Study on the Effect of COQ10 on Heart Recovery After Coronary Intervention in Coronary Heart Disease

 Coronary atherosclerotic heart disease (CHD) is a condition in which yellow, waxy plaque builds up in the coronary arteries (coronary arteries), and the plaque builds up inside the arteries to form atherosclerosis [1]. As the disease progresses, the plaque hardens and ruptures, narrowing the coronary arteries and affecting the blood supply to the heart muscle [2], and the ruptured plaque forms a thrombus that blocks the coronary arteries, narrowing them and causing myocardial ischemia, hypoxia, and myocardial damage. Coronary heart disease has significant regional differences, generally higher in the north than in the south of China, higher in urban than in rural areas, and is on the rise [3].

 

Coronary heart disease (CHD) remains one of the diseases with the highest morbidity and mortality rates. Intervention of risk factors (hypertension, dyslipidemia, obesity, diabetes mellitus, poor lifestyle, etc.) can help in the prevention and treatment of CHD [4]. Intervention is often used in addition to pharmacologic therapy in clinical practice. Aspirin combined with clopidogrel is one of the main therapeutic agents in patients with coronary artery disease and before and after percutaneous coronary intervention. Coenzyme Q10 (CoQ10) is a fat-soluble compound located mainly in the inner mitochondrial membrane of cells, and is involved in electron transport in the respiratory chain, antioxidant, metabolic regulation, and regulation of cell differentiation.

 

Coenzyme Q10 is synthesized by itself and ingested externally, of which exogenous supplementation is the most important. It has the effects of vasodilating, reducing blood viscosity and peripheral resistance, increasing arterial blood flow, inhibiting platelet aggregation, promoting fibrinolysis, and lowering blood lipids, etc., and is used in the treatment of a variety of cardiovascular diseases because of its effects on anti-inflammation and reduction of vascular proliferation through its influence on NF-κB [5]. In this study, 79 cases of coronary heart disease patients admitted to Beijing Anzhen Hospital from August 2017 to August 2018 were studied, and the effect of coenzyme Q10 on cardiac function after coronary intervention was investigated.

 

1 Information and Methodology

1.1 Research Target

Eighty-six cases of coronary heart disease patients admitted to Bejing Anzhen Hospital from August 2017 to August 2018 were selected, of which 7 cases were lost to visit, with a loss of visit rate of 8.14%, and 79 coronary heart disease patients were followed up as the study subjects, which were divided into the control group and the study group according to the randomized numerical table method. The control group consisted of 40 cases, including 18 females and 22 males, aged 53-78 years, with an average age of 63.87±12.34 years. There were 39 cases in the study group, including 20 females and 19 males, aged 52-78 years, with an average age of 64.17±11.78 years. There was no statistical difference between the two groups in terms of gender, age, and other basic information (P＞0.05).

 

This study was approved by the Ethics Committee of our hospital. Inclusion criteria: ① Referring to the "Diagnostic Criteria for Coronary Atherosclerotic Heart Disease (WS319 2010)"[6] , all patients were diagnosed with coronary heart disease and underwent percutaneous coronary intervention, and there were no contraindications to the use of drugs in this study; ② In addition to the clinical symptoms of angina pectoris, they also manifested palpitations, chest tightness, and fatigue, etc.; ③ All the patients agreed to voluntarily participate in the experiment and signed an informed consent form. (iii) All patients agreed to participate in the experiment and signed the informed consent form. Exclusion criteria: ① severe hepatic and renal insufficiency or cardiac failure, hematopoietic and coagulation disorders; ② asymptomatic myocardial ischemia or myocardial infarction in the acute stage of the patient; ③ serious cardiac arrhythmia or antiplatelet drugs within the past month; ④ with surgery or trauma, drug or alcohol dependence history.

