CN101057857B - Composition for preventing and improving Parkinson disease and its preparation method - Google Patents

Abstract

The invention discloses a combination of vitamin B group, which can effectively improve paralysis agitans. The invention also discloses the combination of LA and ALCAR with low dosage, which can effectively inhibit disorder of mitochondria function caused by elliptone, the expression level of synapse I and ubiquitin is increased, the oxidation injury is increased and anti-oxidation GSH level is reduced. The invention also discloses a mitochondria nutrescin compound, combined with low-concentration LA and ALCAR and vitamin B, and is more effective for paralysis agitans prevention and treatment.

Description

A composition for preventing and improving Parkinson's disease and its preparation method

technical field

The invention relates to the field of neurodegenerative diseases, in particular to the prevention and improvement of Parkinson's disease.

Background technique

The pathological mechanism of Parkinson's disease (PD) is multifaceted. From the perspective of dopamine (DA) metabolism, the reduction of DA in DA neurons is an important link in the occurrence of PD, and the rate-limiting enzyme of its anabolic tyrosine hydroxylase (TH) needs tetrahydropteridine and O ₂ , Fe ²⁺ and other cofactors participate together, and TH activity and content are low; dopa decarboxylase (DDC) uses pyridoxal phosphate as its prosthetic group, and the role of free radicals is another In this mechanism, dopamine itself is easily oxidized, and under the action of monoamine oxidase in the body (with the participation of Fe ³⁺ ), H ₂ O ₂ and O ² + are produced. When the body is in a state of oxidative stress, DA is metabolized abnormally and becomes endogenous At the same time, Fe ²⁺ /Fe ³⁺ participates in the formation of neuromelanin, catalase and glutathione peroxidase with free radical scavenging function have functional defects, further inhibit the mitochondrial NADH electron transfer respiratory chain, and block the complex The activity of I leads to the accumulation of free radicals. On the other hand, the change of Fe ²⁺ will affect the synthesis of heme in the mitochondria, leading to the dysfunction of complex IV and further aggravating the damage of mitochondria. Not only the normal tricarboxylic acid The acid cycling process fails and eventually the DA neurons die.

Increasing evidence has shown that Parkinson's disease (PD), whether caused by the toxic MPTP or by genetic damage, is associated with oxidative damage (Betarbet et al., 2002; Ebadi et al., 1996; Kondo, 1996; Schapira et al., 1990). At present, PD is mostly treated with methods of resisting oxidative damage and protecting striatal dopamine (DA) neurons, such as: blocking DA receptors with Mazindol; blocking NMDA receptors with Dizocilpin maleate; Nutritional factors enhance the survival rate of neurons; provide antioxidants such as _VE , _VC ; or inhibit monoamine oxidase with Selegiline. However, all these methods are not very effective because of side effects.

Mitochondria are the source of oxidants and also the targets of their attacks. The decline in the levels of mitochondrial antioxidants and their metabolites may damage the effect of the antioxidant defense mechanism of nerve cells, leading to neurodegenerative diseases such as Alzheimer's disease (AD) and PD. .

When VB5, VB6, VB11, and VB12 acted alone, they all had a certain effect on the improvement of the motor ability and the prolongation of the lifespan of PD fruit flies, but they could not achieve very effective effects on the motor ability and lifespan, and even encountered effects on both. totally opposite phenomenon.

Data show that supplementing the diet of aged rats with acetyl-L-carnitine (ALCAR) and/or R-lipoic acid (LA) can ameliorate the increased mitochondrial apoptosis associated with aging and reduce the degree of oxidative damage suffered by mitochondria , and enhance the consciousness and exercise vitality of aged rats. Moreover, it has been reported that the combined use of ALCAR and LA is more effective than single use in improving mitochondrial apoptosis in aged rats. The combined use of ALCAR and LA is large, and it is believed that the greater the dose, the better the effect.

Therefore, there is an urgent need in this field to provide a composition, which has a small dose and high safety, and can more effectively inhibit the oxidative damage of mitochondria, so as to prevent, treat and improve Parkinson's disease.

Contents of the invention

The present invention aims to provide a composition, its preparation method and application.

In the first aspect of the present invention, a composition is provided, which contains two or more (such as 2-12 kinds) mitochondrial nutrients, or the composition is composed of two or more (such as 2-12 kinds) species) mitochondrial nutrient composition.

In another preferred example, the mitochondrial nutrients are selected from the group consisting of R-lipoic acid, acetylcarnitine, vitamin B5, vitamin B6, vitamin B11, vitamin B12, coenzyme Q10, thiamin, riboflavin, niacin , biotin or creatine.

In another preferred embodiment, the composition contains:

(a) Vitamin B12;

(b) One or more B vitamins selected from the group consisting of vitamin B5, vitamin B6, and vitamin B11.

In another preferred example, the above component (b) is vitamin B5; or the above component (b) is vitamin B5 and vitamin B6.

In another preferred example, the above-mentioned component (b) includes three B vitamins including vitamin B5, vitamin B6 and vitamin B11.

In another preferred embodiment, the above composition also contains

(c) One or more mitochondrial nutrients selected from the group consisting of R-lipoic acid, or acetylcarnitine.

In another preferred embodiment, the composition contains:

(i) 5-150 parts by weight vitamin B5;

(ii) 2-1000 parts by weight vitamin B6;

(iii) 0.4-40 parts by weight of vitamin B11;

(iv) 0.003-1 parts by weight vitamin B12;

And components (i)+(ii)+(iii)+(iv) account for 10-100% of the total weight of the composition. Preferably components (i)+(ii)+(iii)+(iv) account for 20-90% by weight of the total composition.

In another preferred embodiment, the composition contains:

(i) 15-50 parts by weight vitamin B5;

(ii) 50-300 parts by weight vitamin B6;

(iii) 1-10 parts by weight vitamin B11;

(iv) 0.01-0.2 parts by weight of vitamin B12.

In another preference, the composition also contains:

(v) 100-350 parts by weight R-lipoic acid

(vi) 100-2000 parts by weight of acetylcarnitine;

And components (i)+(ii)+(iii)+(iv)+(v)+(vi) account for 15-100% of the total weight of the composition. Preferably components (i)+(ii)+(iii)+(iv)+(v)+(vi) account for 30-90% by weight of the total composition.

In another preference, the composition also contains:

(v) 150-250 parts by weight R-lipoic acid

(vi) 180-500 parts by weight of acetylcarnitine.

In another preferred example, the composition contains (1) R-lipoic acid and (2) acetylcarnitine.

In another preferred embodiment, the composition contains:

(1) 100-350 parts by weight of R-lipoic acid;

(2) 100-2000 parts by weight of acetylcarnitine;

And the components (1)+(2) account for 10-100% of the total weight of the composition. Preferably components (1)+(2) account for 20-90% of the total weight of the composition.

In another preferred embodiment, the composition contains:

(1) 150-250 parts by weight of R-lipoic acid;

(2) 180-500 parts by weight of acetylcarnitine.

In another preference, the composition also contains:

(3) 5-150 parts by weight vitamin B5;

(4) 2-1000 parts by weight vitamin B6;

(5) 0.4-40 parts by weight of vitamin B11;

(6) 0.003-1 parts by weight vitamin B12;

And the components (1)+(2)+(3)+(4)+(5)+(6) account for 15-100% of the total weight of the composition. Preferably components (1)+(2)+(3)+(4)+(5)+(6) account for 30-90% of the total weight of the composition.

In another preference, the composition also contains:

(3) 15-50 parts by weight vitamin B5;

(4) 50-300 parts by weight of vitamin B6;

(5) 1-10 parts by weight of vitamin B11;

(6) 0.01-0.2 parts by weight of vitamin B12.

In another preferred example, the composition further contains a pharmaceutically acceptable carrier.

In the second aspect of the present invention, a method for preparing a composition is provided, which includes the steps of: ① mixing two or more mitochondrial nutrients together to form a composition.

In another preferred example, the mitochondrial nutrients in the preparation method are selected from the group consisting of R-lipoic acid, acetylcarnitine, vitamin B5, vitamin B6, vitamin B11, vitamin B12, coenzyme Q10, thiamine, riboflavin niacin, biotin, or creatine.

In another preferred embodiment, it includes the step: in step ①, (a) vitamin B12 and (b) one or more B vitamins selected from vitamin B5, B6 or B11 are mixed to prepare a composition.

In another preferred embodiment, it includes the step: in step ①, then mix (c) one or more mitochondrial nutrients selected from the group below: R-lipoic acid or acetylcarnitine to prepare a composition.

In another preferred embodiment, it includes the step: In step ①, it also includes mixing one or more additional mitochondrial nutrients selected from the group consisting of coenzyme Q10, thiamine, riboflavin, niacin, biotin or creatine.

In another preferred example, in step ①, (i) 5-150 parts by weight vitamin B5; (ii) 2-1000 parts by weight vitamin B6; (iii) 0.4-40 parts by weight vitamin B11; and (iv) 0.003-1 parts by weight of vitamin B12 are mixed to prepare the composition.

In another preferred embodiment, it includes the step: in step ①, it also includes mixing (v) 100-350 parts by weight of R-lipoic acid and (vi) 100-2000 parts by weight of acetylcarnitine to prepare the composition .

In another preferred embodiment, it includes the step: in step ①, mix (1) R-lipoic acid and (2) acetylcarnitine to prepare the composition.

In another preferred embodiment, it includes the step: In step ①, it also includes mixing one or more additional mitochondrial nutrients selected from the group consisting of coenzyme Q10, thiamine, riboflavin, niacin, biotin or creatine.

In another preferred embodiment, it includes the step: in step ①, mix (1) 100-350 parts by weight of R-lipoic acid and (2) 100-2000 parts by weight of acetylcarnitine to prepare the composition.

In another preferred embodiment, it includes the steps: in step ①, also includes mixing (3) 5-150 parts by weight of vitamin B5; (4) 2-1000 parts by weight of vitamin B6; (5) 0.4-40 parts by weight of vitamin B6; B11; (6) 0.003-1 parts by weight of vitamin B12 are mixed to prepare a composition.

In the third aspect of the present invention, the use of the above-mentioned composition is provided, and the composition is used for preparing a drug for preventing, treating or improving Parkinson's disease, or for preparing a drug for preventing or improving Parkinson's disease dietary supplements.

In the fourth aspect of the present invention, a method for preventing, treating or improving Parkinson's disease is provided, the method is to administer one or more (more preferably two or more) ) mitochondrial nutrients.

In another preferred example, the mitochondrial nutrients are selected from the group consisting of R-lipoic acid, acetylcarnitine, vitamin B5, vitamin B6, vitamin B11, vitamin B12, coenzyme Q10, thiamin, riboflavin, niacin , biotin or creatine.

In another preferred embodiment, the method is to administer an effective amount of:

(a) Vitamin B12;

(b) One or more B vitamins selected from the group consisting of vitamin B5, vitamin B6, and vitamin B11.

In another preferred example, the treatment method is to also apply an effective amount of:

(c) One or more mitochondrial nutrients selected from the group consisting of R-lipoic acid, or acetylcarnitine.

In another preferred example, the treatment method is to administer effective doses of: (1) R-lipoic acid and (2) acetylcarnitine to subjects in need.

Therefore, the present invention provides a composition with small dosage and high safety, which can more effectively inhibit the oxidative damage of mitochondria, and achieve the purpose of preventing, treating and improving Parkinson's disease.

Description of drawings

Figure 1 shows the inhibitory effects of pretreatment with LA, ALCAR and their combination on the decrease of mitochondrial membrane potential induced by rotenone.

Figure 2 shows the inhibitory effect of pretreatment with LA, ALCAR and their combination on the decline of mitochondrial complex I activity induced by rotenone.

