ROS scavenging efficiency and Combination Index (CI) analysis of varying proportions of the [ VC+LA ] mixture (c).

Figure 19 shows the rate constants of DHLA and GSH reduction of DHA in example 8.

FIG. 20 shows the changes in intracellular VC and LA content of the [ VC+LA ] mixture, VC and LA in example 8 after the effect of the VC and LA on cells stimulated by oxidation.

FIG. 21 shows the change in the VC and LA content of the [ VC+LA ] mixture, VC and LA in the blood of mice after gastric lavage in example 8.

FIG. 22 is a schematic diagram showing the mechanism of the regeneration of the active ingredient in the product of the present invention by intracellular circulation.

The specific embodiment is as follows:

the above-described aspects of the present invention will be described in further detail below by way of specific embodiments of the present invention. It should not be construed that the scope of the above subject matter of the present invention is limited to the following examples. Any modifications, equivalent substitutions or improvements made by those skilled in the art, without departing from the spirit and principles of the present invention, should be included within the scope of the present invention.

EXAMPLE 1 preparation of VC and LA containing [ VC+LA ] coated tablets

A 5% polyvinylpyrrolidone solution and a 1% tartaric acid solution were prepared using an ethanol solution. Vitamin C (136 mg), lipoic acid (80 mg), microcrystalline cellulose (67 mg), lactose (50 mg), polyvinylpyrrolidone (20 mg), corn starch (40 mg), tartaric acid (4 mg), magnesium stearate (1 mg) and talcum powder (2 mg) are respectively sieved by a 100-mesh sieve, and the materials are mixed and sieved by a 20-mesh sieve. The wet granules obtained were dried at 30 ℃. Mixing the dried granules with magnesium stearate and talcum powder, tabletting, and making into tablet. Preparing hydroxypropyl methylcellulose phthalate (HPMCP) coating solution containing light-shielding agent, coating and spraying tablet, and drying to obtain coated tablet.

EXAMPLE 2 preparation of [ VC+LA ] capsules

Vitamin C (149 mg), lipoic acid (69 mg), microcrystalline cellulose (70 mg), lactose (80 mg), polyvinylpyrrolidone (15 mg), corn starch (40 mg), tartaric acid (6 mg), magnesium stearate (3 mg) and talcum powder (2 mg) are sieved through a 100-mesh sieve, and microcrystalline cellulose ethanol solution is used as a binder to prepare granules, and the granules are sieved through a 14-mesh nylon sieve and dried at 45 ℃. And (3) filling the obtained granules into capsules, and finally preparing the vitamin capsules.

EXAMPLE 3 [ VC+LA ] in vivo antioxidant studies of coated tablets

Female balb/c mice (6 weeks old) with similar weights were purchased for 20, fed with small mouse quasi-diet and guaranteed free feeding and drinking water, and after one week, formal experiments were carried out. Mice were divided into 4 groups of 5 animals each, each of (1) physiological saline group, (2) bacterial lipopolysaccharide (LPS, 4 mg/kg) treated for 6 hours, (3) [ VC+LA ] coated tablet (15 mg/kg) +LPS (4 mg/kg) and astaxanthin tablet (15 mg/kg) +LPS (4 mg/kg). The [ VC+LA ] coated tablet and astaxanthin tablet are prepared into solution by normal saline, and the solutions are all infused into stomach by mouth. The mice of the corresponding group were respectively gastrically coated with [ VC+LA ] and astaxanthin tablets 7 days before the intraperitoneal injection of LPS, once a day, for a total of 7 times. After day 7, mice were fasted for 12h and the corresponding groups were given intraperitoneal injections of LPS. 6 hours after LPS injection, blood, liver and brain tissues of the mice were collected, and Malondialdehyde (MDA), glutathione peroxide (GSH-Px), hydrogen peroxide catabolic enzyme (CAT) and superoxide dismutase (SOD) detection, and glutamic pyruvic transaminase (ALT) and glutamic oxaloacetic transaminase (AST) detection in serum were performed respectively. The experimental results are shown in fig. 1-3, after LPS stimulates mice, lipid peroxidation products MDA of blood, liver and brain tissues are increased, antioxidases GSH-Px, CAT and SOD are reduced, and glutamic-pyruvic transaminase (ALT) and glutamic-oxaloacetic transaminase (AST) are reduced, which indicates that LPS causes oxidative stress generation of the bodies of the mice and liver function injury. When mice are pre-dosed with [ VC+LA ] coated tablets, the mice obviously inhibit MDA rise, GSH-Px, CAT and SOD decline and ALT and AST decline of blood, liver and brain tissues, and the inhibition effect is stronger than astaxanthin with the strongest antioxidant effect at present (figures 1-3), which shows that the [ VC+LA ] coated tablets have stronger antioxidant effect than astaxanthin and can effectively reduce the damage of oxidative stress to liver functions. The experimental result also shows that VC and LA which can circulate mutually are combined, so that the super-strong antioxidation effect is realized.