 

1.1 Methodologies  

Both groups were given basic treatment before and after the interventional therapy, avoiding factors that trigger angina attacks (such as excessive physical activity, agitation, etc.). In the control group, the patients were treated with conventional coronary heart disease treatment. The study group was given coenzyme Q10 (10mg/bottle, China National Drug Standard H10930021, Eisai China Pharmaceutical Co., Ltd.) 10 mg, 3/d, and the efficacy of the treatment was evaluated after 1, 3, and 6 months of treatment.

 

1.2 Observation Indicators  

Clinical efficacy: obvious effect: clinical symptoms and signs disappeared, cardiac function recovered to class Ι or improved class Ⅱ; effective: cardiac function improved class Ι; ineffective cardiac function has not been significantly improved or symptoms worsened. The total effective rate = (obvious effect + effective)/total number of cases × 100%[7]. 6-min walking experiment: patients were asked to walk as fast as possible in a straight corridor, and if symptoms such as chest pain, chest tightness, sweating, or pallor appeared in the course of the walking, they had to stop, and the cardiac function was evaluated according to the 6-min walking distance. 1-min walking distance <150m indicated that the patient had severe heart failure, and 1-min walking distance between 150~450m indicated that the patient was in serious heart failure, and 1-min walking distance between 150~450m indicated that the patient had severe heart failure. A 1-min walking distance of 150-450 m indicates severe heart failure, and a 1-min walking distance of >450 m indicates mild heart failure, which means that the patient's heart function is still good or the effect of medication is good [8].

 

Cardiac function indexes: left ventricular ejection fraction (LVEF), left ventricular end-systolic volume (LVESV), left ventricular end-systolic diameter (LVESD) and left ventricular end-diastolic diameter (LVEDD) were examined in the patients of the two groups before and after the treatment [9]. Coagulation indices: 5 ml of venous blood was drawn from patients before and 1, 3, and 6 months after the drug was administered, and the patients' prothrombin time (PT), fibrinogen (FIB) content, and maximal platelet aggregation rate (MPAR) were measured [10]. Inflammatory factor levels: Measure the levels of ultrasensitive C-reactive protein (hs-CRP), tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in the serum of the patients before and after the administration of the drug. Adverse drug reactions (ADRs) were observed and recorded during the treatment period[11] . 1.4 Statistical methods SPSS 22.0 was used for statistical analysis. Data were analyzed using SPSS 22.0. The count data were expressed as percentage (%) with χ2 test; the measurement data were expressed as (x ± s) with t test. The difference was considered statistically significant at P < 0.05.

 

2 Results

2.1 Comparison of Baseline Conditions Between the Two Groups of Patients

There was no statistical difference between the two groups in terms of age, gender, blood glucose, blood lipids, smoking, history of hypertension, diabetes mellitus and medication history (P＞0.05), Table 1. 2.2 Comparison of clinical efficacy between the two groups before and after treatment There was an overall efficiency of 94.9% in the study group, which was higher than that of the control group of 82.5%, and the difference had a statistically significant difference (P＜0.05), Table 2.

Table 1 Comparison of the basic data of the two groups of patients

 

particulars	Control group (n=40)	Study group (n=39)	P-value
Age (years)	63.87 ± 12.34	64.17 ± 11.78	0.096
Male (n, %)	18 (45)	19 (48.7)	0.159
Blood glucose (mmol/L)	5.01±0.65	4.98±0.63	0.089
Lipids (mmol/L)	5.21±0.32	5.23±0.31	0.098
ACEI	11 (27.5)	10 (25.6)	0.232
beta-blocker	15 (37.5)	16 (41)	0.474
Smoking history (n, %)	5 (12.5)	6 (15.4)	0.365
History of hypertension (n, %)	8 (20)	8 (20.5)	0.453
History of diabetes (n, %)	4 (10)	3 (7.7)	0.325

Table 2 Comparison of the clinical outcomes of the two groups of patients before and after treatment

 

groups	number of examples	produce an effect (n, %)	validity (n, %)	null (n, %)	Overall effectiveness (%)
control subjects	40	18 (45)	15 (37.5)	7 (17.5)	82.5
research group	39	20 (51.3)	17 (43.6)	2 (5.1)	94.9
P value <0.05

 

2.3 Comparison of Cardiac Function Indexes Before and After Treatment in Two Groups

After 6 months of treatment, the ejection fraction LV EF of both groups increased, while LVEDD, LVESD and LVESV decreased, with statistical differences (P < 0.05). After treatment, the cardiac function indexes of patients in the study group were significantly better than those of the control group, and the difference was statistically significant (P < 0.05), Table 3.