Figure 3 shows the inhibitory effects of pretreatment with LA, ALCAR and their combination on the decrease of ATP level induced by rotenone.

Figure 4 shows the inhibitory effect of pretreatment with LA, ALCAR and their combination on the increase of cytochrome c release induced by rotenone.

Figure 5 shows the inhibitory effect of pretreatment with LA, ALCAR and their combination on the decrease of GSH level induced by rotenone.

Figure 6 shows the inhibitory effects of pretreatment with LA, ALCAR and their combination on the increase of protein oxidative damage induced by rotenone.

Figure 7 shows the inhibitory effect of pretreatment with LA, ALCAR and their combination on oxidative DNA damage induced by rotenone.

Figure 8 shows the inhibitory effect of pretreatment with LA, ALCAR and their combination on the increase of ROS induced by rotenone.

Figure 9 shows the effects of pretreatment with LA, ALCAR and their combination on the expression changes of synaptophysin I (α-synuclein) and its mRNA induced by rotenone.

Figure 10 shows the effects of pretreatment with LA, ALCAR and their combination on the increase of synaptophysin I and ubiquitin levels induced by rotenone.

Figure 11 shows the effect of VB5 on the climbing ability of PD male Drosophila.

Figure 12 shows the effect of VB5 on the lifespan of PD male Drosophila.

Figure 13 shows the effect of VB5 on the climbing ability of PD female Drosophila.

Figure 14 shows the effect of VB5 on the lifespan of PD female Drosophila.

Figure 15 shows the effect of VB12 on the climbing ability of PD male Drosophila.

Figure 16 shows the effect of VB12 on the lifespan of PD male Drosophila.

Figure 17 shows the effect of VB12 on the climbing ability of PD female Drosophila.

Figure 18 shows the effect of VB12 on the lifespan of PD female Drosophila.

Figure 19 shows the effect of VB6 on the climbing ability of PD male Drosophila.

Figure 20 shows the effect of VB6 on the lifespan of PD male flies.

Figure 21 shows the effect of VB6 on the climbing ability of PD female Drosophila.

Figure 22 shows the effect of VB6 on the lifespan of PD female Drosophila.

Figure 23 shows the effect of VB11 on the climbing ability of PD male Drosophila.

Figure 24 shows the effect of VB11 on the lifespan of PD male Drosophila.

Figure 25 shows the effect of VB11 on the climbing ability of PD female Drosophila.

Figure 26 shows the effect of VB11 on the lifespan of PD female Drosophila.

Figure 27 shows the effect of VB5+VB12 on the climbing ability of PD male Drosophila.

Figure 28 shows the effect of VB5+VB12 on the lifespan of PD male Drosophila.

Figure 29 shows the effect of VB5+VB12 on the climbing ability of PD female Drosophila.

Figure 30 shows the effect of VB5+VB12 on the lifespan of PD female Drosophila.

Figure 31 shows the effect of VB6+VB11+VB12 on the climbing ability of PD male Drosophila.

Figure 32 shows the effect of VB6+VB11+VB12 on the lifespan of PD male Drosophila.

Figure 33 shows the effect of VB6+VB11+VB12 on the climbing ability of PD female Drosophila.

Figure 34 shows the effect of VB6+VB11+VB12 on the lifespan of PD female Drosophila.

Figure 35 shows the effect of VB5+VB6+VB11+VB12 on the climbing ability of PD male Drosophila.

Figure 36 shows the effect of VB5+VB6+VB11+VB12 on the lifespan of PD male Drosophila.

Figure 37 shows the effects of VB5+VB6+VB11+VB12 on the climbing ability of PD female Drosophila.

Figure 38 shows the effect of VB5+VB6+VB11+VB12 on the lifespan of PD female Drosophila.

Figure 39 shows the inhibitory effect of LA+ALCAR, VB5+VB6+VB11+VB12 and LA+ALCAR+VB5+VB6+VB11+VB12 pretreatment on the mitochondrial damage caused by MPP, which further leads to cell death.

Figure 40 shows the effect of LA on the climbing ability of PD male flies.

Figure 41 shows the effect of LA on the lifespan of PD male flies.

Figure 42 shows the effect of LA on climbing ability of PD female flies.

Figure 43 shows the effect of LA on the lifespan of PD female flies.

Figure 44 shows the effect of ALCAR on the climbing ability of PD male flies.

Figure 45 shows the effect of ALCAR on the climbing ability of PD female flies.

Figure 46 shows the effect of ALCAR on lifespan in PD flies.

Figure 47 shows the effect of LA+ALCAR on the climbing ability of PD flies.

Figure 48 shows the effect of LA+ALCAR on the lifespan of Drosophila with PD.

Figure 49 shows the expression of tyrosine hydroxylase and synaptophysin I in the Drosophila model of PD.

1. Female PD flies, 2. Male PD flies, 3. Female non-PD flies, 4. Male non-PD flies

Figure 50 shows the effect of LA on synaptophysin I expression in male PD flies.

1. Non-PD fly control, 2. PD fly control, 3. LA10

Figure 51 shows the effect of LA on synaptophysin I expression in female PD Drosophila.

1. Non-PD fly control, 2. PD fly control, 3. LA10

Figure 52 shows the effect of LA on Synaptophysin 1 mRNA expression in male PD Drosophila.

Figure 53 shows the effects of LA and VB6 on dopamine neurons in the nervous system of Drosophila PD.

1. Uas-DDC-gal4 fruit fly (the father fruit fly of PD fruit fly) for 1 day, 2. PD fruit fly control for 1 day, 3. uas-DDC-gal4 fruit fly for 20 days, 4. PD fruit fly control for 20 days 5. PD fruit flies were administered VB6 for 20 days, 6. PD fruit flies were administered LA for 20 days

Figure 54 shows the effect of VB5, VB6, VB11, VB12, LA on the expression of synaptophysin I in male PD Drosophila.

1. Non-PD fly control, 2. PD fly control, 3. VB5, 4. VB6, 5. VB11, 6. VB12, 7. LA

Figure 55 shows the effect of VB5+VB6+VB11+VB12 (ie VB _s ) on the expression of synaptophysin I mRNA in male PD Drosophila.

Detailed ways

After extensive and in-depth research, the inventor unexpectedly found that the combination of small doses of acetylcarnitine (ALCAR) and R-lipoic acid (LA) can effectively inhibit the senile degeneration of nerve cells.

The present inventors have also unexpectedly found that vitamin (Vit) B ₅ is used in combination with one or more B vitamins among vitamins B ₆ , B ₁₁ , and B ₁₂ (especially vitamin (Vit) B ₅ , B ₆ , B ₁₁ and B ₁₂ four vitamins in combination), can produce a synergistic effect, thereby more effectively improving Parkinson's disease.

In another preferred example, the inventor further found that the combined use of small doses of ALCAR and LA, and vitamins (Vit) B ₅ , B ₆ , B ₁₁ and B ₁₂ has a better effect on improving Parkinson's disease .

definition

As used herein, the terms "comprising" or "comprising" include "comprising", "consisting essentially of", and "consisting of".

As used herein, the term "consisting essentially of" means that in addition to essential ingredients or essential components, the composition may also contain a small amount of secondary ingredients and/or impurities that do not affect the active ingredients. For example, sweeteners to improve taste, antioxidants to prevent oxidation, and other additives commonly used in the art may be contained.

As used herein, the term "effective amount" refers to an amount that can produce functions or activities on humans and/or animals and that can be accepted by humans and/or animals.

As used herein, the terms "ALCAR", "acetylcarnitine" and "acetyl-L-carnitine" are used interchangeably and all refer to the acetyl derivative of L-carnitine.

As used herein, the term "pharmaceutically or food acceptable carrier" refers to a carrier used for the administration of therapeutic agents or the consumption of health products, including various excipients and diluents. The term refers to carriers which, by themselves, are not essential active ingredients and which are not unduly toxic upon administration. Suitable vectors are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991). Pharmaceutically acceptable carriers in compositions can contain liquids such as water, saline, glycerol and ethanol. In addition, there may also be auxiliary substances in these carriers, such as wetting agents or emulsifying agents, pH buffering substances and the like. Non-essential ingredients other than mitochondrial nutrients such as LA, ALCAR, VitB ₅ , VitB ₆ , VitB ₁₁ and VitB ₁₂ , as well as other non-essential ingredients (such as other auxiliary medicinal materials or food materials), are also included in pharmaceutical or food science in the definition of acceptable carrier above.

As used herein, the term "essential ingredients" refers to essential chemical substances as active ingredients, ie, mitochondrial nutrients such as LA, ALCAR, VitB5, VitB6, VitB11, and VitB12. The ingredients may also be used in the form of "physiologically acceptable salts" or "physiologically acceptable salts derived from acids or bases". Said salts include (but are not limited to): salts formed with following inorganic acids: such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, and salts formed with organic acids, while organic acids refer to acetic acid, oxalic acid, succinic acid, tartaric acid , methanesulfonic acid and maleic acid. Other salts include those formed with alkali or alkaline earth metals such as sodium, potassium, calcium or magnesium, in the form of esters, carbamates or other conventional "prodrugs" (when administered in this form , which can be converted into active moieties in vivo).

As used herein, the term "composition of the invention" includes pharmaceutical compositions, food compositions and/or dietary supplements as long as they contain or consist essentially of two or more mitochondrial nutrients selected from the group consisting of: R-sulfur Caprylic acid, acetylcarnitine, vitamin B5, vitamin B6, vitamin B11, vitamin B12, coenzyme Q10, thiamin (VB1), riboflavin (VB2), niacin (VB3), biotin (VB7), or creatine.

As used herein, "mitochondrial nutrients" refer to nutrients that can protect mitochondria from oxidative damage and improve mitochondrial function, including those that can ① protect mitochondria from oxidative stress, such as antioxidants and metal chelates; ② repair mitochondrial membranes ; ③ Functionally repair and prevent mitochondrial oxidative damage, such as substrates or coenzymes for mitochondrial enzymes; and ④ Induce the expression of phase 2 antioxidant enzymes.

As used herein, the term "unit dosage form" refers to the preparation of the composition of the present invention into a dosage form required for single administration for convenience of administration, including but not limited to various solid dosage forms (such as tablets), liquid dosage forms, and capsule dosage forms. , Sustained release agent. The unit dosage form contains the composition of the present invention which is effective for preventing, treating or improving Parkinson's disease.

As used herein, "parts by weight" or "parts by weight" can be used interchangeably, and said parts by weight can be any fixed weight expressed in milligrams, grams or kilograms (such as 1 mg, 1 g, 2 g, 5 g, or 1kg, etc.). For example, a composition consisting of 1 part by weight of component a and 9 parts by weight of component b can be 1 gram of component a+9 gram of component b, or 10 gram of component a+90 gram of component b etc. composition. In the composition, the percentage content of a certain component=(parts by weight of this component/sum of parts by weight of all components)×100%. Therefore, in a composition composed of 1 part by weight of component a and 9 parts by weight of component b, the content of component a is 10%, and the content of component b is 90%.

The composition of the present invention can be directly used for preventing, alleviating or treating Parkinson's disease, or can be co-administered with other drugs or dietary additives.

Mitochondrial nutrients

The composition of the present invention may contain one or more mitochondrial nutrients selected from the group consisting of R-lipoic acid, acetylcarnitine, vitamin B5, vitamin B6, vitamin B11 or vitamin B12. The mitochondrial nutrient composition of the present invention contains or consists essentially of ① one or more of VitB12+VitB5, VitB6, and VitB11, or contains or consists essentially of ②R-lipoic acid+acetylcarnitine, or contains or consists essentially of ③ R-lipoic acid + acetylcarnitine + VitB5 + VitB6 + VitB11 + VitB12 composition. Usually, the weight of ① one or more of VitB12+VitB5, VitB6, VitB11, or ②R-lipoic acid+acetylcarnitine, or ③R-lipoic acid+acetylcarnitine+VitB5+VitB6+VitB11+VitB12 accounts for the total composition 15-99% by weight, preferably 30-90%, more preferably 50-90%. The rest of the substances are pharmaceutically or food acceptable carriers. In a preferred example, the composition of the present invention usually does not contain other drugs currently used in the treatment of Parkinson's disease, such as L-DOPA, selegiline and the like.