EXAMPLE 4 in vivo antioxidant comparison of [ VC+LA ] coated tablets with commercially available multicomponent antioxidant products

The Shang Chen-time health grape seed vitamin C and E tablet is an antioxidant product which is widely sold in the market at present, and the antioxidant active ingredients of the soup ministerial health grape seed vitamin C and E tablet are procyanidine, vitamin C and vitamin E.

Female balb/c mice (6 weeks old) with similar weights were purchased for 20, fed with small mouse quasi-diet and guaranteed free feeding and drinking water, and after one week, formal experiments were carried out. Mice were divided into 4 groups of 5 animals each, each of (1) physiological saline, (2) bacterial lipopolysaccharide (LPS, 4 mg/kg) treated for 6 hours, (3) [ VC+LA ] coated tablet (15 mg/kg) +LPS (4 mg/kg) and (4) grape seed vitamin C plus E tablet (15 mg/kg) +LPS (4 mg/kg). The [ VC+LA ] coated tablet and grape seed vitamin C and E tablet are prepared into solution by normal saline, and the solution is orally and gastrolavage. The mice of the corresponding group were respectively subjected to [ VC+LA ] coated tablets and grape seed vitamin C plus E-coated tablets for intragastric administration 7 days before the intraperitoneal injection of LPS, once daily, for a total of 7 times. On day 7, mice were fasted for 12h and the corresponding groups were given intraperitoneal injections of LPS. 6 hours after LPS injection, blood, liver and brain tissues of the mice were collected, and Malondialdehyde (MDA), glutathione peroxide (GSH-Px), hydrogen peroxide catabolic enzyme (CAT) and superoxide dismutase (SOD) detection, and glutamic pyruvic transaminase (ALT) and glutamic oxaloacetic transaminase (AST) detection in serum were performed respectively. The experimental results are shown in fig. 4-6, and after LPS stimulated mice, lipid peroxidation products MDA of blood, liver and brain tissues are increased, antioxidases GSH-Px, CAT and SOD are reduced (fig. 4-6), and glutamic pyruvic transaminase (ALT) and glutamic oxaloacetic transaminase (AST) are reduced (fig. 5), which indicates that LPS causes oxidative stress generation of the bodies of the mice and liver function injury. When mice were pre-dosed with grape seed vitamin C plus E tablets, they significantly inhibited MDA rise in blood, liver and brain tissue, GSH-Px, CAT and SOD decline, and ALT and AST decline, exhibiting excellent antioxidant effects (fig. 4-6). Notably, [ VC+LA ] coated tablets exhibited better antioxidant effects than grape seed vitamin C plus E tablets, and were effective in reducing oxidative stress in blood (FIG. 4), liver (FIG. 5) and brain tissue (FIG. 6). The experimental results show that the [ VC+LA ] coated tablet combining the two antioxidants can achieve better antioxidation effect than the antioxidation products combining various antioxidants which are commercially available at present, and can effectively prevent the generation of oxidative stress. The experimental result also shows that VC and LA which can circulate mutually are combined, so that stronger antioxidation effect is realized compared with the common combination of various antioxidants.