Table 3 Comparison of cardiac function indexes before and after treatment in two groups of patients

 

groups	Observation time	LVEF (%)	LVEDD (mm)	LVESD (mm)	LVESV (ml)
Control group (n=40)	pre-treatment	45.38±3.57	55.86±3.37	52.48±4.08	146.32±34.28
	post-treatment	55.67±2.68a	47.38±2.95a	41.36±3.58a	115.47±14.52a
Study group (n=39)	pre-treatment	46.13±3.15	55.18±3.21	52.85±3.97	146.87±35.02
	post-treatment	60.25±2.42ab	41.39±2.27ab	31.58±3.02ab	101.38±11.76ab

Note: LVEF: left ventricular ejection fraction; LVEDD: left ventricular end-diastolic internal diameter; LVESD: left ventricular end-systolic internal diameter; LVESV: left ventricular end-systolic volume; compared with pretreatment.

Isolation, Purification and Quantitative Analysis of Coenzyme Q 10 from Fermentation Broths

 Coenzyme Q10, also known as ubiquinone and decenoquinone, is a class of fat-soluble quinone compounds, with the chemical name of 2,3-dimethoxy 5-methyl 6-decyloisopentenyl benzoquinone.  Coenzyme Q10 is yellow or orange crystalline powder, odorless and tasteless, insoluble in water, soluble in chloroform, benzene, acetone or petroleum ether, melting point 48 . 0 ~ 50 . 0 °C, relative molecular mass 863.4. 4; in plants and animals, microorganisms and other cell bodies and mitochondrial inner membrane conjugation, is an important organism in the respiratory chain hydrogen delivery body.

 

As a multifunctional biochemical drug, coenzyme Q 10 has a wide range of clinical applications [1, 2]; coenzyme Q10 can be produced by animal and plant tissue extraction, chemical synthesis, and microbial fermentation; compared with other methods of preparation, microbial fermentation has the features of low raw material cost, easy control and large-scale production, which can meet the clinical needs of this drug.

 

Coenzyme Q 10 is an intracellular product of microbial fermentation; therefore, the fermentation product Coenzyme Q 10 must be isolated and purified. High performance liquid chromatography (HPLC) is a demanding, expensive and time-consuming analytical method. Therefore, a simple, accurate and relatively rapid method for the determination of coenzyme Q10 in fermentation broth is required. The separation and determination of coenzyme Q10 in the fermentation broth were carried out by organic solvent extraction, thin-layer chromatography and ultraviolet spectrophotometry, which can be used as a reference for further large-scale fermentation production control and compositional analysis.

 

1 Materials and Methods

1 . 1 Instruments

Chromatography silicon GF254-plate (10 cm × 20 cm); EZ585Q Freeze Dryer: manufactured by FT's systems Inc; High-speed Centrifuge; Ultrasonic Cell Breaker; TU-1800 Automatic Scanning Visible Ultraviolet Spectrophotometer: manufactured by Beijing Pudian General Instrument Company Limited; High Performance Liquid Chromatograph: shimadzu model LC-6AD liquid chromatograph. High Performance Liquid Chromatograph: shimadzu model LC- 6AD .

 

1 . 2 Main reagents

Anhydrous ethanol, methanol, acetone, petroleum ether, chloroform, benzene, ethyl ether, hexane, iso-octane, isopropanol, etc. are analytically pure.

 

1 . 3 Samples

Imported Coenzyme Q10 standard: product of siGma, a gift from Zhoushan Hailixiang Pharmaceutical Co.