Representative other mitochondrial nutrients suitable for use in the present invention include, but are not limited to: Coenzyme Q10, Thiamin, Riboflavin, Niacin, Biotin, and Creatine.

1. Coenzyme Q10 (CoQ): An electron carrier located in the inner mitochondrial membrane, it can stabilize respiratory chain components and can function as a mitochondrial antioxidant (Ebadi et al., 2001; Ernster, 1994; Frei et al., 1990). In a variety of mitochondrial disorders, including PD, Huntington's disease and ataxia, CoQ has a positive impact on clinical treatment and changes in biochemical indicators (Beal et al., 1998; Ebadi et al., 2001; Shults et al., 2002). In MPTP-treated mice, CoQ attenuated MPTP-induced neurotoxicity, increased dopamine levels in the striatum, increased the number of striatal mitochondria and ATP synthesis, and enhanced the activity of mitochondrial complexes in the striatum (Beal, 2003; Ebadi et al., 2001; Ebadi et al., 1996), attenuates the increase in oxidants induced by MPTP and the depletion of dopamine caused by MPTP in the striatum (Beal et al., 1998; Ebadi et al., 2001). Clinical studies have shown that the application of high doses of CoQ in PD patients can slow down the process of disabling symptoms, and the dose can reach up to 1200mg/d. Therefore CoQ can safely slow down the worsening of PD symptoms (Shults et al., 2002).

The levels of CoQ (Ernster, 1994), LA, and carnitine (Liu et al., 2002) in the body will decline with age, and supplementation with LA, ALCAR, or CoQ can improve age-related decline in cognitive function and neurodegeneration phenomenon (Beal, 2003; Liu et al., 2002.a).

2. Thiamine (VB1): Thiamine (VB1) is the precursor of thiamine pyrophosphate coenzyme, and VB1 is phosphorylated to form thiamine pyrophosphate, which is a cofactor for various enzymes including mitochondrial enzymes; VB1 plays an important role in the metabolism of acetylcholine and its release from presynaptic neurons. It has been reported that in neurodegenerative diseases and other forms of dementia, for example, the activity of VB1-dependent enzymes in the brain and peripheral tissues of AD patients is reduced (Higdon, 2003), including pyruvate dehydrogenase and α-ketoglutarate dehydrogenase (Butterworth and Besnard, 1990; Gibson et al., 1988; Rao et al., 1993), which causes apoptosis of mitochondria, leading to oxidation of proteins in mitochondria, which in turn affects thiamine pyrophosphate The Km value of vitamin B1 forms a vicious circle, leading to brain dysfunction, and this functional defect can be reversed by high doses of VB1 (Harman, 1988; Heroux et al., 1996). Therefore, normal and pathological aging-specific brain dysfunction may be alleviated by high doses of VB1. It can be seen that taking VB1 may be an effective candidate drug for the prevention and treatment of PD.

3. Riboflavin (VB2): Riboflavin (VB2) is the precursor of the coenzymes FMN and FAD of mitochondrial complexes I and II. Because the VB2 derivative FAD is glutathione reductase (catalyzes the production of reduced glutathione), xanthine oxidase (catalyzes the production of uric acid) and methylenetetrahydrofolate reductase (can catalyze the production of formazan from homocystine). So VB2 deficiency may have an indirect effect on brain function by affecting the electron transport chain, antioxidant enzymes and homocystine metabolism. Simultaneously elevated levels of homocysteine (Duan et al., 2002; Kruman et al., 2002; Seshadri et al., 2002) and reduced glutathione (Shukitt-Hale et al., 1998) and uric acid (Ames et al., 1981) are associated with aging and cognitive impairment. Mitochondrial complex I with FMN and FAD as cofactors is defective in PD patients. Recent physiological and pathological experiments have restricted the main role of the oxygen production site to the FMN group of mitochondrial complex I, unlike the previous view that the oxygen production site is generally believed to be on the ubiquinone of mitochondrial complex III, This finding further demonstrates the importance of ameliorating the mitochondrial complex I deficiency in PD (Liu, Y., Fiskum, G., and Schubert, D. 2002d). Clinical studies have shown that taking high-dose VB2 (oral, 30mg every 8 hours) while removing red meat from food can improve the exercise capacity of some PD patients (Coimbra and Junqueira, 2003), which suggests that this therapy can improve the performance of PD patients. VB2 sensitive mechanisms, such as glutathione depletion, cumulative mitochondrial DNA mutations, disordered mitochondrial protein complexes and abnormal iron metabolism, etc. VB2 may stimulate the activity of mitochondrial enzymes and change the Km value of FMN/FAD. However, VB2 is an important coenzyme of monoamine oxidase (MAO) on the outer mitochondrial membrane. Its increase may increase the decomposition of dopamine in the dopamine system, which is not conducive to the improvement of PD symptoms. Therefore, the application of VB2 must be carefully matched with other mitochondrial nutrients to prevent side effects.

4. Nicotinic acid (VB3): Niacin and nicotinamide are collectively referred to as nicotinic acid, which is the precursor of NAD and NADP. In mitochondria and cytoplasm, high doses of nicotinic acid can increase the level of NAD/NADP (Ames et al., 2002), increase the chance of affinity of mitochondrial complex I for the substrate, and further enhance the enzyme activity. NADH and NADPH are not only substrates and coenzymes of mitochondrial complex I, but also endogenous antioxidants. The application of VB3 in MPTP-treated mice and poly ADP-ribose polymerase knockout mice can resist neurotoxicity, and can reduce neurological injury and focal cerebral ischemia, ATP consumption caused by malonate and MPTP (Beal, 2003). NADH can also stimulate the synthesis of endogenous dopamine and reduce the muscle motor impairment and recognition dysfunction in PD patients (Birkmayer and Birkmayer, 1989; Kuhn and Muller, 1997). It has also been reported that VB3 and VB2 have a certain effect on mitochondrial complex I deficiency diseases, such as mitochondrial encephalopathy, lactic acidosis and cerebral shock. However, the intake of VB3 in PD patients is significantly reduced (Hellenbrand et al., 1996), which requires appropriate supplementation.

5. Biotin (VB7): Mitochondrial enzymes that use VB7 as a cofactor include pyruvate carboxylase, acetyl-CoA carboxylase, propionyl-CoA carboxylase, and carboxylase holoenzyme synthetase, etc. Sugar metabolism, fatty acid synthesis and metabolic processes of various amino acids. VB7 deficiency is quite common in the population (especially in pregnant women), (Mock et al., 2002.a; Mock et al., 2002b), and directly leads to the aging of mitochondria and the production of oxidants (Atamna, Erlitski, & Ames , 2005). VB7 is a compensatory group for the biotin-dependent carboxyl groups of the above four enzyme molecules (Mock, 1989), among which the first three are mitochondria-specific enzymes, all of which catalyze the metabolite feedback reaction in the tricarboxylic acid cycle and provide sub- Precursor of heme - succinyl-CoA. Lack of VB7 reduces the activity of these enzymes, and the β-methylcrotonyl-CoA accumulated in mitochondria upon VB7 deficiency reacts with glycine (Mock, 1989), leading to amino acid depletion in the mitochondrial matrix. Therefore, the lack of VB7 will lead to the lack of mitochondrial succinyl-CoA and aminoacetic acid, further causing the lack of heme.

6. Creatine: Creatine is a substance synthesized from three amino acids, arginine, glycine and methionine. Creatine can be synthesized in the liver, kidney, pancreas and other organs; it can also be ingested from the diet. The creatine/phosphocreatine conversion system between the cytoplasm and mitochondria is able to utilize a unique recombinant isoform of mitochondrial creatine kinase (CK) and function as a spatial energy buffer: intracellular creatine, phosphate It can be converted with phosphocreatine according to the metabolic level of cells and the concentration of ATP. Creatine increases creatine/phosphocreatine conversion and inhibits the opening of the mitochondrial permeability transport pore (Beal, 2003). ATP is an energy supply material for anaerobic metabolism of the body, but its molecular weight is large and its stability in vivo and in vitro is poor, while creatine has a small molecular weight and good chemical stability, and has extensive storage significance in the body. In a double-blind study of 45 young vegetarians, a placebo-controlled cross-over study showed that oral creatine supplementation for six weeks (5g/day) was equally effective for both mental work and high-efficiency work. role (Rae et al., 2003). Likewise, creatine has also been used in the treatment of several neurological diseases including PD and Huntington's disease.

In the present invention, the mitochondrial nutrient composition that can be effectively used to prevent, improve and treat Parkinson's disease includes but is not limited to: ① one or more of VitB12+VitB5, VitB6, and VitB11; ②R-lipoic acid+acetyl carnitine Alkali; ③R-lipoic acid + acetylcarnitine + VitB5 + VitB6 + VitB11 + VitB12. For mammals suffering from Parkinson's disease, administration of the composition prepared from the above-mentioned mitochondrial nutrients can significantly improve the effect. The composition is a pharmaceutical composition; or a food composition.

In a preferred mode of the present invention, the composition is formulated using components selected from the following: VitB12, VitB5, VitB6, VitB11, and the components can be combined in any combination of two, three or four, A more preferred combination is VitB12+VitB5+VitB6+VitB11.

In a preferred example of the present invention, the joint application of R-lipoic acid + acetylcarnitine is used to greatly improve the effect and greatly reduce the dosage compared with the single application.

In addition, in a preferred example of the present invention, when the combination of ①VitB12+VitB5+VitB6+VitB11 is used, adding R-lipoic acid+acetylcarnitine to ① can achieve a better effect.

Therefore, the composition for preventing, improving or treating Parkinson's disease of the present invention contains vitamin B12 and any one or more of vitamins B5, B6 or B11. When the composition contains vitamin B12, it also contains one or more of the components vitamin B5, vitamin B6 or vitamin B11. For example, the composition includes but is not limited to: vitamin B12+vitamin B5 composition, A combination of vitamin B12+vitamin B6, a combination of vitamin B12+vitamin B11, a combination of vitamin B12+vitamin B5+vitamin B6, a combination of vitamin B12+vitamin B5+vitamin B11, a combination of vitamin B12+vitamin B6+vitamin B11, or a combination of vitamin B12+ Composition of vitamin B5+vitamin B6+vitamin B11.

In a preferred example, the composition contains:

(i) 5-150 parts by weight of vitamin B5, preferably 15-50 parts by weight, more preferably 25-35 parts by weight;

(ii) 2-1000 parts by weight of vitamin B6, preferably 50-300 parts by weight, more preferably 80-150 parts by weight;

(iii) 0.4-40 parts by weight of vitamin B11, preferably 1-10 parts by weight, more preferably 2.5-5.5 parts by weight;

(iv) 0.003-1 parts by weight of vitamin B12, preferably 0.01-0.2 parts by weight, more preferably 0.07-0.15 parts by weight;

And component (i)+(ii)+(iii)+(iv) accounts for 10-100% of the total weight of the composition; preferably, component (i)+(ii)+(iii)+(iv) It accounts for 20-90% of the total weight of the composition; more preferably, components (i)+(ii)+(iii)+(iv) account for 40-90% of the total weight of the composition.

The composition for preventing, improving or treating Parkinson's disease of the present invention contains R-lipoic acid and acetylcarnitine.

