Selection of a High-Yielding Strain of Coenzyme Q10

 Abstract: Rhizobium radiobacter WSH2601 was used as the starting strain, and a genetically stable actinomycin D-resistant mutant strain WSH-F06 was obtained by UV light and nitrosoguanidine mutagenesis. The effects of nutrients such as carbon and nitrogen sources, as well as environmental conditions such as inoculum volume, loading volume, and initial pH on the cell growth and accumulation of coenzyme Q10 of the mutant strain were investigated in shake flasks. F06 on cell growth and coenzyme Q10 accumulation.  Through mutagenesis and optimization of fermentation conditions, the coenzyme Q10 production and intracellular content of the mutant strain WSH-F06 reached 34 mg/L and 2.4 mg/g, respectively, which were 16% higher than that of the starting strain under the same conditions.

 

1 Preface

Coenzyme Q10 (CoQ10), also known as ubiquinone, is a derivative of 2,3-dimethoxy-5-methyl-1,4-dibenzoquinone with 10 isoprenoid derivatives in the side chain. It is a natural antioxidant synthesized by cells and an activator of cellular metabolism, and has the function of enhancing immunity.  It is a natural antioxidant synthesized by cells and an activator of cellular metabolism, and has the function of enhancing the immunity of the body, thus it is a biochemical drug with high clinical value.

 

Recent studies have shown that Coenzyme Q10 has anti-tumor effects and has been used clinically in the treatment of advanced metastatic cancer.  It can also lower blood pressure and is used in the treatment of congestive heart failure.  It has also been shown to be effective in the treatment of scurvy, duodenal ulcer, necrotizing periodontitis, viral hepatitis, and in promoting pancreatic function and secretion[2-4] .  Coenzyme Q10 also has anti-aging properties[5] , so it has a wide range of applications in cosmetics.

 

Coenzyme Q10 can be synthesized by biological extraction, chemical synthesis and microbial fermentation.  The production of coenzyme Q10 by microbial fermentation is the most promising method because of its abundant raw materials, mild reaction conditions, relatively simple isolation and purification process, and high clinical value of the product[1] .

 

At present, the main factor limiting the industrial production of coenzyme Q10 by fermentation is the low fermentation yield, which leads to the high production cost.  There are few research reports on the production of coenzyme Q10 by fermentation method in China, but there is a method in Japan which involves the selection and breeding of high-yielding strains of coenzyme Q10 and their use in the production of coenzyme Q10[6] , and the coenzyme Q10 yield of the mutant strain of Soilobacillus can be up to 2.50 mg/g. The metabolic engineering research on the biosynthesis of coenzyme Q10 is under way, but due to the complexity of the synthesis pathway of coenzyme Q10 and its many steps, no breakthrough has been made[7] . However, due to the complexity of the coenzyme Q10 synthesis pathway and the many steps involved, a breakthrough has not yet been achieved[7] .

 

In the present work, a mutant strain of WSH-F06 was obtained by complex mutagenesis using Rhizobium radiobacter WSH2601 as the starting strain, and its fermentation culture conditions were initially investigated.

 

2 Materials and Methods

2.1 Strains

The starting strain was Rhizobium radiobacter WSH2601, preserved in the Environmental Biotechnology Laboratory, School of Bioengineering, Jiangnan University.

 

2.2 Culture media

Seed medium (g/L): glucose 20, peptone 10, yeast 10, sodium chloride 5; pH 7.2, sterilized at 121oC for 15 min. Inclined medium with 20 g/L of agar.

Bacterial basal medium (g/L): glucose 5 (NH4)2SO4 2 s sodium citrate 1 s KH2PO4 6, K2HPO4 4 s agar 20; pH 7.2 121oC sterilized for 15 min.

Basic fermentation medium (g/L): glucose 30, peptone 10, yeast paste 10, KH2PO4 0.4, Na2HPO4.12H2O 1.5, MgSO4.7H2O 0.3; pH 7.2, sterilized at 121oC for 15 min.

 

2.3 Strain culture methods

2.3.1 Slope activation and seed culture

Inoculate the activated slant with the inoculated strains stored in the refrigerator and incubate for 24 h at 30oC; add the activated slant into the seed medium and incubate for 24 h at 30oC with shaking at 200r/min. Shaking flasks were filled with 50 mL of 250 mL triangular flasks if not specified.