EXAMPLE 5 in vivo antioxidant comparative study of [ VC+LA ] coated tablets with [ VC+LA+grape seeds (PC) ] coated tablets, [ VC+LA+vitamin E (VE) ] coated tablets

(1) Preparation of [ VC+LA+PC ] coated tablets

A 5% polyvinylpyrrolidone solution and a 1% tartaric acid solution were prepared using an ethanol solution. Vitamin C is added separately

(136 mg), lipoic acid (80 mg), grape seeds (50 mg), microcrystalline cellulose (47 mg), lactose (35 mg), polyvinylpyrrolidone (20 mg), corn starch (25 mg), tartaric acid (4 mg), magnesium stearate (1 mg) and talcum powder (2 mg) are sieved by a 100-mesh sieve, and the materials are mixed and sieved by a 20-mesh sieve. The wet granules obtained were dried at 30 ℃. Mixing the dried granules with magnesium stearate and talcum powder, tabletting, and making into tablet. Preparing hydroxypropyl methylcellulose phthalate (HPMCP) coating solution containing light-shielding agent, coating and spraying tablet, and drying to obtain film

[ VC+LA+PC ] coated tablets.

(2) Preparation of [ VC+LA+VE ] coated tablets

A 5% polyvinylpyrrolidone solution and a 1% tartaric acid solution were prepared using an ethanol solution. Vitamin C (136 mg), lipoic acid (80 mg), vitamin E (50 mg), microcrystalline cellulose (47 mg), lactose (35 mg), polyvinylpyrrolidone (20 mg), corn starch (25 mg), tartaric acid (4 mg), magnesium stearate (1 mg) and talcum powder (2 mg) are respectively sieved by a 100-mesh sieve, and the materials are mixed and sieved by a 20-mesh sieve. The wet granules obtained were dried at 30 ℃. Mixing the dried granules with magnesium stearate and talcum powder, tabletting, and making into tablet. Preparing hydroxypropyl methylcellulose phthalate (HPMCP) coating solution containing a light-shielding agent, carrying out tablet coating spraying, and drying to form a film to obtain the [ VC+LA+VE ] coated tablet.

(3) In vivo antioxidant assay

Female balb/c mice (6 weeks old) of similar body weight were purchased 25, fed with small mouse quasi-diet and guaranteed free feeding and drinking water, and after one week, formal experiments were carried out. Mice were divided into 5 groups of 5 animals each, which were respectively (1) physiological saline group, (2) LPS (4 mg/kg) treated for 6 hours group, (3) [ VC+LA ] coated tablet (15 mg/kg) +LPS (4 mg/kg) group, (4) [ VC+LA+PC ] coated tablet (15 mg/kg) +LPS (4 mg/kg) group and (5) [ VC+LA+VE ] coated tablet (15 mg/kg) +LPS (4 mg/kg) group. The [ VC+LA ] coated tablet, [ VC+LA+PC ] coated tablet and [ VC+LA+VE ] coated tablet are prepared into solutions by normal saline, and the solutions are all orally irrigated. The mice of the corresponding group were respectively subjected to intragastric administration of [ VC+LA ], [ VC+LA+PC ] and [ VC+LA+VE ] for 7 days before intraperitoneal injection of LPS, once daily, for a total of 7 times. On day 7, mice were fasted for 12h and the corresponding groups were given intraperitoneal injections of LPS. 6 hours after LPS injection, blood, liver and brain tissues of the mice were collected, and Malondialdehyde (MDA), glutathione peroxide (GSH-Px), hydrogen peroxide catabolic enzyme (CAT) and superoxide dismutase (SOD) detection, and glutamic pyruvic transaminase (ALT) and glutamic oxaloacetic transaminase (AST) detection in serum were performed respectively. The experimental results are shown in fig. 7-9, and after the mice are stimulated by LPS, lipid peroxidation products MDA of blood, liver and brain tissues are increased, antioxidases GSH-Px, CAT and SOD are reduced (fig. 7-9), and glutamic pyruvic transaminase (ALT) and glutamic oxaloacetic transaminase (AST) are reduced (fig. 8), which indicates that LPS causes oxidative stress generation of the bodies of the mice and liver function injury. After the mice were pre-dosed with [ VC+LA ] coated tablets, [ VC+LA+PC ] coated tablets and [ VC+LA+VE ] coated tablets, respectively, the three tablets effectively inhibited the MDA rise, GSH-Px, CAT and SOD fall, and ALT and AST fall (FIGS. 7-9), and the antioxidant effect of the [ VC+LA ] coated tablets was close to that of the [ VC+LA+PC ] coated tablets and [ VC+LA+VE ] coated tablets combined with the multi-antioxidants. Experimental results show that VC+LA which can be regenerated in a mutually circulating way can realize stronger antioxidation effect without introducing other antioxidants for combined use.