In a preferred example, the composition contains:

(1) 100-350 parts by weight of R-lipoic acid, preferably 150-250 parts by weight, more preferably 180-220 parts by weight;

(2) 100-2000 parts by weight of acetylcarnitine, preferably 180-500 parts by weight, more preferably 180-300 parts by weight;

And component (1)+(2) accounts for 10-100% of composition gross weight; Preferably, component (1)+(2) accounts for 20-90% of composition gross weight; More preferably, composition Parts (1)+(2) account for 40-90% of the total weight of the composition.

In a preferred mode of the present invention, components (1) and (2) exist in an appropriate ratio, and the weight ratio of (1) and (2) is 1:10-10:1; more preferably, the components The weight ratio of (1) to (2) is 1:5-5:1; most preferably, the weight ratio of components (1) to (2) is 1:1-3:1.

As a particularly preferred composition of the present invention, it contains the following components: (a) 5-150 parts by weight of vitamin B5, preferably 15-50 parts by weight, more preferably 25-35 parts by weight; (b) 2 -1000 parts by weight of vitamin B6, preferably 50-300 parts by weight, more preferably 80-150 parts by weight; (c) 0.4-40 parts by weight of vitamin B11, preferably 1-10 parts by weight, more preferably 2.5- 5.5 parts by weight; (d) 0.003-1 parts by weight vitamin B12, preferably 0.01-0.2 parts by weight, more preferably 0.07-0.15 parts by weight; (e) 100-350 parts by weight R-lipoic acid, preferably 150 parts by weight -250 parts by weight, more preferably 180-220 parts by weight; and (f) 100-2000 parts by weight of acetylcarnitine, preferably 180-500 parts by weight, more preferably 180-300 parts by weight; and component (a )+(b)+(c)+(d)+(e)+(f) account for 15-100% of the total weight of the composition; preferably, component (i)+(ii)+(iii)+ (iv) accounts for 30-90% of the total weight of the composition; more preferably, components (i)+(ii)+(iii)+(iv) account for 50-90% of the total weight of the composition.

The composition of the present invention may also contain one or more representative other mitochondrial nutrients: 8-1200 mg parts by weight of coenzyme Q ₁₀ , 4-200 mg parts by weight of thiamine, 2-240 mg parts by weight of riboflavin, 20-800 mg parts by weight Niacin by weight, 1-20 mg by weight biotin and 0.5-12 mg by weight creatine.

In another preferred embodiment, the composition contains:

(i)VB5 15mg

(ii)VB6 50mg

(iii)VB11 2mg

(iv) VB12 0.05mg.

In another preferred embodiment, the composition contains:

(1) R-lipoic acid 100mg,

(2) Acetylcarnitine 100mg.

In another preferred embodiment, the composition contains:

(a) R-lipoic acid 100mg,

(b) Acetylcarnitine 100mg

(c)VB5 15mg

(d)VB6 50mg

(e)VB11 2mg

(f) VB12 0.05 mg.

As a particularly preferred composition of the present invention, the composition further contains a pharmaceutically or food acceptable carrier or excipient: 50-1000 parts by weight.

The pharmaceutical composition or dietary supplement of the present invention can be made into any conventional preparation form by conventional methods, preferably a sustained-release dosage form, which can make the necessary components release slowly, stably and continuously.

The sustained-release dosage form can be prepared by a conventional method, and a preferred method is to use an excipient as a coating material, and coat the necessary components in it to form a coating slow/controlled release preparation, or to use the necessary components Loaded into a sustained-release matrix to form a matrix-type sustained/controlled release preparation. Another preferred method is to make the essential ingredients into their respective salts, and use the difference in solubility of different salts to gradually release each essential ingredient to produce a long-lasting effect.

The composition provided by the present invention can be determined according to the dosage form to be prepared and the route of administration. Those skilled in the art, after referring to the composition and the proportioning provided by the present invention, adopt the formula of conventional pharmaceutical composition or food composition. The method of preparation enables the preparation of the composition of the present invention. A preferred method is to dissolve the fat-soluble essential components first with a cosolvent and then add water to dissolve them to form a solution, and directly dissolve the water-soluble essential components to form a solution, and then mix the solution of the fat-soluble essential components with the water-soluble A solution of the essential ingredients is mixed to obtain the composition provided by the present invention.

The dosage of the composition provided by the invention for preventing and improving Parkinson's disease is 100-350mgLA and 100-2000mgALCAR per day; or _4-150mgVitB5 , _{2-1000mgVitB6} , _{0.4-40mgVitB11} and 0.003-1mgVitB per day ₁₂ ; or 100-350 mg LA, 100-2000 mg ALCAR, 4-150 mg VitB ₅ , 2-1000 mg VitB ₆ , 0.4-40 mg VitB ₁₁ and 0.003-1 mg VitB ₁₂ daily.

The compositions for preventing and improving Parkinson's disease provided by the present invention may also include, but are not limited to, one or more of the following other representative mitochondrial nutrients: coenzyme Q10, thiamine, riboflavin, niacin , biotin and creatine, the usage and dosage are oral coenzyme Q ₁₀ 8-1200mg/day, oral thiamine 4-200mg/day, oral riboflavin 2-240mg/day, oral niacin 20-800mg/day, oral Biotin 1-20mg/day, oral creatine 0.5-12mg/day.

A preferred method is to supplement energy when taking it, such as taking carbohydrates and fruit juice drinks at the same time, so as to provide auxiliary energy for preventing and improving Parkinson's disease and promote the synthesis of mitochondrial ATP. The energy needed to supplement is about 50-150 calories.

When used to prepare pharmaceutical or food compositions, the effective dosage of the mitochondrial nutrient composition used may vary with the mode of administration and the severity of the disease to be treated.

There is no particular limitation on the dosage form of the composition of the present invention, and it can be any dosage form suitable for mammals; preferably, the dosage form can be selected from: granules, capsules, tablets, powders, oral liquids , suspension, or emulsion.

In another preferred mode of the present invention, the composition is used as a dietary additive, added to water, non-orange juice drinks, solid food, cooking dishes, such as grape juice or apple juice. Preferably added to meals that provide at least 50-150 calories.

In another preferred mode of the present invention, the food-acceptable carrier or excipient is selected from: fillers, disintegrants, lubricants, glidants, effervescent agents, flavoring agents, coating materials, dietary products, or sustained/controlled release agents.

In another preferred mode of the present invention, the composition is in unit dosage form, each dose contains 100-350mgLA and 100-2000mgALCAR; or contains 4-150mgVitB ₅ , 2-1000mgVitB ₆ , 0.4-40mgVitB ₁₁ and 0.003-1mgVitB ₁₂ ; or containing 100-350 mg LA, 100-2000 mg ALCAR, 4-150 mg VitB ₅ , 2-1000 mg VitB ₆ , 0.4-40 mg VitB ₁₁ and 0.003-1 mg VitB ₁₂ . Each dose may also contain one or more representative other mitochondrial nutrients: 8-1200mg coenzyme Q ₁₀ , 4-200mg thiamine, 2-240mg riboflavin, 20-800mg niacin, 1-20mg biotin and 0.5-12mg creatine.

When the composition is prepared in a unit dosage form, 1-3 doses of the composition in the unit dosage form are taken every day; more preferably, 1-2 doses of the composition in the unit dosage form are taken every day.

The composition provided by the present invention can be administered orally. Solid carriers include: starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose, and kaolin, while liquid carriers include: culture medium, polyethylene glycol, nonionic surfactants, and edible oils (such as corn oil, peanut oil, etc.) and sesame oil). Adjuvants commonly used in the preparation of pharmaceutical compositions may also advantageously be included, such as flavourings, colours, preservatives and antioxidants such as Vitamin C, BHT and BHA.

From the standpoint of ease of preparation and administration, preferred pharmaceutical compositions are solid compositions, especially tablets and solid-filled or liquid-filled capsules. Oral administration is preferred.

More preferred dosage forms are those that can ensure that nutrients are supplied to mitochondria slowly and stably for 24 hours. One way is to use a suitable carrier to carry the mitochondrial nutrient composition, so that the mitochondrial nutrient composition is released slowly. For example, using excipients (such as hydroxypropyl methylcellulose) as a coating material, encapsulating mitochondrial nutrients in it to form spherical particles (coated slow/controlled release preparations); or loading mitochondrial nutrients into matrix sustained-release tablets ( Such as hydroxyethyl cellulose) (skeleton type sustained/controlled release preparations).

It should be understood that the mitochondrial nutrient composition of the present invention may also contain other ingredients that are necessary for the human body or beneficial to the human body but do not affect or produce direct medicinal effects. In addition, according to the doctor's guidance, the composition of the present invention can also be co-administered with other conventional drugs for treating or preventing Parkinson's disease.

The composition of the present invention can be used to prepare medicines for preventing, improving or treating cell mitochondrial metabolic disorders; or, the composition of the present invention can be used for preparing medicines for preventing, improving or treating Parkinson's disease.

The present inventor utilizes the Drosophila Parkinson's disease model (obtained by the hybridization of synaptophysin I transgenic fruit flies and uas-DDC fruit flies) transferred into the synaptophysin I gene, and the Parkinson's fruit fly model expresses synaptophysin I, And the loss of dopamine neurons occurs in adulthood, filamentous inclusions containing synaptophysin I appear in neurons, and motor function is impaired. Because this fruit fly model has the basic characteristics of human Parkinson's disease, it is possible to use this genetic method to study Parkinson's disease.

The inventors found that when VB5, VB6, VB11 and VB12 were used in combination on the PD fruit fly model, the motor ability and lifespan of the PD fruit fly were significantly improved.

When acting alone, LA in the 50X dose group is effective on the motor ability and lifespan of PD flies, while ALCAR only prolongs the lifespan of PD flies, but has no effect on the motor ability. When the two act together, the 10X dose group has a stronger effect on the motor ability of PD flies than the 10X dose of LA alone.

The combined effect of VB5, VB6, VB11, VB12, LA and ALCAR is stronger than that of single drug, and the curative effect is more stable and safe under the condition of combined action. In the experiment of mitochondrial damage caused by MPP treatment of cells, the inventors found that the cell survival rate after LA+ALCAR+VB5+VB6+VB11+VB12 pretreatment was higher than that of LA+ALCAR pretreatment or VB5+VB6+VB11+VB12 pretreatment high cell viability.

The present inventors used the chronic rotenone model (5nM, 4 weeks) of SK-NM-C as an in vitro PD model to reproduce the toxic effects of rotenone on mitochondrial function, and the effects on PD pathological features, mitochondrial function, oxidation and anti-oxidative damage biomarkers, etc. Several aspects were investigated, pretreatment with LA (0.1, 1, 10, 100 uM), ALCAR (0.1, 1, 10, 100 uM) and their combinations of different components for 4 weeks. The molecular pathology of PD is a defect in mitochondrial function. In this cellular model, rotenone-treated mitochondria had decreased complex 1 activity, increased ATP depletion, and increased cytochrome C release on the inner mitochondrial membrane. Appropriate doses of LA and ALCAR, two mitochondrial nutrients, had therapeutic effects on mitochondria treated with rotenone. The most effective doses are 10uM LA and 100uM ALCAR, and when the two are used in combination, the effective doses appear at 0.1 and 1uM. This optimal ratio is much more effective than either alone. Mitochondrial nutrients such as LA and ALCAR can effectively inhibit the generation of cellular oxygen free radicals and oxidative damage caused by rotenone.