 

2.3.2 Fermentation cultures

The seed culture was added to the fermentation medium at 5%(j) inoculum and incubated at 30oC for 50 h with 200r/min shaking (unless otherwise specified).  Three parallel samples were taken from each sample.

 

2.4 Coenzyme Q10 Extraction

15 mL of fermentation broth was centrifuged at 3000 r/min for 20 min, and the supernatant was poured off.  Add 15 mL of acetone into the fermentation broth, and centrifuge the supernatant at 3000 r/min for 10 min with ultrasonic shaking for 0.5 h. The supernatant was analyzed by HPLC[8] .

 

2.5 Cell dry weight determination

4 mL of the fermentation broth was centrifuged at 10,000 r/min for 10 min and the organisms were collected and washed twice with distilled water, dried at 60oC for 48 h until constant weight and then weighed.

 

2.6 Mutagenesis methods

The bacteria were inoculated into seed medium, incubated in shake flasks for 24 h, and then centrifuged at 10 000 r/min for 5 min. 5 mL of the culture solution was collected, and the bacteria were washed twice with 0.1 mol/L sodium phosphate buffer (pH 6.5) to form a bacterial suspension with a concentration of 107 organisms/mL. 10 mL of the suspension was placed in a sterile dish, irradiated with ultraviolet light (UV) for 5 s, and then added with nitrosoguanidine (NTG) solution to make the concentration of NTG in the treatment solution increase.  10 mL of the suspension was placed in a sterile dish, irradiated with ultraviolet light (UV) for 5 s, and then nitrosoguanidine (NTG) solution was added to make the final concentration of NTG in the treatment solution 300 mg/L. The cells were washed three times with 0.1 mol/L phosphate buffer (pH 6.5) after NTG had been in action for 20 min.  After 12 h of incubation in the seed medium, the cells were centrifuged and washed three times, diluted and spread directly on the bacterial basal medium. Then, a certain amount of actinomycin D was added in the middle of the plate and the plate was incubated at 30oC for 4~5 d. After the appearance of obvious inhibition circle, the single colony with good growth within the inhibition circle was selected as the mutant strain of actinomycin D-resistant bacteria.

 

3 Results and discussion

3.1 Acquisition of high yielding mutant strains

Actinomycin D (ActD) is a teratogen and carcinogen. Its structure contains a planar phenoxazine ring and two cyclic pentapeptides, which can be inserted into the DNA molecule and can selectively inhibit transcription and protein synthesis[9] . ActD is highly cytotoxic, and the addition of a certain amount of ActD to the culture medium can cause stress and toxicity to bacterial cells.  Coenzyme Q10 is a physiologically active substance that enhances immunity. If an ActD-resistant mutant strain is obtained after mutagenesis, which indicates that the mutant strain is more resistant to teratogens, the intracellular accumulation of physiologically active substances, such as coenzyme Q10, may be increased.

 

R. radiobacter WSH2601 was used as the starting strain, and 70 actinomycin D mutant strains were obtained by combined mutagenesis with ultraviolet light and nitrosoguanidine.  The mutant strains were inoculated into basic fermentation medium and screened in shake flasks. 7 mutant strains were found to increase coenzyme Q10 production by more than 10% compared with the starter strain.  These 7 mutant strains were each inoculated into 3 shake flasks for re-screening, and a mutant R. radiobacter (ActDr) WSH-F06 was found to have a coenzyme Q10 yield of 11±0.2 mg/L, which was 16% higher than that of the starting strain (9.5±0.2 mg/L).  The coenzyme Q10 yield of this strain was stabilized above 10.8 mg/L in each generation after five consecutive passages, which confirmed the good genetic stability of this mutant strain, and therefore it was selected as a further research strain.

 

3.2 Effect of carbon source on fermentation of R. radiobacter WSH-F06 auxin Q10

3.2.1 Effect of different carbon sources on coenzyme Q10 fermentation

The effect of different carbon sources on the production of coenzyme Q10 by R. radiobacter WSH-F06 fermentation was investigated by keeping the other components of the fermentation medium unchanged at a concentration of 30 g/L, and the results are shown in Fig. 1. It is shown that glucose and sucrose are favorable for the accumulation of coenzyme Q10, and the highest production of coenzyme Q10 of 11.1 mg/L was obtained when glucose was used as the carbon source.