EXAMPLE 6 evaluation of coated tablets for biocompatibility/biosafety

Acute toxicity experiment: evaluation of [ VC+LA ] by acute toxicity test]In vivo biocompatibility/biosafety of coated tablets. BALB/c mice weighing about 20g were purchased and kept under standard conditions so that they were free to obtain food and water. After one week of observation, the corresponding experiment was performed. Mice were randomly divided into 3 groups of 10 mice (5 males, 5 females) each, and [ VC+LA ] were administered by gavage]Coated tablet (1000 mg/kg, physiological saline solution). Meanwhile, we recorded body weight every 2 days for 2 weeks after administration using normal saline-treated and untreated (blank) mice as controls. After the observation, 1000 μl blood samples of mice were collected and divided into two parts: 150. Mu.L of blood was collected in EDTA-K containing solution ₂ Is arranged in the blood collection tube; 850. Mu.L of blood was collected in a 1.5mL EP tube. The collected EP blood was left at room temperature for 30min and then centrifuged for 15min (3000 rpm/min), and subjected to centrifugation for 2 times,the above serum was collected each time. The liver function index of mice was measured by a fully automatic hematology analyzer: alanine aminotransferase, glutamate ammonia-transferase, and alkaline phosphatase, renal function index: urea nitrogen and creatinine and white blood cell count: leukocytes, lymphocytes, monocytes and neutrophils. After the end of the observation, none of the three groups of mice had died, and as shown in FIG. 10a, [ VC+LA ] even at a dose of 1000mg/kg]The mice in the coated tablet group did not lose weight and slightly increased in weight as in the saline group and the blank group. Mice were weighed for heart, liver, spleen, lung and kidney, [ VC+LA ]]No significant difference between the organ weights of the coated tablet mice and those of normal saline and blank mice (fig. 10 b), indicating [ vc+la ]]The coated tablets did not cause significant toxic damage to mice. As shown in fig. 10c, [ vc+la ]]Alanine aminotransferase, glutamate ammonia transferase and alkaline phosphatase were all at normal levels in coated panel mice, indicating that they did not cause liver function changes. FIG. 10d shows [ VC+LA ]]Urea nitrogen and creatinine were both at normal levels in the coated panel mice, indicating that they did not cause renal function changes. As shown in fig. 10d, [ vc+la ]]Coated tablet group mice white blood cell count: white blood cells, lymphocytes, monocytes and neutrophils were at normal levels, indicating that no significant immune response was elicited in vivo. Taken together, it is shown that [ VC+LA ]]The coated tablet has excellent biocompatibility and does not cause a significant immune response.

Immunogen reaction test: to determine whether the [ VC+LA ] coated tablets elicit an immune response in vivo, further evaluation was performed by detecting mouse tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) in serum. Female BALB/c mice weighing about 20g were purchased and kept under standard conditions so that they were free to gain access to food and water. After one week of observation, the corresponding experiment was performed. The mice were randomly divided into 3 groups of 3 mice each, one group of mice was given a [ VC+LA ] coated tablet by gavage, and the other group of mice was gavaged with sterile physiological saline. Mice that were not treated at all served as blank. 24h after administration, blood was collected by taking the eyeball and collecting the blood in a 1.5mL EP tube. The blood was left at room temperature for 30min and centrifuged for 15min (3000 rpm/min), and the above serum was collected 2 times. Cytokine detection in serum was performed by ELISA kit instructions for mouse IL-6 and TNF- α. TNF- α is a cytokine involved in systemic inflammation, is secreted primarily by macrophages, and has the function of modulating immune cells. IL-6 is a pro-inflammatory cytokine, produced primarily by Coulomb cells and macrophages, and is a marker of inflammation. Thus, it can be assessed by TNF- α and IL-6 whether [ VC+LA ] elicits an immune response. As shown in fig. 11b, TNF- α and IL-6 in the serum of mice in the [ vc+la ] group were not significantly different from those in the saline and blank groups, indicating that [ vc+la ] did not significantly elicit an immune response in the body.