The present invention proves that the mitochondrial nutrients VB5, VB6, VB11, VB12, LA and ALCAR and their different combinations will have different curative effects on the PD fruit fly model, and the combined application of VB5, VB6, VB11 and VB12, or the combined application of LA and ALCAR The effect is stronger than that of the drug alone. LA and ALCAR have certain effects on inhibiting mitochondrial dysfunction, oxidative damage and preventing the accumulation of synaptophysin I and ubiquitin in PD cell models. Moreover, the mixture of LA and ALCAR at low concentrations (0.1-1uM) exhibited a positive synergistic effect, which was 10 times stronger than either drug alone on mitochondrial dysfunction and oxidation induced by rotenone treatment. Damage works. In addition, the protective effect of LA+ALCAR+VB5+VB6+VB11+VB12 on cells was stronger than the combination of LA+ALCAR or VB5+VB6+VB11+VB12.

The main advantages of the present invention are:

1. By organically combining the necessary ingredients, multi-target and multi-directional synergistic effects are exerted;

2. The ingredients are natural, non-toxic and side effects, suitable for long-term use;

3. Low cost, practical and effective;

4. The effect is obvious.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. For the experimental methods without specific conditions indicated in the following examples, usually follow the conventional conditions or the conditions suggested by the manufacturer. All percentages and parts are by weight unless otherwise indicated.

Example 1-3

Preparation of Composition No. 1-3

Example 1 Example 2 Example 3 Composition No.1 Composition No.2 Composition No.3 R-lipoic acid (mg) 100 200 350 Acetyl-L-carnitine (mg) 120 200 1500

Mix LA and ALCAR according to the above formula to prepare composition No.1-3.

Example 4-6

Preparation of Composition No. 4-6

Example 4 Example 5 Example 6 Composition No.4 Composition No.5 Composition No.6 VitB ₅ (mg) 5 30 150 Vitamin _B6 (mg) 2 100 800 VitB ₁₁ (mg) 0.4 4 40 VitB ₁₂ (mg) 0.003 0.1 1

Mix VitB ₅ , VitB ₆ , VitB ₁₁ and VitB ₁₂ according to the above formula to prepare composition No.4-6.

Example 7-9

Preparation Composition No.7-9

Example 7 Example 8 Example 9 Composition No.7 Composition No.8 Composition No.9 R-lipoic acid (mg) 100 200 350 Acetyl-L-carnitine (mg) 120 200 1500 VitB ₅ (mg) 5 30 150 Vitamin _B6 (mg) 2 100 800 VitB ₁₁ (mg) 0.4 4 40 VitB ₁₂ (mg) 0.003 0.1 1

Mix LA, ALCAR, VitB ₅ , VitB ₆ , VitB ₁₁ and VitB ₁₂ according to the above formula to prepare composition No.7-9.

Example 10

Synergistic effect of LA and ALCAR (cytological experiment)

Material

ALCAR (purchased from Sigma-Tau, Germany) and LA (R-a-lipoic acid tris salt, purchased from Viatris, Germany); ADP and rotenone (purchased from sigma); DCF-DA, JC-1, Hoechst33258 and TRITC coupling Mouse IgG antibody (purchased from Molecular Probes); protein oxidative damage detection kit (purchased from Chemicon International Inc. Temecula); anti-8-hydroxyguanosine antibody (purchased from TrevigenGaithersburg MD); BCA protein quantification kit (purchased from from PIERCE Company); anti-a-synuclein monoclonal antibody (purchased from Calbiochem Company); anti-ubiquitin modification antibody (purchased from DAKO Company); IQTM SYBER Green (purchased from Bio-Rad Company); FITC and TRITC conjugated goat antibody Rabbit antibody (purchased from Sino-American Biotech Company); western blot analysis reagent (purchased from Santa Cruz Biotechnology Company); calf serum (purchased from Hyclone Company); double antibody (purchased from Invitrogen Company); GSH analysis kit ( purchased from Nanjing Jiancheng Bioengineering Institute).

Cell Culture and Handling

SK-N-MC neurofibroblasts, cultured in MEM medium of Earle's buffer, added 15% calf serum, 5mM glucose, 2mM glutamic acid, 1mM sodium pyruvate, non-essential amino acids, 50U/ ml of penicillin and streptomycin. LA and ALCAR and the combination of these two drugs were added to the culture medium for four weeks. The concentrations of LA and ALCAR are: 0.1, 1.0, 10, 100uM and combinations of different concentrations: 0.1uM LA+ALCAR0.1uM, 1uM+1uM, 10uM+10uM, 0.1uM+1uM, 0.1uM+10uM. Wash with MEM After 3 times, the culture medium was changed to 5nM or without 5nM rotenone for 4 weeks (Sherer et al., 2002). LA and ALCAR stock solutions were dissolved in sterile PBS buffer. Rotenone was dissolved in ethanol. Control cells were supplemented with ethanol and PBS. Routine cells are cultured in 24-well plates, the medium is changed three times a week, and the cells are subcultured every week after the cells are overgrown.

Mitochondrial Membrane Potential Analysis (MMP)

Mitochondrial membrane potential was measured with JC-1 (Tirosh et al, 2000). JC-1 is a dual-emission, potential-sensitive molecular probe that emits green when the mitochondrial potential is low or in the presence of low concentrations of monomers. Light (529nm), high concentration will self-polymerize and emit red light (590nm). Both components are very sensitive to electrical potential, and the ratio of red and green light provides analysis of mitochondrial electrical potential. 5X10 ⁴ cells were cultured in a special 96-well plate (blk/clrbtm, Costar) to detect fluorescence. Cells were incubated with 10uM/ml JC-1 at 37°C for 15 minutes. After incubation, wash twice with PBS, and detect with a dual-wavelength/dual-light microplate reader (Flex Station_384, purchased from Molecular Devices, USA)

Mitochondrial complex I activity assay

The mitochondria of SK-N-MC cells were separated by differential centrifugation (Humphries and Szweda, 1998). 3x107 cells were washed twice with pre-cooled PBS, and the cells were resuspended in extraction buffer (0.25M sucrose, 5mM Tris-HCl buffer solution, pH 7.5, and 1mM EDTA pH 8.0) and ground with a glass grinder, the unbroken cells were centrifuged at 600g for 15 minutes, the suspension was centrifuged at 10000g for 25 minutes, the mitochondrial pellet was washed once with extraction medium, and the mitochondrial complex I The kinetics of activity were determined by measuring the decrease in the absorbance value of DCPIP at 600 nm (Smith 1967).

ATP level detection

ATP levels were measured using digitonin-permeable cells and a bioluminescence kit to quantify protein concentrations.

After the cells are confluent in the 96-well plate. Aspirate the buffer, add 100ul of 0.25M sucrose, 20mM MOPS, and 0.05mg/ml digitonin to each space, adjust the pH to 7.4, and place at room temperature for 3 minutes. Change to digitonin buffer solution containing 0.25M sucrose, 20mM MOPS, 20mM EDTA, adjust the pH to 7.4 and let it stand for 5 minutes, dry and use 100ul of buffer solution containing 0.25M sucrose, 20mM MOPS, 20mM EDTA, 5mM inorganic phosphate, 1mM ADP buffer, adjust the pH value to 7.4 and add as follows: some add buffer only; some add 5mM pyruvate and 1mM L-malate; some add 5mM glutamic acid and 1mM L-malate; some add 1.0uM Rotenone and 10 mM succinic acid; finally 2 mM antimycin A, 2 mM ascorbic acid and 0.1 mM TMPD. Incubate at 37°C for 1 hour, then add 20ul of 1.6M perchloric acid and 15u16N potassium carbonate and centrifuge at 3000g for 10 minutes to pellet cell debris. The ATP level was analyzed with a bioluminescence analysis system, and the protein concentration was measured with a BCA protein quantification kit.

Cytochrome C Release Assay

Cells were confluent on coverslips in MEM medium. Remove the buffer and add MTGFM (1:100, 1 uM) preheated at 37 degrees Celsius. After incubation for 1 hour at 37°C, wash the cells with fresh pre-warmed growth buffer. Carefully remove the wash buffer, add the pre-prepared growth buffer containing 3.7% formaldehyde, and incubate at 37°C for 15 minutes. Cells were washed several times with PBS after fixation. The fixed cells were permeabilized with 100% methanol at -20°C for 5 minutes. After infiltration, wash with PBS 3 times for 10 minutes each time. Treat with 5% BSA at room temperature for 1 hour, then dilute to 1% BSA with cytochrome C antibody (mouse 1:1000) and incubate for 1 hour at room temperature. The cells were washed with PBS three times for 5 minutes each, and incubated with BBS diluted with FITC-conjugated mouse antibody (1:500) for 1 hour at room temperature. Wash 3 times with PBS for 10 minutes each, and incubate with 2ug/ml DAPI for 1 hour. Cells were washed and analyzed by confocal microscopy.

GSH level test

The cells were grown on a 100 mm plate, and the cells (1×10 ⁷ ) were broken with 0.4 ml of 0.9% NACL solution, and the cell pellets were evenly distributed. 3000g at 4°C; centrifuge for 10 minutes and collect the supernatant. GSH was analyzed with a GSH assay kit based on DTNB and measured at 412 nm with a spectrophotometer. GSH is an indicator of cellular proteins and expressed as a percentage of the GSH level of the reference cell.

Protein carbonylation detection

For protein carbonylation assays, cells were grown on 100 mm plates. Soluble and insoluble protein fragments were collected by Western-Blotting as described above. Protein carbonylation was measured with a protein oxidative damage detection kit.

Brief steps: 5ul (3ug/ul) of protein is mixed and dissolved with an equal amount of 12% SDS and twice the amount of 1xDNPH. The reference reaction replaced the DNPH reference solution with 1x primers. The reaction at room temperature was carried out for 15 minutes and then stopped by adding 1.5 times of neutralizing solution. After 12% SDS-PAGE electrophoresis, 15ug of protein was transferred to nitrocellulose membrane, added 1% BSA/PBS-T (PBS contains 0.05% mixed 20) and shaken at room temperature for 1 hour. Membranes were incubated overnight at 4°C in a rabbit anti-dinitrophenylated hydrazone antibody at 1:150. After washing three times with PBS-T, the membrane was incubated in horseradish peroxidase goat anti-rabbit conjugated antibody for 1 hour at room temperature. Expose film for autoradiography.

DNA oxidative damage

The 8-hydroxyguanosine antibody formulated by the manufacturer (1:300) was used to detect DNA oxidative damage. Cells were grown on 12mm coverslips and mixed with 70% ethanol at minus 20 degrees Celsius for 10 minutes. TRITC-conjugated anti-mouse antibody was used as secondary antibody. Imaged with confocal laser microscopy. Red fluorescence enables quantification of each cell. Data from 3 independent experiments represent mean +/- standard deviation. Detect a minimum of 200 cells.

DNA oxidative damage was also analyzed by comet electrophoresis. This conventional method has been validated by predecessors (Olive et al, 1990, 2005; Tice et al, 2000). Cells were spread by treatment with 0.25% insulin and diluted medium. 10ul of cells (1x104) and 90ul of 0.6% low-melting point agarose were mixed at 37 degrees Celsius, and a layer of 1% normal melting point agarose was quickly coated on a microscope slide. Immerse the slide in 100ml mixed solution (2.5MNaCl, 100mM EDTA, 10mM Tris, pH10), add 10ml Triton X-100 and 10ml DMSO. Then put it into the electrophoresis buffer (300Mm NaOH, 1Mm EDTA, pH greater than 13) at 4 degrees Celsius for 20 minutes to loosen the DNA before electrophoresis. Electrophoresis was run for 20 minutes at 4°C, 0.73V/cm, 28mA. Slides were immersed in 0.4M Tris, pH 7.5, faded with 2ug/ml DAPI and covered with a coverslip. Cells were observed with Olympus BX61 a_60 oil lens (aperture value=1.25) connected with Olympus DP70 microscope. Nuclei were decolorized with DAPI and excited with a UV laser (380 nm). Images were analyzed with Image-ProPlus software. Fifty were randomly selected from each model and comet tails were measured as an indicator of DNA oxidative damage in this model. Data from 3 independent experiments represent mean +/- standard deviation.