 

3.2.2 Effect of glucose concentration on coenzyme Q10 fermentation

The effect of glucose concentration in the medium on the production of coenzyme Q10 is shown in Figure 2.  The highest dry weight and coenzyme Q10 production were observed at a glucose concentration of 30 g/L, which reached 10.2 g/L and 10.8 mg/L, respectively. Further increase of glucose concentration inhibited the growth of the bacteria and the accumulation of coenzyme Q10.

3.2.3 Effect of Complex Carbon Sources on Coenzyme Q10 Fermentation

Although the yield of Coenzyme Q10 was not high when molasses was used as the sole carbon source, this study investigated the effect of combining glucose and molasses as carbon sources on the production of Coenzyme Q10, considering that there are many microfactors in molasses that can stimulate the growth of microorganisms.  As shown in Figure 3, the addition of molasses to the glucose medium increased the production of coenzyme Q10.  When 30 g/L glucose and 60 g/L molasses were added to the medium, Coenzyme Q10 production increased by 13% compared with that of 30 g/L glucose as the sole carbon source, and further increase in the amount of molasses was not favorable to the accumulation of Coenzyme Q10.

 

3.3 Effect of nitrogen source on fermentation of R. radiobacter WSH-F06 auxin Q10

3.3.1 Effect of different nitrogen sources on coenzyme Q10 fermentation

The effects of different nitrogen sources at a concentration of 160 mmol/L on the production of CoQ10 were investigated by using 30 g/L glucose and 60 g/L molasses as carbon sources, and the results are shown in Fig. 4. R. radiobacter (ActDr) WSH-F06 has a broad-spectrum of nitrogen utilization, with higher CoQ10 yields when using either organic nitrogen sources (peptone, yeast paste, and corn syrup) or inorganic nitrogen sources [(NH4)2SO4 and NH4NO3] as the nitrogen source s. The highest CoQ10 yield was achieved when using corn syrup as the nitrogen source, reaching 13 8 mg/L. 2SO4 and NH4NO3] were used as nitrogen sources, and the production of coenzyme Q10 was higher than that of organic nitrogen sources (peptone, yeast paste, corn syrup) and inorganic nitrogen sources [(NH4) 2SO4 and NH4NO3].

 

3.3.2 Nitrogen source orthogonal experiments

R. radiobacter (ActDr) WSH-F06 Higher coenzyme Q10 yields were achieved when peptone, yeast paste, corn syrup and (NH4)2SO4 were used as single nitrogen sources.  In order to investigate whether a combination of nitrogen sources can further increase the yield of coenzyme Q10, an orthogonal experiment was conducted to investigate the effect of different nitrogen ratios on coenzyme Q10 yield.  The orthogonal design is shown in Table 1. 30 g/L glucose and 60 g/L molasses were used as the carbon sources, and the rest of the conditions were unchanged.

 

In the concentration range of the experiment, peptone had the greatest effect on the accumulation of coenzyme Q10, and (NH4)2SO4 had the least effect (NH4)2SO4 7 g/L peptone 5 g/L yeast 5 g/L, corn syrup 30 g/L was the optimal nitrogen source ratio.  Under this nitrogen ration, the highest yield of Auxin Q10 reached 24.1 mg/Ls with 30 g/L glucose and 60 g/L molasses as the carbon sources, and the highest yield of Auxin Q10 reached 75% higher than that of a single nitrogen source (Fig. 4) with shake flask incubation for 50 h. The highest yield of Auxin Q10 reached 24.1 mg/Ls with 30 g/L glucose and 60 g/L molasses as the carbon sources.

 

3.4 Effect of environmental conditions on the fermentation of R. radiobacter WSH-F06 auxin Q10

3.4.1 Effect of initial pH on coenzyme Q10 fermentation

The effect of initial pH on growth and coenzyme Q10 synthesis is shown in Fig. 5.  The bacterium did not grow at the acidic initial pH, and the coenzyme Q10 production was close to zero.  In the range of pH 6.8~7.5, the intracellular coenzyme Q10 content remained unchanged at about 1.9 mg/g, and there was little change in the amount of coenzyme Q10 production and the bacterial volume, so the initial pH could be chosen as 7.2.

 

3.4.2 Effect of loading volume on coenzyme Q10 fermentation

The different filling volumes of fermentation flasks can reflect the level of dissolved oxygen in the fermentation medium to a certain extent.  The effects of different filling volumes of 500 mL flasks on the growth of the bacteria and the synthesis of coenzyme Q10 were investigated.  Figure 6 shows that the highest coenzyme Q10 production was achieved at 50 mL, which indicates that R. radiobacter (ActDr) WSH-F06 has an optimal requirement of dissolved oxygen for coenzyme Q10 production, which is consistent with the findings of a previous study using the starting strain in our laboratory[10] .