 

1 . 4 Preparation of Coenzyme Q 10

Preparation procedure: Fermentation broth (prepared in the laboratory, strain Rhizobi- um radiobacter WsH2061) → centrifugation at 6,500 r/min for 15 min → freeze-drying → extraction with stirring of organic solvents → centrifugation to remove the bacterial body → evaporation and drying under pressure → residue extraction with a small amount of petroleum ether, concentration under reduced pressure of the collected petroleum ether layer and then washed with water → dried with anhydrous sodium sulfate → dissolution with a fixed amount of anhydrous ethanol → thin layer chromatography separation → scraping off spots with the same R Dissolve with a fixed amount of anhydrous ethanol → thin layer chromatography → scrape off the spots with the same R『 value as the standard → petroleum ether extraction → evaporation and drying → dissolve the residue with about 0.5 mL of ethanol → low pressure evaporation and drying → remove the bacteria by centrifugation. Dissolve with about 0.5 mL of ethanol → store at low temperature for use.    

 

1 . 5 Analytical Methods for Coenzyme Q 10

UV analysis: UV scanning of the sample to be measured at a wavelength of 190-300 nm, determination of its maximum UV absorption wavelength, and quantitative determination at 275 nm.

 

High performance liquid chromatography (HPLC): the column was sPherisorbC18 (10 cm×4.6 mm ID). The column was sPherisorbC18 (10 cm×4.6 mm ID), filled with octadecylsilane-bonded silica gel, methanol/anhydrous ethanol (1:1, v/v/v) as the mobile phase, with a column temperature of 35 ℃, a 10,000-checksquare-meter wavelength of 275 nm, and a sample volume of 20 μL.

 

2 Results and Discussion

2 . 1 Determination of Coenzyme Q10 Extraction and Preparation Methods

Since coenzyme Q10 is an intracellular substance, which mainly exists in the inner membrane of mitochondria, its extraction effect has a direct impact on the purity and yield of the product. The most commonly used extractant for the extraction of intracellular quinones is organic solvents [3, 4]. The results of the extraction of intracellular quinones from bacterial cells using 1) methanol and ether at a volume ratio of 2:1; 2) iso-octane and isopropanol at a volume ratio of 3:1; 3) methanol and chloroform at a volume ratio of 1:2; 4) acetone direct extraction; and 5) ultrasonic crushing of acetone suspension are shown in Fig. 1. The results were shown in Fig. 1. The results showed that the methanol-chloroform mixture was easy to separate multiple layers after extraction, and the subsequent treatment was difficult, so we finally decided to use acetone suspension ultrasonication method.

 

Fig. 1 Effect of different solvent treatments on the extraction rate of coenzyme Q10

 

2 . 1 . 1 Determination of Solvent Stirring Extraction Time

Freeze-dried cells were ultrasonicated with acetone suspension at 0.5, 1, 3, 5 and 8 h. The results are shown in Fig. 2 . 5, 1, 3, 5 and 8 h of continuous stirring to compare the extraction effect, the results are shown in Figure 2 . The finalized organic extraction time was 5 h .

 

Fig.2 Effects of extracting time on the extract yields of CoQ10

 

2 . 1 . 2 The Effect of Extraction Temperature on the Extraction Effect

Adopting room temperature, 40, 60 ℃ for extraction, the results are shown in Figure 3 . Considering the continuous heat preservation treatment on the volatilization of organic solvents and the purpose of the composition of the possible impact, and finally determine the organic extraction temperature for room temperature (25 ℃) combined with intermittent heating method of extraction .