Active oxygen detection

Cells were confluent in 24-well plates. Aspirate the culture solution, add 500ul of 0.25M sucrose, 20mM MOPS, and 0.05mg/ml digitonin to each well, adjust the pH to 7.4, and let it stand at room temperature for 3 minutes. Then use digitonin buffer solution containing 0.25M sucrose, 20mM MOPS, and 20mM EDTA to adjust the pH to 7.4, leave it at room temperature for 5 minutes, and then dry it. Then add the respiratory buffer containing 0.25M sucrose, 20mM MOPS, 20mM EDTA, 5mM inorganic phosphate, 1mM ADP, 5mM glutamic acid, 1mM L-malic acid, adjust the pH to 7.4 and add 10uM carboxyl H ₂ DCFDA at 37 degrees Celsius Mitochondrial ROS was measured for 30 min under the conditions. Replace buffer, wash with PBS, transfer cells to Eppendorf tubes, and perform cytometric analysis.

Synaptophysin I and ubiquitin level detection

Fluorescent immunolabeling and Western blotting were used to detect the distribution and level of synaptophysin I and ubiquitin. Detected by fluorescent immunolabeling method, the cells were grown on a 12mm coverslip, and synaptophysin I and ubiquitin had been done (Arima et al1999, Zhang et al, 2004). Mix with methanol for 10 minutes, wash with PBS and treat with 5% BSA for 1 hour at room temperature. After washing three times, add synaptophysin I mouse antibody (1:200) and ubiquitin (1:500) rabbit antibody to the mixed cell solution, incubate at room temperature for half an hour, and then wash three times with PBS. Afterwards, FITC-coupled goat anti-mouse antibody (1:100) and TRITC-coupled goat anti-rabbit antibody (1:100) were added, incubated at 37°C for half an hour, and then washed three times. Then add 5ug/ml Hoechst33342 and incubate at room temperature for 10 minutes, wash 3 times with PBS, place cells in glycerol/PBS (9:1) mixture, and then use LSM510META_Germany_Zeiss laser confocal microscope with 63x/1.4HCxPlanAPO Oil immersion observation. Synaptophysin I was excited with an argon laser (488nm), ubiquitin was excited with a helium laser (543nm), and DNA was excited with a UV laser (364nm).

With Western blotting detection, cells are collected, proteins are extracted, modified on the basis of previous experience in Western blotting detection, the brief steps are as follows: cells are dissolved in buffer (50mM Tris-HCl, 250mM NaCl, 5mM EDTA, 50mM NaF, 1% NP40, 2ug/ml aprotinin, 2ug/mlleupeptin, 1mM phenylmethylsulfonyl fluoride, 700U/ml DNaseI, 1% beta-mercaptoethanol and the above reagents were purchased from Sigma). Soluble proteins were quantified with BCA protein quantification kit, and insoluble proteins were detected with Coomassie Brilliant Blue. The same amount of protein was electrophoresed with 15% SDS-PAGE and then transferred to a nitrocellulose membrane, and fixed with 5% BSA in TBS-T (20mM Tris, 500mM NaCl, 0.1% mixed 20) at room temperature for 2 hours. Membranes were incubated overnight at 4°C in synaptophysin I mouse antibody (1:2000) or ubiquitin (1:500) rabbit antibody during immersion. Then wash six times with TBS-T, 5 minutes each time. Add horseradish peroxidase coupled with secondary antibody (synaptophysin I1: 4000 goat anti-mouse antibody, ubiquitin 1:1000 goat anti-rabbit antibody), room temperature for 1-2 hours. Fluorescent autoradiography sensitizes the film.

Reverse transcription primer setting and quantification of reverse transcription PCR

The cDNA chain was synthesized from 1 ug of total RNA and a small amount of dNTP using reverse transcriptase XL (purchased from Takara, Shiga, Japan). For primers, refer to Sherer et al., 2002: the forward primer for β-actin is tcaccatggatgatgatatcgcc; the reverse primer for β-actin is ccaacacgcagctcattgtagaagg; the forward primer for synaptophysin I is aggactttcaaaggccaagg; the reverse primer for synaptophysin I is tcctccaacatttgtcacttgc. Both pairs of primers can cross the boundaries of the tumor-like protrusions. Amplification was performed using multiple cycles of an RT-PCR machine for quantitative purposes (Bio-Rad, Hercules, CA). The amount of these two PCR products is tracked and quantified by the IQTM SYBER fluorescence receiver at the final annealing step of each cycle. The reaction was completed in a 25ul system containing 200nmol positive and negative factors. The expression level of each template gene was calculated using the 2- ^CT method. Data collection begins when the target sequence is amplified to a certain size. 2— ^CT depicts the curve changes of gene expression in treated cells. Use the expression level of actin and untreated cells (control) as a standard: _C _T =_C _{T, Rotenone} -_C _{T, Control} = (C _{T, synuclein} -C _{T, actin} ) _Rotenone -(C _Tsynuclein -C _{T , actin} ) _Control . The result of mRNA level is represented by the ratio of treated cells to Control cells.

result

Effects of MMPs

As shown in Figure 1A (distribution state of JC-1 under the laser confocal microscope) and Figure 1B (fluorescence reading analysis of the number of JC-1), compared with the reference system, rotenone treatment caused the excitation fluorescence of mitochondrial membranes to change from red to green, red light The ratio of intensity/green light intensity decreased, indicating that rotenone induced mitochondrial depolarization. The pretreatment of LA, ALCAR and their combination can effectively inhibit this effect, and LA+ALCAR (0.1μM or 1μM) is the most effective. LA10μM or ALCAR100μM are as effective. Pretreatment of rotenone-treated cells with LA or ALCAR in 200 μM H ₂ O ₂ for 2 hours had the same effect as that done by Shere et al. (2002) (Fig. 1C). Since the effect of adding or not adding H ₂ O ₂ after treatment with rotenone is the same, so we do not add H ₂ O ₂ in all the following analyses.

The results showed that low doses of LA+ALCAR (0.1 μM or 1 μM) most effectively inhibited mitochondrial depolarization.

Analysis results of complex I activity

The kinetics of mitochondrial complex I activity was determined with different concentrations of DCPIP. As shown in Figure 2, it was found that rotenone treatment reduced the activity of complex I by 30% compared with the reference system, while pretreatment of LA (10, 100 μM), ALCAR (100 μM), and LA+ALCAR (0.1, 1 μM) could prevent this one drop. And LA+ALCAR (0.1, 1μM) is more effective than any single concentration of LA and ALCAR.

The results showed that low doses of LA+ALCAR (0.1 μM or 1 μM) were most effective in preventing the decline in the activity of complex I.

ATP analysis results

Rotenone can effectively reduce the ability of cells to produce ATP (Figure 3). Both LA (10μM) and ALCAR (100μM) could effectively inhibit the decrease of ATP level. LA+ALCAR (0.1, 1 μM) also significantly inhibited the reduction of ATP levels, but the higher concentration of the mixture (both 10 μM) lost the inhibitory effect, and the high concentration of LA (100 μM) also lost the inhibitory effect.

The results showed that high concentrations of LA+ALCAR had no inhibitory effect on the decline in the ability of cells to produce ATP, while low doses of LA+ALCAR (0.1 μM or 1 μM) could effectively inhibit the decline in this ability.

Results of cytochrome c release assay

The release of cytochrome C can be judged by the image: (Figure 4)

1) The reference cells show that the mitochondria are well protected and cytochrome c is on the mitochondria. The orange color compounded on the image shows that mitochondria are enriched in cytochrome c. Rotenone-treated cells were less orange than control cells;

2) Cells treated with rotenone showed that cytochrome C was released from the mitochondria into the cytosol, making the staining spots less and more dispersed.

The results showed that both LA (10 μM) and ALCAR (100 μM) could inhibit the release of cytochrome C, and low doses of LA+ALCAR (both 1 μM) could effectively inhibit the release of cytochrome C.

Antioxidant GSH Level Results

Rotenone treatment resulted in a significant loss of GSH. LA (0.1-100 μM except 1 μM), ALCAR (1-100 μM except 10 μM) protected against rotenone-induced GSH loss ( FIG. 5 ). LA (10μM) and ALCAR (100μM) are the two most effective concentrations acting alone.

The results showed that low doses of LA+ALCAR (both 1 μM) were most effective in protecting against rotenone-induced GSH loss.

The effect of protein carbonylation

Protein carbonylation is an important indicator of protein oxidative damage. We use Western blotting analysis and DNPH reaction to measure insoluble protein fragments. As shown in Figure 6, protein carbonylation was increased in rotenone-treated cells (b) compared to the reference line (a). Treatment with LA had no effect on protein carbonylation. ALCAR100μM treatment protein carbonylation was significantly inhibited.

The results showed that low-dose LA+ALCAR (0.1 μM or 1 μM) treatment had obvious inhibitory effect on protein carbonylation, while higher concentration combined use (10 μM+10 μM) had no inhibitory effect.

Effects of DNA Oxidative Damage

Antibody to 8-hydroxyguanosine depigmented cells, maker of oxidative DNA damage (red). The same cells were labeled with Hoechst33342 for nuclear morphology. As shown in Figures 7A and 7B, 8-hydroxyguanosine immunoreactivity was increased in rotenone-treated cells. Many cells with oxidatively damaged DNA display a fragmented nuclear morphology that is characteristic of apoptosis. LA (10 μM) and ALCAR (100 μM) decreased 8-hydroxyguanosine immunoreactivity.

The results showed that low-dose LA+ALCAR (0.1 μM or 1 μM) could inhibit the increase of 8-hydroxyguanosine immunoreactivity induced by rotenone.

The effect of ROS detection

Because mitochondria are the source and target of ROS, DCF was used to detect whether mitochondrial functional impairment and oxidative damage were accompanied by increased ROS after rotenone treatment. As shown in Figure 8.

The results showed that the ROS of the samples treated with rotenone significantly increased, while LA (10uM) and ALCAR (100uM) had a significant protective effect on this. In addition, the combined use of low concentrations of the two (0.1+0.1μM and 1+1μM) also has a protective function against the increase of ROS.

Analysis results of expression and distribution status of synaptophysin I and ubiquitin

PD symptoms are manifested in the accumulation of synaptophysin I and ubiquitin in the cytoplasm, which we call Lewy bodies. The expression levels of synaptophysin I and ubiquitin can be checked by Western blotting analysis. The image and quantitative results of Western blotting As shown in Figure 9A and 9B. Compared with Control (a). The model after rotenone treatment showed the soluble level of synaptophysin I, and pretreatment with 0.1-100uM LA or 0.1-100uM ALCAR can reduce the effect of rotenone The resulting increase in synaptophysin I expression has an inhibitory effect. The two also have significant effects when mixed at concentrations of 0.1+0.1uM and 1+1uM.

The mRNA level of Synaptophysin I was detected by RT-PCR method, as shown in Figure 9C, the amount of mRNA of Synaptophysin I in cells treated with rotenone was significantly reduced compared with the reference, and in 10uM LA and 100uM ALCAR, both at concentrations of 0.1+0.1uM and 1+1uM restore the mRNA level of synaptophysin I to the level of the reference system.

Analysis of the state distribution of synaptophysin I and ubiquitin by immunofluorescence assay Rotenone caused an increase in the levels of synaptophysin I and ubiquitin in the cytoplasm ( FIG. 10 ). 10uM LA and 100uM ALCAR and their mixtures at concentrations of 0.1+0.1uM and 1+1uM could inhibit the increase of synaptophysin I and ubiquitin levels in the cytoplasm.