 

3.4.3 Effect of Inoculum Volume on Coenzyme Q10 Fermentation

The effects of inoculum size on the growth and synthesis of coenzyme Q10 were investigated by adding 3%, 5%, 7%, 9% and 11% (j) to the fermentation medium at pH 7.2 (data not shown).  In the range of inoculum levels, the biomass did not change much and the intracellular content and yield of coenzyme Q10 were higher at 7%~9%, so 7% inoculum level was preferred.

 

3.5 Shake flask fermentation of R. radiobacter WSH-F06 for coenzyme Q10 production

The optimal nutrient and environmental conditions for the fermentation of coenzyme Q10 as determined from the above shake flask experiments were 30 g/L glucose and 60 g/L molasses as the carbon source, 7 g/L (NH4)2SO4 and 5 g/L peptone, 5 g/L yeast paste, and 30 g/L maize syrup as the nitrogen source at an initial pH of 7.2, with a volume of 50 mL and an inoculum volume of 7%, and incubated in shake flasks at 30oC at 200 r/min. The culture was shaken at 200 r/min at 30oC to examine the accumulation of coenzyme Q10 by R. radiobacter WSH-F06 as shown in Figure 7.

 

During R. radiobacter WSH-F06 fermentation, coenzyme Q10 synthesis and cell growth were partially coupled. A small amount of coenzyme Q10 was synthesized during the logarithmic growth phase and the maximum coenzyme Q10 production and intracellular coenzyme Q10 content reached 34.0 mg/L and 2.4 mg/g, respectively, at 83 h of fermentation. When the strain was cultured in basic fermentation medium, the production reached a maximum of 11.0 mg/L at 50 h of fermentation, and then showed a decreasing trend (data not given).  After optimization of the medium and environmental conditions, the fermentation time was extended to 83 h, but the coenzyme Q10 yield increased by 210% compared with the pre-optimization period, and the production intensity increased from 0.22 mg/(L.h.) to 0.41 mg/(L.h.).

 

The coenzyme Q10 yields of the starting strain R. radiobacter WSH2601 and the actinomycin D-resistant mutant strain R. radiobacter(ActDr)WSH-F06 were 29.3 and 34.0 mg/L, respectively, and the intracellular coenzyme Q10 contents were 2.1 and 2.4 mg/g, respectively, after incubation with the optimized medium for 83 h. This indicates that the combination of UV and NTG mutagenesis combined with the screening of actinomycin D resistance was effective. The intracellular coenzyme Q10 content was 2.1 mg/g and 2.4 mg/g, respectively. The coenzyme Q10 production of the mutant strain was increased by about 16% compared with that of the starting strain, which indicated that the combination of UV and NTG mutagenesis combined with the screening of actinomycin D resistance was effective.  The effect of optimization of fermentation conditions on coenzyme Q10 yield was greater than that of mutation breeding, indicating that optimization of fermentation conditions is still an important tool to exploit the potential of strains.

 

Yoshida et al[6] developed a mutant strain of Agrobacterium tumefaciens AU-55 that produced 110 mg/L of coenzyme Q10 and 2.5 mg/g of intracellular coenzyme Q10, with a production intensity of 0.76 mg/(L.h.).  The actinomycin-resistant D mutant strain R. radiobacter (ActDr) WSH-F06 obtained in this study showed comparable intracellular coenzyme Q10 content to that of A. tumefaciens.  However, due to the high-density culture of A. tumefaciens AU-55 by replenishment during the fermentation process, its coenzyme Q10 production was higher than that of R. radiobacter (ActDr) WSH-F06.

 

4 CONCLUSIONS

(1)  A high yielding mutant strain of coenzyme Q10, WSH-F06, was obtained by combined mutagenesis with ultraviolet light and nitrosoguanidine using Rhizobium radiobacter WSH2601 as the starting strain and actinomycin D resistance as the screening model. The nutrient and environmental conditions of the mutant strain were optimized for the production of coenzyme Q10 in shake flask experiments, and the final yield was 34 mg/L, which was 3.6 times higher than that before mutagenesis and optimization. Nutritional and environmental conditions were optimized for the production of coenzyme Q10 by the mutant strain in shake flask experiments, and the final coenzyme Q10 yield was 34 mg/L, which was 3.6 times higher than that of the mutant strain before mutagenesis and optimization.  The intracellular coenzyme Q10 content of the mutant strain WSH-F06 was 2.4 mg/g, which was comparable to that of A. tumefaciens AU-55, a mutant strain of A. tumefaciens developed by Yoshida[6] .