EXAMPLE 7 teratogenic and mutagenic

Micro-verification: micronucleus assay is a genotoxicity test method for detecting chromosomal or mitotic lesions, whereby the genotoxicity of [ VC+LA ] coated tablets is assessed by micronucleus assay. BALB/c mice weighing about 20g were purchased and kept under standard conditions so that they were free to obtain food and water. After one week of observation, the corresponding experiment was performed. Mice were randomly divided into 3 groups of 10 mice (male 5, female 5). One group of mice was given a [ VC+LA ] coated tablet (1000 mg/kg) by gavage, and the other group of mice was given a corresponding dose of physiological saline by gavage once a day for 2 days. The third group of mice was injected with cyclophosphamide (100 mg/kg) by intraperitoneal pulse 24h before the end of the treatment. At the end of treatment, mice were sacrificed by cervical dislocation and bone marrow cells of the mouse femur were collected into a small (1 mL) amount of Fetal Bovine Serum (FBS). The femoral bone marrow suspension was aspirated using a 21G needle syringe and dropped onto a slide glass to prepare a smear. After the smear was dried, the smear was further placed in a methanol solution to fix cells for 10 minutes, followed by staining in Giemsa staining solution (10%, v/v) for 30 minutes. After dyeing, smear cleaning is carried out by clear water, and then natural air drying is carried out. The prepared smears were observed under a microscope, 2000 red blood cells (per animal) were recorded, and micronuclei scoring was performed on multi-stained red blood cells (PCEs) and on positively-stained red blood cells (ncts) to evaluate the genotoxicity of [ vc+la ] coated tablets. The ratio of PCEs to ncts was also determined as an indicator of bone marrow toxicity. Micronucleus tests are tests for detecting chromosomal lesions, essentially mutations in genetic material, which have now been defined by the Ministry of health as a necessary indicator of the toxicological detection of foreign compounds, and we therefore evaluated whether [ VC+LA ] coated tablets are genotoxic by micronucleus tests. Mice (male and female) of the [ vc+la ] coated tablets and saline (negative control) group formed significantly lower numbers of micronuclei than cyclophosphamide (positive control) group (fig. 12 a) and the PCE/NCE ratio was significantly increased (fig. 12b, the normal range of PCE/NCE was 0.6-1.2, if the ratio < 0.1, it indicated that PCE formation was severely inhibited), indicating that the [ vc+la ] coated tablets hardly caused chromosome breakage, forming micronuclei.

Chromosome aberrations: chromosome aberration (chromosomal aberration) refers to the change in number and structure of chromosomes in a biological cell, and thus the genotoxicity of [ VC+LA ] coated tablets was determined by the Chromosome Aberration (CA) method. BALB/c mice weighing about 20g were purchased and kept under standard conditions so that they were free to obtain food and water. After one week of observation, the corresponding experiment was performed. Mice were randomly divided into 3 groups of 10 mice (male 5, female 5). One group of mice was given [ VC+LA ] coated tablets (1000 mg/kg) by gavage, and the other group of mice was given the same dose of physiological saline by gavage once daily for 2 days. The third group of mice was injected with cyclophosphamide (100 mg/kg) intraperitoneally 24h before the end of the treatment. All animals were given colchicine (4 mg/kg) via the abdominal cavity 2h before the mice were sacrificed at cervical dislocation. Bone marrow cells of the mouse femur were collected/washed with PBS (pH 6.8,5 mL) and centrifuged for 10min (1000 rpm/min), and cell pellet was obtained. The collected cell pellet was placed in 0.075MKCl hypotonic solution, incubated at 37℃for another 30min, and then fixed with glacial acetic acid/methanol fixative (glacial acetic acid/methanol, 1:3, v/v) for 1min. After fixation, the cells were centrifuged for another 10min (1000 rpm/min) to obtain a cell pellet. The collected cell pellet was further centrifuged (this step was repeated 1 more time) after further fixation for 20 min. After centrifugation, the supernatant was removed, resulting in about 0.5mL of cell suspension. The cell suspension was added dropwise to a pre-chilled (4 ℃) slide, dried and stained with Giemsa stain (10%, v/v) for 30min. The frequency of chromosomal aberration was determined by scoring 100 well-dispersed metaphase cells (per animal) and used as an indicator of genotoxicity. The breaks of the various chromosomes and chromatids, gaps, deletions of the chromosomes and chromatids, loss and exchange of centromeres were recorded. As shown in fig. 13, [ vc+la ] coated tablets did not significantly increase the percent chromosomal variation in both male and female mice compared to the negative control, whereas the percent chromosomal variation was significantly increased in both male and female mice of cyclophosphamide. In conclusion, the [ VC+LA ] coated tablets have almost no genotoxicity and can be applied in vivo.