 

 

Fig.3 Effects of extracting temperature on the extract effectiveness of CoQ10

 

2 . 2 Determination of Coenzyme Q10 by TLC-UV Method

2 . 2 . 1 Thin-layer chromatography for the separation of coenzyme Q10 Spreading agent selection

Thin-layer chromatographic separation of the crude extract of coenzyme Q10 was carried out using chloroform and benzene (1:1, v/v) or petroleum ether and ether (8:2, v/v) as the unfolding agent, and iodine vapor as the color rendering agent or under ultraviolet light directly [5]. 5 μL of sample was used as the sample volume, and the reference concentration of the standard sample was 100 μg/mL, and the spots displayed were compared with the R『 value of the standard. At the same time, a series of standard solutions with mass concentrations of 10 ~ 150 μg/mL were prepared, and their minimum detection mass concentrations were determined, and the results are shown in Table 1.

Table 1 Selection of unfolding agents for thin layer chromatography and minimum mass concentration for detection

Developer	Chloroform/benzene Petroleum ether/ ether Benzene/acetone (volume ratio 85 : 15 1 : 1) 8 : 2) 93 : 7)
R『   Value Minimum detectable mass concentration/ (μg/mL)		0 . 5   35	0 . 67   24	0 . 85   50	-   -

 

Benzene/acetone was colorless by iodine vapor and UV light; the size of color spots of other developers was not related to the mass concentration, but only the color shade was related to the mass concentration of the samples, so it was necessary to combine with the UV method for the determination. According to the suitable range of R『 value and detection sensitivity, chloroform and benzene were selected as the unfolding agents and analyzed by TLC.

 

2 . 2 . 2.2.2 Preparation of UV Spectrophotometric Standard

Curve 20 mg of Coenzyme Q10 standard was accurately weighed, dissolved in anhydrous ethanol and concentrated to 50 mL, and then the solution was diluted to a certain volume to obtain a series of mass concentrations of 8, 20, 40, 80, 120, 160, 200 μg/mL, etc., and the absorbance was measured at 275 nm. The results showed that the linearity of coenzyme Q10 in the range of 80~120 μg/mL was good, and the regression curve equation was y = 59 . 111x - 1 . 577 5, correlation coefficient R2 = 0 . 999 6 .

 

2 . 2 . 3 Comparison of UV Absorption Curves of Coenzyme Q10 and Establishment of Quantitative Methods

The UV absorption curves of the coenzyme Q10 samples prepared from the above data, compared with the standards, were scanned at 190-300 nm with anhydrous ethanol as a blank, and the UV absorption curves are shown in Fig. 4.

 

Fig. 4 UV scanning absorption curves of the standard (upper panel) and sample (lower panel)

As can be seen in Figure 4, the scanned peak shapes of the crude extract and coenzyme Q10 sample obtained by organic solvent extraction and petroleum ether extraction were basically the same, with maximum absorption at (207 ± 2) nm and (275 ± 1) nm, and the peaks of the sample liquid were deviated from the peaks at 275 nm, and there were also some small peaks of impurities. This is due to the extraction of the sample by a variety of organic solvents and the dissolution of intracellular impurities, which have a large absorption near (207 ± 2) nm and (275 ± 1) nm.

 

Therefore, direct UV quantitative determination of the crude extract would bring some errors, so the content of Coenzyme Q10 was quantified by thin-layer chromatography separation, further purification and UV detection. The method is as follows: Crude extract → Spotting, unfolding on a silica gel plate → scraping off the spot with the same R『 value as the standard product → the target material is washed with a small amount of petroleum ether → drying on a rotary evaporator → the residue is dissolved in 0.3 mL of ethanol → the sample is dissolved with 0.3 mL of ethanol. Dissolve the residue with about 0.3 mL of ethanol → Sample. The sample was diluted with alcohol solvent according to the concentration of the collected sample and the absorbance value was measured at 275 nm.

 

2 . 3 Comparison of the Results of TLC-UV method and HPLC Method

The extracts obtained by organic solvent extraction, petroleum ether elution and TLC separation were dissolved in a certain amount of anhydrous ethanol and then analyzed by HPLC and compared with the results of the above experiments, as shown in Table 2 .

 

The mass concentration of Coenzyme Q10 measured by the TLC-UV method was 92% of that by the HPLC method, which may be due to the partial loss of the spots developed by TLC in the re-extraction process.