 

(2) Actinomycin D resistance was used as a screening model for selecting mutant strains with high coenzyme Q10 production, which provides a new idea for mutation breeding of coenzyme Q10 and other physiologically active substances-producing bacteria.

 

References.

[1] Zhang Huizhan.  Pathway Engineering [M].  Beijing: China Light Industry Press, 2002. 130-136.

[2] WU Zufang WENG Peifang CHEN Jian.  Progress of functional studies on coenzyme Q10 [J].  Journal of Ningbo University, 2001,14(2): 85-88.

[3] Ernster L, Dallner G. Biochemical, Physiological and Medical Aspects of Ubiquinone Function [J]. Biochim. Biophys. Acta, 1995, 1271: 195-204.

[4] Kawamukai M. Biosynthesis, Bioproduction and Novel Roles of Ubiquinone [J]. J. Biosci. Bioeng., 2002, 94: 511-517.

[5] Gingold E B, Kopsidas G, Linnane AW. Coenzyme Q10 and Its Putative Role in the Ageing Process [J]. Protoplasma, 2002, 214: 24-32.

[6] Yoshida H, KotaniY, Ochiai K. Production ofUbiquinone-10 Using Bacteria [J]. J. Gen. Appl. Microbiol. 1998, 44: 19-26.

[7] WU Zufang: WENG Peifang, LI Yin et al.  Breeding concepts and optimization strategies of fermentation conditions for coenzyme Q10 production [J].  Food and Fermentation Industry, 2001, 27(7): 49-53.

[8] WU Zufang, GAO Guocheng, CHEN Jian.  Isolation, purification and quantitative analysis of coenzyme Q10 in fermentation broth [J].  Journal of Wuxi University of Light Industry, 2002, 21(4): 420-423.

[9] Ni Jingman, Yang Xiaowu, Pan Xinfu.  In vitro DNA binding characterization of the anticancer drug actinomycin D and its new analogues [J].  Journal of Lanzhou University, 2002, 38(3): 87-89.

[10] WU Zu-Fang, SANG Guo-Cheng, CHEN Jian.  Shake flask fermentation conditions for the production of coenzyme Q10 by Rhizobium radiodurans WSH260l [J].  Journal of Wuxi University of Light Industry, 2003, 22(1): 65-69.

Study of the Therapeutic Effects of COQ10 in Alzheimer's Disease

 With the increase of life expectancy, Alzheimer's disease (AD) has become one of the major diseases affecting the quality of life in the later years of human beings, with Alzheimer's disease (AD) accounting for about 70% of the cases of Alzheimer's disease[1] . AD is a common degenerative disease of the central nervous system, whose main pathological features are β-amyloid deposits and neurofibrillary tangles composed of highly phosphorylated Tau proteins.

 

AD is a common degenerative disease of the central nervous system, and its main pathological features are age spots composed of β-amyloid deposits and neurofibrillary tangles composed of highly phosphorylated Tau proteins. The prevalence of AD increases gradually with age, and the severe cognitive impairment obviously affects the quality of life of patients and brings great pressure to families and society. The exact etiology of AD has not yet been fully elucidated, but studies have pointed out that oxidative stress, mitochondrial dysfunction, neuroinflammation and cerebrovascular dysfunction may be involved in the occurrence and development of AD, among which oxidative stress and mitochondrial dysfunction have received extensive attention from scholars at home and abroad.

 

As a fat-soluble antioxidant, COQ10 regulates the body's antioxidant enzyme system, scavenges oxygen free radicals, and reduces oxidative stress [2], and has been found to have neuroprotective effects as well. A brief review of the therapeutic effects of coenzyme Q10 on AD is presented.

 

1 Antioxidant Effects of COQ10

Mitochondria as the main place of cellular energy production, energy production is mainly realized through the transfer of high-energy electrons in the mitochondria. However, the production of adenosine triPhosPhate (ATP) by electron transfer is accompanied by the generation of reactive oxygen species (ROS). Normally, antioxidant enzymes (e.g., proteins such as thiols, reduced glutathione, α-tocopherol, etc.) are present in the body to scavenge ROS[3] .