EXAMPLE 8 VC and LA joint mechanism Studies

1. Different ratios of VC+LA mixture to remove various free radicals [ solution experiments ]

(1) Scavenging 1-diphenyl-2-trinitrophenylhydrazine free radical (DPPH-): DPPH absolute ethanol solution of 0.1M concentration was prepared and added to a 96-well plate at 100. Mu.L per well. Adding VC and LA or [ VC+LA ] with different proportions into the 96-well plate respectively]Aqueous mixture (n) _VC :n _LA =10:1, 5:1, 2:1, 1:1, 1:2, 1:5, 1:10) (such that the final concentration is from 0, 1, 2.5, 5, 10, 25, 50, 100, 250 to 500 μg/mL), incubation for 30min at 37 ℃ protected from light. The UV absorption of each well at 517nm was then detected by a microplate reader. As shown in FIGS. 14a and 14b, DPPH-scavenging efficiency was low for VC and LA alone, and its half maximal effect concentration (EC ₅₀ ) 75 and 102 μg/mL, respectively. As shown in fig. 14c, when VC and LA are combined, they exhibit synergistic DPPH-clearing effects, with CI values of each combination being less than 1. And when n _VC :n _LA When=10:1 and 1:10, VC and LA exhibited a weaker synergistic effect, and both groups had CI values greater than 0.6. And when n _VC :n _LA When=2:1, VC and LA exhibit optimal clearance, whichAnd->And the CI values are respectively 5.4 and 2.7 mug/mL, are smaller than 0.15 under the condition of each inhibition rate, and have optimal synergy.

(2) Scavenging hydroxyl radical (OH): 50. Mu.L of FeSO was added to each of the 96-well plates ₄ Solution (9 mmol/L), 50. Mu.L salicylic acid-ethanol solution (9 mmol/L), 50. Mu.L VC, LA or different ratios of [ VC+LA ]]Mixture (n) _VC :n _LA =5:1, 2:1, 1:1, 1:2, 1:5) (concentrations from 0, 1, 2.5, 5, 10, 25, 50, 100, 250 to 500 μg/mL). After mixing for 10min, 50. Mu.L of H was added to each well ₂ O ₂ Solution (8.8 mmol/L). After mixing, incubation was carried out at 37℃for 30min. The UV absorbance at 510nm was measured for each group of materials using distilled water as a reference, and the experimental results are shown in FIG. 20. VC (FIG. 15 a) and LA (FIG. 15 b) alone have a certain capability of scavenging OH, and EC thereof ₅₀ 69.4 and 60.7. Mu.g/mL, respectively. As shown in fig. 15c, when VC and LA are combined, they exhibit synergistic scavenging of OH, with CI values of less than 1 for each group. When n is _VC :n _LA When=2:1, VC and LA exhibit optimal clearance, whichAnd->And the CI values are respectively 5.1 and 2.6 mug/mL, and are smaller than 0.2 under the condition of each inhibition rate, so that the optimal synergistic effect of removing OH is shown.

2 different concentration [ VC+LA ] mixture for eliminating active oxygen in cell and protecting cell

(1) H of different concentrations ₂ O ₂ And [ VC+LA ]]Toxicity of the mixture to normal cells: human Umbilical Vein Endothelial Cells (HUVECs) in logarithmic growth active phase are selected and inoculated in 96-well plates, and H with different concentrations is added into the cells after 24H of culture ₂ O ₂ And [ VC+LA ]]5 replicates were set for each concentration, and a control group was set. After 4h incubation, the old medium was removed by pipetting, washing 3 times with PBS, adding 200. Mu.L of medium containing 10% (v/v) MTT for further incubation for 2h, then pipetting the old medium again, adding 150. Mu.L of DMSO per well, shaking on a shaker for 2min, and finally measuring absorbance at 490nm with an ELISA reader to calculate cell viability. Experimental results show that [ VC+LA ]]The mixture was non-toxic to normal cells even at high concentrations (1000. Mu.g/mL) (FIG. 16), indicating excellent biocompatibility. H ₂ O ₂ Exhibit a concentration-dependent cytotoxicity of the cells,cell viability was about 60% at a concentration of 250. Mu. Mol/mL (FIG. 17).