The results show:

Low-dose LA+ALCAR (0.1μM or 1μM) can significantly inhibit the increase of synaptophysin I expression;

Low-dose LA+ALCAR (0.1 μM or 1 μM) can restore the mRNA level of synaptophysin I to the level of the reference system;

Low doses of LA+ALCAR (0.1 μM or 1 μM) could inhibit the increase of levels of synaptophysin I and ubiquitin in the cytoplasm.

Example 11

Synergy between LA and ALCAR (PD Drosophila Behavioral Experiment)

Materials and methods

Drosophila Parkinson's disease model (obtained by crossing synaptophysin I transgenic fruit flies with Uas-DDC-gal4 fruit flies) (purchased from the United States) into which the synaptophysin I gene was transferred, and the Parkinson's fruit fly model expressed synaptic Synaptophysin I, and the loss of dopamine neurons occurs in adulthood, filamentous inclusions containing synaptophysin I appear in neurons, and motor function is impaired (see Figure 49). This Drosophila model has the basic features of human Parkinson's disease (Feny, 2000).

Since the eclosion of fruit flies, male and female were separated into experiments, and fruit flies were randomly divided into 0X, 0.2X, 1X, 5X, 10X, 20X, 50X and other experimental groups, each group including about 170-300 male and female flies, PD The female parent of Drosophila (ie, α-syn Drosophila) itself does not express synaptophysin I, and its motor ability test value is the same as that of PD Drosophila 3 days after eclosion, which can be used as a non-PD control (non-PD).

All flies were cultured in culture tubes at 25°C, 50-60% humidity, and 12 hours of light.

Drosophila medium adopts corn yeast medium (the dissolved yeast powder is added and stirred when the temperature drops to 60-70°C), and propionic acid is added to inhibit mold growth; the medium is heated to boiling and then cooled to 45°C Next, add relevant drugs to the desired concentration (OX, 0.2X, 1X, 5X, 10X, 20X, 50X and other doses). The prepared culture medium was covered with gauze, and after the water evaporated, it was corked and stored in the refrigerator. Shelf life 6 days

Medium dosing method: (a) The final solvent amount is based on 10ml/100g medium, and the control group only adds 10ml of solvent; water-soluble drugs are directly dissolved in 6ml of water (can be made into a concentrated solution in advance); fat-soluble drugs First use sucrose ester to aid in dissolution and then add 6ml of water (due to the slight difference in the method of different drugs), the aid solvent can be dimethyl sulfoxide, DMSO (<10%), sucrose ester (<0.5%), Tween 20 etc.; (b) According to the physical and chemical characteristics of various drugs, such as heat resistance temperature, light reaction, solubility, etc., make corresponding adjustments when dispensing, and do not add water-soluble drugs and fat-soluble drugs at the same time when they are mixed. (c) Wherein 5X, 10X, 20X, 50X, 100X dosage groups respectively represent the mg number of the medicine contained in 100g culture medium.

Climbing ability test experiment: from 3 days after eclosion, carry out once every 6 days. Use a glass tube with a diameter of 27mm and a length of 110mm as the test tube, mark a height 80mm from the bottom of the bottle, put 10 fruit flies into the tube, shake it lightly to make the fruit flies fall to the bottom of the tube, and record for 10 seconds The number of fruit flies that climbed to a height of 80mm and above was tested continuously for 10 times, and the probability value of each fruit fly climbing to a height of 80mm was calculated according to the number of fruit flies recorded at a height of 80mm and above in these 10 times, as The tube climbing index of Drosophila.

At the same time, the medium of the fruit flies was changed every 3 days, and the number of dead flies was recorded every 3 days. Based on this, the survival rate-eclosion age curve was drawn by using the EXCEL software, and the life span was investigated.

The test data of Drosophila longevity and climbing ability were all processed by EXCEL software. .

result

See Figures 40-48.

The results showed that the 20X and 50X dose groups of LA could improve the climbing ability of PD fruit flies and prolong their lifespan to a certain extent; the 5X, 10X and 20X dose groups could better inhibit synaptophysin I at the mRNA level and protein level , but the 50X dose group is more prominent in protecting dopamine neurons. The 5X, 10X, and 20X dose groups of ALCAR can effectively prolong the lifespan of PD flies, but have no effect on the climbing ability of PD flies.

The 10X and 20X dose groups of LA+ALCAR can effectively prolong the lifespan of PD fruit flies, and the effect is stronger than that of LA alone in the 10X dose group, and the 5X and 20X dose groups of LA+ALCAR can effectively improve the climbing of PD fruit flies. ability to climb.

Example 12

Synergy of VitB ₅ , VitB ₆ , VitB ₁₁ and VitB ₁₂

The experimental method was the same as described in Example 11. Only the dosage expression methods of VitB5, VitB6, VitB11 and VitB12 are slightly different (see Table 1-7).

The doses of VitB5, VitB6, VitB11 and VitB12 are shown in Table 1-7 below:

Table 1

Table 2

table 3

Table 4

table 5

Table 6

Table 7

result

1. See Figure 11-14.

The results showed that VB5 alone extended the lifespan of PD male flies, but had no effect on their climbing ability. For female PD flies, VB5 has no effect on the climbing ability except that low doses (1X, 5X) have no significant effect on lifespan, while high doses can prolong lifespan (20X, 100X).

2. See Figure 15-18.

The results showed that VB12 alone could improve the climbing ability of PD male flies, but had no effect on their lifespan. For female PD flies, the effect of VB12 on improving climbing ability was not as good as that of males, and had little effect on their lifespan.

3. See Figure 19-22.

The results showed that VB6 alone could prolong the lifespan of PD male flies, but had no effect on their climbing ability, while VB6 could protect dopamine neurons. For female PD flies, VB6 can prolong their lifespan, but has no effect on their climbing ability.

4. See Figures 23-26.

The results showed that VB11 alone had no significant effect on the climbing ability and lifespan of male PD flies. In female PD flies, VB11 slightly improved their climbing ability and lifespan.

5. See Figures 27-30.

The results showed that the combined effect of VB5+VB12 had no significant effect on the climbing ability and lifespan of male PD flies. Under the combined effect of VB5+VB12, the effect on the climbing ability and lifespan of female PD flies is not clear.

6. See Figures 31-34.

The results showed that the combined effects of VB6, VB11 and VB12 had no significant effect on the climbing ability and lifespan of male PD flies. Similarly, the combined effects of VB6, VB11, and VB12 had no significant effect on the climbing ability and lifespan of female PD flies.

7. See Figures 35-38.

The results showed that when VB5, VB6, VB11, and VB12 were combined, the three dose combinations of 1X, 20X, and 100X had significant effects on the climbing ability and lifespan of male PD flies. For female PD flies, when VB5, VB6, VB11, and VB12 acted together, the four dose combinations of 1X, 10X, 20X, and 100X significantly improved climbing ability, but had little effect on lifespan.

In summary, although VB5, VB6, VB11, and VB12 all have inhibitory effects on the expression of synaptophysin I to varying degrees (see Figure 54), their effects on exercise capacity and lifespan are weak when they act alone, and two pairs of 1. When the three components are combined, there is a certain effect. When the four components work together, it has a significant effect on improving the climbing ability and prolonging the lifespan of PD fruit flies. The mechanism may be related to the inhibition of synaptophysin I expression. effect (see Figure 55).

Example 13

Synergy of LA, ALCAR, VitB ₅ , VitB ₆ , VitB ₁₁ and VitB ₁₂

Materials and methods

SK-N-MC neurofibroblasts were treated with LA, ALCAR, VB5, VB6, VB11, and VB12 for 72 hours, then added MTTP (250 μm) for 72 hours, removed the medium, added 5 mg/mL MTT and incubated at 37°C for 3 hours After 1 hour, the light absorption value at 550 nm was measured, and the light absorption value at 550 nm was used as an indicator of cell activity.

Table 8: Dosage table of each experimental group

LA (μmol/L) ALCAR (μmol/L) VB5(mg/L) VB6(mg/L) VB11(mg/L) VB12(mg/L) control group 0 0 0 0 0 0 LA+ALCAR 1 1 0 0 0 0 VB5+VB6+VB11+VB12 0 0 10 10 10 14 LA+ALCAR+VB5+VB6+VB11+VB12 1 1 10 10 10 14

result

See Figure 39.

The results showed that the combination of LA, ALCAR, VitB5, VitB6, VitB11 and VitB12 had a protective effect on the Parkinson's disease cell model that was 3% stronger than that of LA and ALCAR, and 8% stronger than that of VitB5, VitB6, VitB11 and VitB12. %.

Example 14

Effects of LA, VitB ₅ , VitB ₆ , VitB ₁₁ and VitB ₁₂ and their combined effects on the expression of synaptophysin I gene in Drosophila PD and their protective effects on dopamine neurons

Materials and methods

The Drosophila Parkinson's disease model using the same synaptophysin I gene as described above was used as the research object, and the female parent Drosophila (α-syn Drosophila) or the male Drosophila (uas-DDC-gal4 Drosophila) of PD Drosophila Flies, ie DDC Drosophila) were used as non-PD control, and the feeding, administration, and dosage of Drosophila were all carried out in the same way as in previous examples 10-13. When the fruit flies reached the corresponding age, a certain number (about 200) of fruit flies were randomly selected in each experimental group, and their heads were cut off at 4°C, and the heads were homogenized and centrifuged to separate the protein and RNA components. Carry out the RT-PCR experiment of synaptophysin Iwestern-blotting or mRNA according to the method similar to the cytology experiment part; or dissect the head of the fruit fly, take out the nerve tissue for immunohistochemical experiment, and refer to the experiment content of the cytology part.

result

See Figures 50-51.

The results showed that LA at a dose of 10X had an inhibitory effect on the expression of synaptophysin I.

See Figure 52.

The results showed that 20X dose of LA had an inhibitory effect on the expression of synaptophysin I gene.

See Figure 53.

The results showed that VitB ₆ and 20X doses of LA were able to prevent the loss of dopamine neurons in PD flies.

See Figure 54.

The results showed that LA, VitB ₅ , VitB ₆ , VitB ₁₁ and VitB ₁₂ had inhibitory effects on the expression of synaptophysin I respectively.

See Figure 55.

The results showed that VitB ₅ +VitB ₆ +VitB ₁₁ +VitB ₁₂ had an inhibitory effect on the expression of synaptophysin I gene.

discuss

Aiming at the multifactorial pathogenesis of PD, the present invention selects 6 kinds of nutrients that have a certain effect on the repair of mitochondrial damage as the main components. Prevention and treatment effect of PD.

Specifically, pantothenic acid (VB5) is a coenzyme A (CoA) precursor, composed of CoA and the phosphopantetheinine moiety of fatty acid synthase, and thus is important for the production of acetyl CoA. Pantothenic acid deficiency decreased heme synthesis in monkeys and caused anemia (Plesofsky-Vig, 1996). Mitochondrial complex IV is reduced in cephalosporins deficient in pantothenic acid (Brambl and Plesofsky-Vig, 1986), as is the iron content of complex IV (Brambl and Plesofsky-Vig, 1986), which results in the absence of mitochondrial complex IV. Fatty acids and pantothenic acid are trace raw materials that ensure the normal supply of heme biosynthetic precursor, succinyl-CoA, and the deficiency of either will reduce heme synthesis through a mechanism similar to biotin deficiency. More than 70 enzymes have been found to use coenzyme A or derivatives produced by pantothenic acid as reaction substrates.