 

In AD, the antioxidant enzyme system has been found to be weakened. When the antioxidant enzyme system is weakened to the extent that it is insufficient to scavenge the generated ROS, oxidative stress occurs and oxidative damage is caused to cellular components, which includes mutation of mitochondria DNA (mtDNA), carboxylation of proteins, lipid peroxidation, etc. [4]. Mutations of mtDNA damage the electron transport chain, thus causing respiratory chain complexes, which can lead to the formation of a complex of respiratory chains. Mutations in mtDNA damage the electron transport chain, resulting in reduced activity of the respiratory chain complex and reduced proton pumping, leading to a decrease in the mitochondrial membrane potential (ΔΨm). When ΔΨm falls below a certain threshold, the mitochondrial transition Pore (mPTP) opens, triggering cell death through apoptosis or necrosis [5].

 

Coenzyme Q10 has been found to have a number of molecular bases for the treatment of neurodegenerative diseases. Coenzyme Q10 has been shown to inhibit oxidative damage to mitochondria and can act as an antioxidant on several levels. Coenzyme Q10 scavenges oxygen radicals by interacting with α-tocopherol. In addition, the activation of coenzyme Q10 as a cofactor of mitochondrial uncoupling proteins reduces the production of free radicals[6] .

 

In addition to its radical scavenging activity, Coenzyme Q10 prevents apoptotic cell death by blocking the binding of the pro-apoptotic gene Bax to mitochondria and by inhibiting the activation of the mitochondrial permeability transition (MPT)[7] . Further, Coenzyme Q10 blocked the release of cytochrome c and the activation of cysteine-9, but not cysteine-8, which are the substrates for the activation of endogenous and exogenous apoptotic pathways, respectively, suggesting that Coenzyme Q10 blocks apoptosis through the inhibition of endogenous pathways, but not exogenous pathways [8].

 

2 Neuroprotective Effects of COQ10

2.1 Coenzyme Q10 Reduces β-Amyloid Neurotoxicity

The deposition of β-amyloid (Aβ) can cause damage to neuronal and synaptic functions and lead to neuronal degeneration.L235P PS-1 transgenic old mice overproduced β-amyloid polypeptide-42 (Aβ42) and oxidative stress, and also deposited a large amount of Aβ42 in the cells.An intervention study of L235P PS-1 transgenic old mice using coenzyme Q10 showed that coenzyme Q10 effectively reduced Aβ42 production and accumulation and suppressed oxidative stress[9] .

 

Coenzyme Q10 effectively reduced Aβ42 production and intracellular accumulation and suppressed oxidative stress in L235P PS-1 transgenic aged mice[9] . In vivo and in vitro studies have shown that Aβ is located in the mitochondrial cristae of neurons[10] , and that Aβ accumulation can cause elevated ROS and mitochondrial dysfunction, leading to mPTP opening and cytochrome c release, and resulting in cellular damage and death[11,12] . A novel mitochondria-targeted ubiquinone derivative (MitoQ) prevented the excessive production of reactive substances and the decrease of Δψm in Aβ-induced cortical neurons, suggesting that MitoQ is protective against Aβ-induced cortical neuronal damage[13] .

 

2.2 Coenzyme Q10 Restores the Activity of Mitochondrial Energy Metabolizing Enzymes   

Respiratory chain complexes (I, II, III and IV) are essential for mitochondrial production of life-sustaining ATP, and it has been found that respiratory chain complex IV is decreased in the mitochondria of patients with AD, which is one of the reasons for energy hypometabolism in AD patients [14].

 

Ng LF et al. found that MitoQ treatment group showed a significant increase in respiratory chain complexes IV and I compared with the untreated group, suggesting that MitoQ has a protective effect on the mitochondrial respiratory chain [15]. Ng LF et al. showed that the respiratory chain complexes IV and I were significantly increased in the MitoQ-treated group compared with the untreated group, indicating that MitoQ has a protective effect on the mitochondrial respiratory chain[15] . In another study, Singh A et al. found that 40 mg/kg of coenzyme Q10 restored the reduced activity of mitochondrial respiratory enzyme complexes (I, II, III, IV) in the hippocampus and cerebral cortex of Aβ1-42-treated rats [16].