(2)[VC+LA]The mixture scavenges normal intracellular reactive oxygen species: human Umbilical Vein Endothelial Cells (HUVECs) in logarithmic growth active phase are selected and inoculated in a 96-well plate, and after 24 hours of culture, VC and LA with different concentrations and [ VC+LA ] with different proportions are respectively added into the culture medium]Mixture solution (n) _VC :n _LA =5:1, 2:1, 1:1, 1:2, 1:5) (concentrations from 0, 1, 2.5, 5, 10, 25, 50, 100, 250 to 500 μg/mL), 5 replicates per concentration were set, and control groups were set. After further culturing for 4 hours, the old medium was aspirated, and 250. Mu. Mol/L H was removed ₂ O ₂ Added to the above cells. After further incubation for 4h, old medium was aspirated and washed 3 times with PBS. 200. Mu.L of serum-free medium containing DCFH-DA (1. Mu.M) was added to the cells and incubation was continued for 30min. After incubation, PBS was washed 3 times. Fluorescence of DCF in each group of cells was detected using a fluoromicroplate reader (λex=488 nm, λem=525 nm). Wherein the fluorescence intensity of DCF in blank cells is 100%, only H ₂ O ₂ The cells acting are positive control. ROS clearance (%) = [ FL _{(Material group)} -FL _(blank) ]/[FL _(Positive) -FL _(blank) ]. Experimental results indicate that VC (FIG. 18 a) and LA (FIG. 18 b) alone have a certain ability to scavenge intracellular ROS, and that their EC ₅₀ 150.6 and 138.3 μg/mL, respectively. As shown in fig. 18c, when VC and LA are combined, they exhibit synergistic effects of scavenging intracellular ROS, with CI values of less than 1 for each combination. It was also found that, in the cell, when n _VC :n _LA At =2:1, VC and LA still exhibit optimal clearance, whichAnd->8.1 and 4.05 μg/mL, respectively, and at each inhibition rate, the CI was less than 0.2 (FIG. 18 d), exhibiting an optimal synergistic effect of scavenging intracellular ROS.

Combination mechanism study of [ VC+LA ] mixtures

With n _VC :n _LA For example, =2:1, study [ vc+la]Combination mechanism of mixture

(1) Non-enzymatic reduction of dehydroascorbic acid (DHA): DHA is reduced by Glutathione (GSH) and dihydrolipoic acid (DHLA) to generate vitamin C. The experimental protocol was as follows: DHA solution (0.2 mM, PBS, pH 7.4) was incubated with GSH (0.2 mM or 0.02mM,PBS,pH 7.4) or DHLA (0.2 mM or 0.02mM,PBS,pH 7.4), and then the characteristic UV absorption of VC at 265nm was immediately detected. The results show (fig. 19) that DHLA is significantly more reducing than GSH, indicating that intracellular DHA is more easily reduced to VC by DHLA.

(2) Intracellular VC and LA content detection: the intracellular VC and LA concentrations were determined by high performance liquid chromatography. Briefly, HUVEC cells in the log-growth active phase were selected (5X 10 ⁴ cells/mL) were seeded in 6-well plates at 37 ℃/5% co ₂ And (5) culturing. After 24H incubation, H was used ₂ O ₂ Cells were stimulated for 4h. After stimulation, [ VC+LA ]]Mixture ([ VC)]=0.2 mM), VC (0.2 mM) and LA (0.1 mM) were incubated with cells for 0, 10, 20, 30, 40, 60, 120, 180, 240, 300 and 360min. After incubation, cells were washed 3 times with PBS centrifugation, collected and cell pellet was lysed with lysis solution (containing 1mM EDTA). The lysate was centrifuged at 4℃for 30min (10000 rpm/min) and the supernatant was collected and stored at-20 ℃. And finally, detecting VC/LA by adopting a high-efficiency liquid phase. As shown in FIGS. 25a, b, intracellular VC/LA are rapidly metabolized after a period of time when VC and LA act on the cells, respectively. However, when VC and LA were incubated simultaneously, intracellular VC and LA content was increased (FIGS. 20a, b). The VC and LA can realize mutual circulation regeneration, so that the intracellular high-level VC/LA content is maintained, and the corresponding effect is promoted.