VB6 is the precursor of pyridoxal 5-phosphate (PLP). PLP is an essential coenzyme for DA synthesis and is also directly involved in the synthesis of heme (Scholnick et al., 1972). About 10% of people in the United States Intake of vitamin B6 is less than half of the recommended daily allowance (RDA) (Wakimoto and Block, 2001), so VB6 is an important limiting factor for metabolism. Heme deficiency may be a factor in mitochondrial and neuronal decline during aging (Atamna et al., 2002.a). Heme, a major functional form of iron in cells, is synthesized in mitochondria by the insertion of ferrous protoporphyrin IX by ferrochelatase. Heme deficiency reduces mitochondrial complex IV in brain cells, activates nitrate oxide synthase, alters amyloid precursor proteins, and disrupts iron and zinc homeostasis. Common deficiencies of iron and B6 lead to cognitive difficulties, and iron deficiency in children delays CNS development (Benton, 2001; Tamura et al., 2002). Thus, heme deficiency or dysregulation may be an important and preventable part of the neurodegenerative process (Atamna et al., 2002.a), but this can still be prevented by vitamin B6 supplementation.

Folic acid (VB11) is a raw material for the synthesis of various one-carbon tetrahydrofolate derivatives, and plays a very important role in nucleic acid synthesis and methylation reactions, which are crucial to the normal function of the brain. Folate deficiency will affect the synthesis of nuclear DNA or mtDNA, reducing the availability of purine and dTMP. Mitochondria contain higher levels of folate coenzyme compared to cytoplasmic tetrahydrofolate synthase. The mitochondria of the folic acid coenzyme in the brain contain more long chains of glutamate, which is significantly different from the distribution in the liver. Decreased folic acid levels or elevated homocystine levels are associated with cognitive impairment, Parkinson's disease, Alzheimer's It is closely related to Murray's disease and other types of dementia. (Carl et al., 1996)

VB12 has a wide range of physiological effects, but it needs to be converted into methylcobalamin and coenzyme B12 to be active. In human tissues, there are two biochemical reactions that require the participation of vitamin B12: one is the synthesis from homocysteine The reaction of methionine, in which the produced tetrahydrofolate participates in the synthesis of DNA, indirectly participates in the synthesis of thymidine deoxynucleotide; the other is the conversion of methylmalonate coenzyme A to succinate coenzyme A, which participates in the tricarboxylic cycle , thus playing an important role in the formation of lipoproteins in the nerve myelin and protecting the functional integrity of central and peripheral myelinated nerve fibers. At the same time, it participates in a wide range of protein and fat metabolism. VB12 has a strong affinity for nerves, and can repair nerve myelin and promote regeneration.

In an animal model of Parkinson's syndrome (Duan et al., 2002) and in patients treated with L-dopa, plasma homocysteine levels were found to be increased, and this homocysteine level was correlated with VB11, VB12 Inversely correlated with pyridoxal-5-phosphate levels (Miller et al., 2003).

α-lipoic acid (LA) and its reduced form dihydrolipoic acid are the coenzymes of α-ketoglutarate dehydrogenase and pyruvate dehydrogenase; they are multifunctional antioxidants that can scavenge free radicals, Recycling produces other antioxidants (including glutathione, vitamin C, CoQ, and thioredoxin, all of which recycle to produce vitamin E), as well as chelating catalytic metals (such as iron) to prevent free radicals produce; induce the production of phase II enzymes.

Acetyl-L-carnitine (ALCAR), an acetyl derivative of L-carnitine, can pass through the mitochondrial membrane, transfer long-chain fatty acids into the mitochondria for energy, increase the level of diphosphatidylglycerol, increase respiration and Considered as a secondary antioxidant. Carnitine levels in tissues of animals, including humans, decline with age (Costell et al., 1989; Liu et al., 2002a; Maccari et al., 1990) thereby reducing mitochondrial membrane integrity. Studies on rats, dogs and other animals have shown that supplementing ALCAR can improve cognitive impairment related to aging, promote nerve regeneration, protect nerve cells from mitochondrial uncoupler and inhibitor poisoning, and reduce cerebral ischemia and reperfusion After nerve injury, it increases glutathione and GABA levels in the brain.

The present invention uses the Drosophila Parkinson's disease model (obtained by the hybridization of synaptophysin I transgenic fruit flies and uas-DDC fruit flies) transferred into the synaptophysin I gene, the Parkinson's fruit fly model expresses synaptophysin I, and Loss of dopamine neurons occurs in adulthood, filamentous synaptophysin I-containing inclusions appear in neurons, and motor function impairment occurs. Because this fruit fly model has the basic characteristics of human Parkinson's disease, it is possible to use this genetic method to study Parkinson's disease.

The present invention shows that when VB5, VB6, VB11, and VB12 act alone, they have a certain effect on the improvement of the motor ability and the prolongation of the life span of PD fruit flies, but they cannot be achieved for each specific dose. Effective effect, and even encounter the phenomenon of completely opposite effects on the two; when VB5 and VB12 are combined, there is a slight joint effect, and the impact on exercise ability and lifespan tends to be consistent, and 1X and The effect of the 20X dose group is relatively good, but the degree of influence is small; when VB6, VB11 and VB12 are combined, no obvious effect of the drug on exercise ability and lifespan was observed on PD fruit flies; only in VB5, VB6, VB11 When combined with VB12, we observed a significant improvement in the locomotor capacity and lifespan of PD flies, at a level close to that of non-PD control flies.

When acting alone, LA in the 50X dose group is effective on the motor ability and lifespan of PD flies, while ALCAR only prolongs the lifespan of PD flies, but has no effect on the motor ability. When the two act together, the 10X dose group has a stronger effect on the motor ability of PD flies than the 10X dose of LA alone.

Since VB5, VB6, VB11, VB12, LA and ALCAR have different ways of acting in the body, their combined effects are stronger than those of single drugs, and the curative effect is more stable and safe under the condition of combined effects. In the present invention, the cell survival rate in the MTT environment after LA+ALCAR+VB5+VB6+VB11+VB12 pretreatment is higher than the cell survival rate of LA+ALCAR pretreatment or VB5+VB6+VB11+VB12 pretreatment.

In the further study of the mechanism of action of the drug, the present invention uses the chronic rotenone model (5nM, 4 weeks) of SK-NM-C as an in vitro PD model to reproduce the toxic effect of rotenone on mitochondrial function. (decreased complex enzyme 1 activity, decreased MMP and ATP levels, increased cytochrome c release) oxidative stress (loss of glutathione, increased ROS, protein acyl and 8-OXO-dG), and pathological features of PD (synaptic accumulation of ubiquitin I and ubiquitin levels). The inventors used LA (0.1, 1, 10, 100uM), ALCAR (0.1, 1, 10, 100uM) and their different components in several aspects of PD pathological characteristics, mitochondrial function, and oxidation and anti-oxidative damage biomarkers. Effect of combined pretreatment for 4 weeks.

The molecular pathology of PD is a defect in mitochondrial function. In this cellular model, rotenone-treated mitochondria had decreased complex 1 activity, increased ATP depletion, and increased cytochrome C release on the inner mitochondrial membrane. Appropriate doses of LA and ALCAR, two mitochondrial nutrients, had therapeutic effects on mitochondria treated with rotenone. The most effective doses are 10uM LA and 100uM ALCAR, and when the two are used in combination, the effective doses appear at 0.1 and 1uM. This optimal ratio is much more effective than either alone. Mitochondrial nutrients such as LA and ALCAR can effectively inhibit the generation of cellular oxygen free radicals and oxidative damage caused by rotenone.

The most obvious manifestation of PD symptoms is the accumulation of synaptophysin I and ubiquitin. This shows that the regulation of complex I is the key to the accumulation level of synaptophysin I, and the high expression of synaptophysin I induces oxidative damage. In addition, oxidative damage and reduction of cytochrome c can also accumulate synaptophysin I. The specific change in the PD cell model after rotenone treatment was the significant increase of synaptophysin I and ubiquitin proteins in the cytoplasm. Therefore, whether LA and ALCAR can be used to prevent and treat PD depends on whether they can prevent the expression and distribution of these proteins.

In conclusion, the present invention proves that the mitochondrial nutrients VB5, VB6, VB11, VB12, LA and ALCAR and their different combinations will have different therapeutic effects on the PD fruit fly model, and the combined application of VB5, VB6, VB11 and VB12, or the combination of LA and ALCAR The effect of the application is stronger than the effect of the drug alone. LA and ALCAR have certain effects on inhibiting mitochondrial dysfunction, oxidative damage and preventing the accumulation of synaptophysin I and ubiquitin in PD cell models. Moreover, the mixture of LA and ALCAR at low concentrations (0.1-1uM) exhibited a positive synergistic effect, which was 10 times stronger than either drug alone on mitochondrial dysfunction and oxidation induced by rotenone treatment. Damage works. In addition, the protective effect of LA+ALCAR+VB5+VB6+VB11+VB12 on cells was stronger than the combination of LA+ALCAR or VB5+VB6+VB11+VB12.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (5)

1. a compositions is characterized in that, the chondriosome nutrient in the described compositions is made of following manner;

Described chondriosome nutrient is:

(i) 5-150 weight portion vitamin B5;

(ii) 2-1000 weight portion vitamin B6;

(iii) 0.4-40 weight portion VB11; With

(iv) 0.003-1 weight portion vitamin B12;

Or:

(i) 5-150 weight portion vitamin B5;

(ii) 2-1000 weight portion vitamin B6;

(iii) 0.4-40 weight portion VB11;

(iv) 0.003-1 weight portion vitamin B12;

(v) 100-350 weight portion R-thioctic acid; With

(vi) 100-2000 weight portion acetyl-L-carnitine.

2. compositions as claimed in claim 1 is characterized in that, described chondriosome nutrient is:

(i) 15-50 weight portion vitamin B5;

(ii) 50-300 weight portion vitamin B6;

(iii) 1-10 weight portion VB11; With

(iv) 0.01-0.2 weight portion vitamin B12;

Or:

(i) 15-50 weight portion vitamin B5;

(ii) 50-300 weight portion vitamin B6;

(iii) 1-10 weight portion VB11;

(iv) 0.01-0.2 weight portion vitamin B12;

(v) 150-250 weight portion R-thioctic acid; With

(vi) 180-500 weight portion acetyl-L-carnitine.

3. preparation of compositions method as claimed in claim 1 is characterized in that it comprises step:

1. chondriosome nutrient is mixed in the following manner, form compositions;

Described chondriosome nutrient is: (i) 5-150 weight portion vitamin B5; (ii) 2-1000 weight portion vitamin B6; (iii) 0.4-40 weight portion VB11; (iv) 0.003-1 weight portion vitamin B12;

Or: (i) 5-150 weight portion vitamin B5; (ii) 2-1000 weight portion vitamin B6; (iii) 0.4-40 weight portion VB11; (iv) 0.003-1 weight portion vitamin B12; (v) 100-350 weight portion R-thioctic acid; (vi) 100-2000 weight portion acetyl-L-carnitine.

4. method as claimed in claim 3 is characterized in that, step 1. in, with (i) 15-50 weight portion vitamin B5; (ii) 50-300 weight portion vitamin B6; (iii) 1-10 weight portion VB11; (iv) 0.01-0.2 weight portion vitamin B12 is mixed, and makes compositions;

Or with (i) 15-50 weight portion vitamin B5; (ii) 50-300 weight portion vitamin B6; (iii) 1-10 weight portion VB11; (iv) 0.01-0.2 weight portion vitamin B12; (v) 150-250 weight portion R-thioctic acid; (vi) 180-500 weight portion acetyl-L-carnitine is mixed, and makes compositions.

5. the purposes of a compositions as claimed in claim 1 is characterized in that, described compositions is used to prepare prevention, treat or improve the medicine of Parkinson's disease, or is used to prepare the dietary supplement that prevents or improve Parkinson's disease.

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