 

2.3 Coenzyme Q10 Inhibits the Inflammatory Response

Inflammation has been found to play an important role in the pathogenesis of AD. Choi H et al. found that coenzyme Q10 protected neuronal cells from Aβ25-35-induced neurotoxicity in a concentration-dependent manner by increasing the expression of phosphatidylinositol 3-kinase P85α (P85αPI3K), phosphorylated Akt, phosphorylated glycogen synthase kinase-3β, and heat shock transcription factors, all of which are related to neuronal cell survival, in cortical neurons treated for 48 hr with different concentrations of coenzyme Q10 in response to Aβ fragment 25-35-induced damage.

 

COQ10 protected neuronal cells from Aβ25-35-induced neurotoxicity in a concentration-dependent manner by increasing the levels of phosphatidylinositol 3-kinase P85α (P85αPI3K), phosphorylated Akt, phosphorylated glycogen synthase kinase-3β, and heat-shock transcription factors, which are associated with neuronal cell survival, and by decreasing death signals, including cytoplasmic cytochrome c and activated cysteine-3, which were mediated by the enzyme phosphatidylinositol 3-hydroxy kinase (PIHK), and the cytosolic cytosolic cytosol. This protective effect was blocked by the phosphatidylinositol 3-hydroxy kinase (PI3K) inhibitor LY294002, suggesting that the neuroprotective effect of coenzyme Q10 on Aβ25-35-induced neurotoxicity can be mediated by the activation of the PI3K-Akt signaling pathway[17] .

 

In addition, nuclear factor kaPPa-B (NF-κB) has been reported to be closely related to Aβ-induced neuroinflammation. Li et al. found that coenzyme Q10 reduced neuroinflammation in Aβ25-35-induced PC12 cells by preventing Aβ25-35-induced degradation of IκBα (one of the members of the NF-κB family of inhibitory proteins) and nuclear translocation of P65, inhibiting prostaglandin E2 (PGE2) production and cyclooxygenase-2 (COX-2) protein expression. The study showed that coenzyme Q10 reduced neuroinflammation by preventing Aβ25-35-induced degradation of IκBα (a member of the NF-κB inhibitory protein family) and nuclear translocation of P65, and by inhibiting the production of prostaglandin E2 (PGE2) and the expression of cyclo-oxygenase-2 (COX-2), suggesting that the inhibition of NF-κB signaling pathway by COQ10 in PC12 cells may result in the down-regulation of pro-inflammatory mediators, and thus produce anti-inflammatory effects [18].

 

3 Coenzyme Q10 and the Treatment of AD

Although the exact etiology and pathogenesis of AD are not fully understood, there is a large body of evidence suggesting that oxidative stress-induced production of reactive substances and mitochondrial dysfunction are involved in the onset and progression of AD. So derberg et al. analyzed ubiquinone levels in 10 different brain regions in patients with AD/SDAT, and found that ubiquinone levels were significantly increased in most of the brain regions [19]. The elevated levels of ubiquinone in AD/SDAT may reflect increased oxidative stress.  Isobe et al. reported that the percentage of oxidized/total coenzyme Q10 in the cerebrospinal fluid of patients with early to mid-stage AD was significantly higher than that of controls, and that this percentage was negatively correlated with disease duration, suggesting that an increased percentage of oxidized/total coenzyme Q10 is associated with the pathogenesis of early AD [20].

 

Current researchers are divided on the therapeutic effects of coenzyme Q10 on AD. Dumont et al. found that COQ10 reduced brain oxidative stress and β-amyloid levels and improved cognitive behaviors in Tg19959 mice [21]. McManus et al. found that MitoQ reduced oxidative stress, Aβ accumulation, astrocyte proliferation, synaptic loss, and cysteoaspartic enzyme activation, preventing the decline in cognitive performance in 3xTg-AD mice [22].

 

McManus et al. found that MitoQ reduced oxidative stress, Aβ accumulation, astrocyte proliferation, synaptic loss and cysteinyl asparagin activation, and prevented cognitive decline in 3xTg-AD mice [22]. Singh et al. studied β-amyloid 1-42 (Aβ1-42)-treated AD rats, and found that 40 mg/kg of CoQ10 significantly attenuated oxidative damage, restored mitochondrial respiratory enzymes and histopathological alterations, and reduced acetylcholinesterase (AChE) activity, and improved cognitive performance [16]. Muthukumaran et al. studied the effects of a water-soluble preparation of coenzyme Q10 (Ubisol-Q10) in transgenic AD mice and found that Ubisol-Q10 significantly reduced circulating Aβ peptide and inhibited the formation of cerebral Aβ plaques compared with the untreated group, as well as improved long-term memory and preserved workspace memory [23].