(3) Detection of VC and LA content in blood of mice: the intracellular VC and LA concentrations were determined by high performance liquid chromatography. Briefly, SD rats of about 6 weeks of age were selected and administered by gavage [ VC+LA ] mixtures ([ VC ] = 20 mg/kg), VC (20 mg/kg) and LA (12 mg/kg), respectively, and blood was collected in blood collection tubes through the eyesockets after various times of administration. The blood was centrifuged to obtain plasma. Acetonitrile was added to plasma, sonicated for 1min with shaking, and finally centrifuged at high speed (10000 rpm/min) for 10min, and the supernatant was collected and stored at-20 ℃. And finally, detecting VC/LA by adopting a high-efficiency liquid phase. As shown in fig. 26a, b, VC or LA is readily metabolized in vivo after administration of VC or LA by intragastric administration alone. When the VC and the LA are combined, the concentration of the VC and the LA in blood can be obviously improved, which indicates that the VC and the LA can be recycled and regenerated mutually when being combined, the metabolism speed of the VC and the LA is reduced, and the absorption of the VC and the LA by organisms is further promoted.

Example 9

The capsule prepared in example 2 was evaluated for in vivo antioxidation in mice by the method described in examples 3-5, and the results showed that the capsule showed a comparable antioxidation effect to the coated tablet under the same VC and LA quality conditions, showing a remarkable synergistic antioxidation effect.

Example 10

Referring to the experimental methods described in examples 1 and 2, tablets and capsules containing vitamin C and sodium lipoic acid were prepared by replacing lipoic acid with sodium lipoic acid. The resulting tablets and capsules were also subjected to antioxidant evaluations with reference to examples 3-5, and the results obtained were consistent with those presented in examples 3-5.

Example 11

Referring to the experimental methods described in examples 1 and 2, tablets and capsules containing LA and ascorbyl palmitate were prepared by substituting vitamin C with ascorbyl palmitate. The results obtained for the antioxidant evaluation of the tablets and capsules were also conducted with reference to examples 3 to 5, and were consistent with those of examples 3 to 5.

The above embodiment is only one of the preferred embodiments of the present invention, and should not be used to limit the scope of the present invention, but all the insubstantial modifications or color changes made in the main design concept and spirit of the present invention are still consistent with the present invention, and all the technical problems to be solved are included in the scope of the present invention.

Claims

1. A health product or medicine with antioxidation effect is characterized in that the main active components are vitamin C and lipoic acid.

2. The health product or medicament according to claim 1, wherein the molar ratio of vitamin C to lipoic acid is in the range of 10:1 to 1:10.

3. The health product or medicament according to claim 1, wherein the molar ratio of vitamin C to lipoic acid is 2:1.

4. The health product or medicament according to claim 1, further comprising an auxiliary material.

5. The health product or medicine according to claim 1, wherein the dosage form of the health product or medicine is powder, tablet, capsule or injection agent.

6. The health product or medicine according to claim 5, wherein the tablet is a normal tablet, which is directly taken orally.

7. The health product or medicine according to claim 5, wherein the tablet is a coated tablet, which is directly taken orally.

8. The health product or medicine according to claim 4, wherein the auxiliary materials comprise microcrystalline cellulose, lactose, polyvinylpyrrolidone 30, corn starch, tartaric acid, magnesium stearate, and talcum powder.

9. The health product or medicament according to claim 1, comprising the following components in weight percent: 5-50% of vitamin C, 5-50% of alpha-lipoic acid, 5-25% of microcrystalline cellulose, 10-25% of lactose, 0.1-3% of 5% of polyvinylpyrrolidone 30, 3-15% of corn starch, 0.5-1.5% of tartaric acid, 0.1-1% of magnesium stearate and 0.1-3% of talcum powder.

10. Use of a vitamin composition comprising vitamin C and lipoic acid for the preparation of an antioxidant health product or medicament.