Table 2 Comparison of two assays for Coenzyme Q 10

 

Methodology	Mass concentration of coenzyme Q10/(μg/mL)
TLC-UV method	80 . 1
HPLC method	87 . 3

 

2 . 4 Coenzyme Q10 Identified by High Performance Liquid Chromatography Analysis

The crude extract was analyzed by thin plate chromatography and then by high performance liquid chromatography (HPLC) to identify the extraction effect.［6］ . The concentration of the sample was 200 μg/mL, the detection wavelength was 275 nm, and the chromatograms are shown in Figs. 5 and 6.

 Fig. 5 Chromatogram of the standard sample (200 μg/mL) analyzed by high performance liquid chromatography (detection wavelength: 275 nm).

 

As can be seen in Figures 5 and 6, the retention time of coenzyme Q10 in the chromatograms of both standard and sample was 20.6 min. 6 min. After chromatography of the sample solution separated by this coenzyme Q10 extraction method, HPLC analysis showed that there were few impurities, which did not interfere with the determination of coenzyme Q10. In addition, by changing the detection wavelength to 206 nm, the HPLC analysis of the standard sample and the test sample was carried out, and the results showed that the retention time of the standard sample and the test sample was 20.6 min, and the retention time of the standard sample and the test sample was 20.6 min. The results showed that there was one main peak at the retention time of 20.6 min for both standard and test samples, which proved that they were the same substance.

 

The sample was further reduced with 0.01 mL of sodium borohydride solution. The sample was further reduced with 0.01 mL of sodium borohydride solution and detected under the same chromatographic conditions and wavelength, and the absorption peak of the target disappeared at the original retention time, confirming that it was coenzyme Q10.

 

Fig. 6 Chromatogram of sample (crude extract) analyzed by high performance liquid chromatography (HPLC)

 

3 Summary

(1) By comparing the extraction and isolation methods of coenzyme Q10, it was determined that the method of collecting the logarithmic-phase organisms was to rotate at 6,000 r/min for 15 min; and the method of ultrasonic treatment of acetone suspension was adopted, and the extraction time was 5 h. The extraction time was 5 hours.

 

(2) The oxidative damage of coenzyme Q10 should be avoided during the separation and extraction process, especially under alkaline conditions, and antioxidants, such as pyrogallic gallic acid, should be added if necessary; the samples to be tested should be fresh or stored at low temperatures. By comparing the effects of different organic solvents on the extraction yield, the extraction method with relatively low toxicity, low cost and high extraction rate was selected; the HPLC analytical method was used to identify and confirm that the ideal fermentation method was identified method for the isolation and extraction of Coenzyme Q10 .

 

(3) Compared with HPLC quantitative analysis method, TLC-UV method is a more practical method for quantitative determination of coenzyme Q10 with simpler method, shorter operation time, lower cost, smaller relative error, and simple sample processing.

 

References:

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［2］MORTENSEN S A, VADHANAVIKIT S, FOLKERS K .  Deficiency of coenzyme Q10 in myocardial failure [J].  Drugs Exptl Clin Res, 1984, (7): 497 - 502 .

[3] KRIVANKOVA L, DADAK V .  Methods in enzymology [M]. London: Academic Press Inc, 1980 .   [4] WANG Chunlin . Extraction, isolation and characterization of soybean coenzyme Q10 in China［J］.  Chinese Journal of Pharmaceutical Industry, 1996, 27(3):102 - 104 . [5] Zhuang Xiaolei, Yu Shuhong.  Quantitative determination of paclitaxel by TLC-ultraviolet spectrophotometry［J］.  Biotechnology, 2001, 11(1): 45 - 47 .

［6］MIKATA K, yAMADA .  The ubiquinone system in Hasegawaea japonica (yukaw et Maki) yamada et Banno: A new method for identifying ubiquinone homologs from yeast cells [J].  IFO Res C0mm, 1999, 19:41 - 46 .