 

COQ10 has also been clinically tested in the treatment of AD. Galasko et al. evaluated the efficacy of coenzyme Q10 in 78 patients with mild-to-moderate AD treated with 400 mg of coenzyme Q10 three times a day for 16 weeks using oxygenated stress markers and cognitive function scores, and found that no significant improvement was observed compared with the placebo group [24]. This may be related to the uneven distribution of coenzyme Q10 and its difficulty in crossing the blood-brain barrier and reaching neuronal mitochondria. Idebenone, a synthetic analog of coenzyme Q10, has been used in neuroprotective studies in AD. Senin et al. studied the effects of twice-daily ibuprofen 45 mg for 4 months in 102 elderly AD patients and found that ibuprofen significantly improved memory, attention, and behavior in AD patients [25].

 

Bergamasco et al. conducted a multicenter, randomized, placebo-controlled, double-blind trial of ibenzoquinone treatment for 90 d in 92 patients with AD and found that ibenzoquinone improved memory, attention, and orientation and slowed the progressive deterioration of the disease.26 Weyer et al. studied the effects of ibenzoquinone treatment in 300 patients with mild-to-moderate AD dementia who were randomly assigned to either ibenzoquinone (30 mg, 90 mg) or a placebo 3 times daily for 6 months and found an overall improvement in the Clinical Global Impression Scale (CGI) in the 3-times daily ibenzoquinone 90 mg group. Weyer et al. examined the effects of a 6-month treatment of 300 patients with mild-to-moderate AD dementia randomly assigned to 3 times daily ibenzoquinone (30 mg, 90 mg) or placebo, and found that the 3 times daily ibenzoquinone 90 mg group showed an overall improvement in Clinical Global Impression (CGI), and significant improvements in AD Cognitive Assessment Scale (ADAS-Cog) and Non-Cognitive Functioning (ADAS-Noncog) scores[27] .

 

Gutzmann et al. conducted a 2-year study of patients with mild-to-moderate AD. Gutzmann et al. conducted a two-year randomized, double-blind study in patients with mild-to-moderate AD, and found that the two ibenzoquinone groups (90 and 120 mg three times daily) scored significantly higher than the placebo group on a number of cognitive measures, and that the 120 mg group scored better than the 90 mg group [28]. However, not all studies have been positive.A 1-year multicenter, double-blind, placebo-controlled study by Thal LJ et al. conducted a randomized controlled trial of 3 daily doses of ibuprofen 120, 240, or 360 mg in 536 patients with suspected AD, and found no significant differences in ADAS-Cog and Clinical Gross Impression Change (CGIC) scores among the 4 groups [29].

 

4 Safety of Coenzyme Q10

The high safety profile of coenzyme Q10 in the treatment of AD has been demonstrated in clinical trials. Coenzyme Q10 has been shown to be relatively well tolerated at doses of 200-3000mg/day. When plasma levels of coenzyme Q10 reach 2400 mg/d, mild side effects such as headache, heartburn and other gastrointestinal symptoms may occur, in addition to fatigue, increased involuntary movements and asymptomatic elevation of liver enzymes [30].

 

AD is a common degenerative disease of the central nervous system, and its symptoms such as cognitive decline, personality change and behavioral impairment seriously affect the quality of life of AD patients, and bring great pressure to the family and the society, so it is very urgent to find effective drugs to treat AD patients. Coenzyme Q10, with its anti-oxidative stress and neuroprotective effects, has been shown to improve cognitive decline in animals with AD in both cellular and animal studies.

 

Due to the uneven distribution of exogenous coenzyme Q10, it is not easy to cross the blood-brain barrier and penetrate into the mitochondria of neurons, which leads to the poor therapeutic effect in AD patients. Idebenone, a synthetic analog of coenzyme Q10, has been proved to have good therapeutic efficacy in patients with early to middle stage of AD, and MitoQ, as a ubiquinone derivative targeting the mitochondria, shows powerful antioxidant effects, and it also has shown good pharmacokinetic behaviors in the phase I clinical trials of AD patients. MitoQ, as a mitochondria-targeting ubiquinone derivative, showed strong antioxidant effects and good pharmacokinetic behavior in a phase I clinical trial in AD patients. Therefore, more basic and clinical studies are needed to provide strong evidence that coenzyme Q10 and its analogs are effective in the treatment of AD.

 

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