Additive impact of soy protein dietary intake and exercise on visceral fat mass reduction and mitochondrial complex I activation in skeletal muscle.

Soy protein has shown remarkable effectiveness in reducing fat mass compared with other protein sources, and exercise has the potential to further enhance this fat loss effect. Previous studies have demonstrated that soy protein intake leads to decreased fatty acid synthesis, which contributes to its fat-loss properties. However, the exact mechanism by which these lipids are consumed remains unclear. To investigate this, we conducted a comprehensive study using C57/BL6 male mice, comparing the effects of soy and casein proteins with and without exercise (Casein-Sed, Casein-Ex, Soy-Sed, and Soy-Ex groups) under high- and low-protein conditions (14% or 40% protein). Our findings revealed that combining soy protein intake with exercise significantly reduced epididymal white adipose tissue (eWAT) weight, particularly in the high-protein diet group. Further analysis revealed that exercise increased the expression of lipid oxidation-regulatory proteins, including mitochondrial oxidative phosphorylation protein (OXPHOS) complexes, in the plantaris muscle regardless of the protein source. Although soy protein intake did not directly affect muscle mitochondrial protein expression, the activity of OXPHOS complex I was additively enhanced by exercise and soy protein under the 40% protein condition. Notably, complex I activity inversely correlated with eWAT weight in the soy protein diet group. These results highlight the potential link between improved complex I activity induced by soy protein and fat mass reduction, which emphasizes the promising benefits of combining soy protein with exercise in promoting fat loss.NEW & NOTEWORTHY The findings revealed that soy protein intake combined with exercise resulted in reduced adipose tissue weight compared with that obtained with casein protein intake. Furthermore, the joint impact of exercise and soy protein consumption resulted in enhanced activity of oxidative phosphorylation protein (OXPHOS) complex I in fast-twitch muscles, which appears to be associated with fat mass reduction. These findings elucidate the potential additive effects of soy protein and exercise on body weight management.

Standardization and validation of novel ex-vivo method for mitochondrial bioenergetics using mitochondrial modulators.

Mitochondria is an essential organelle; it produces 95% of the adenine triphosphate (ATP) of cells, their dysfunction is related to the pathogenesis of multiple diseases, such as diabetes mellitus, cardiovascular and neurological disorders. Various pharmacologic agents are known to target mitochondrial function. Moreover, the toxic side effects of multiple drugs used to treat diseases are related to the impairment of mitochondrial function. Thus, there is a need to develop a method to evaluate the effect of pharmacologic agents for their potential and side effects to identify effective mitochondrial-modulating agents. Therefore, the objective of this study was to develop and validate an ex-vivo method for studying the effect of pharmacologic agents on mitochondrial function and rescue of dysfunction. Dimethyl sulfoxide (DMSO) concentrations that drugs were soluble in and maintained mitochondrial function were determined. Metformin (MET) is a known mitochondrial complex-1 inhibitor tested for its ability to compromise mitochondrion function. Coenzyme Q10 (Q10) and Resveratrol (RSV), which are known to enhance mitochondrial function, were added alone and dose-dependent, tested for the ability to rescue metformin-induced mitochondrial dysfunction. Ex-vivo liver and brain mitochondrial function was assessed using an oxytherm Clark-type oxygen electrode. DMSO was found to be toxic above 10% and drugs insoluble below 5%. The addition of 0.5 mg/ml MET decreased liver and brain mitochondrial respiratory control rate (RCR). At the same time, Q10 improved RCR in normal mitochondria and a concentration-dependent manner in MET-induced dysfunctional mitochondria. RSV was added in the last step of the experiment to confirm that compromised function is due to MET. Hence this method can be used to screen pharmacological agents for their potential therapeutics or toxic effect on mitochondria.

Effect of human umbilical cord mesenchymal stem cell exosomes on aerobic metabolism of human retinal pigment epithelial cells.

PURPOSE: To investigate the effect of exosomes secreted by human umbilical cord mesenchymal stem cells (HUCMSC-Exo) on aerobic metabolism of cobalt chloride (CoCl2)-induced oxidative damage in the human retinal pigment epithelial cell line (ARPE-19), and to explore the protective mechanism of HUCMSC-Exo on oxidative damage in ARPE-19 cells. METHODS: HUCMSC-Exo were extracted and identified; CCK-8 assay was used to established the oxidative damage mode of ARPE-19 cells induced by CoCl2; JC-1 flow cytometry was used to detect the effects of exosomes with different concentrations (0, 25, 50, or 100 µg/mL) on the mitochondrial membrane potential (MMP) of oxidatively damaged ARPE-19 cells. The effects of exosomes with different concentrations on the activity of oxidative metabolic enzymes (oxidative respiratory chain complexes I, III, IV, and V) and ATP synthesis in oxidatively damaged ARPE-19 cells were detected by spectrophotometry. RESULTS: Under transmission electron microscope, HUCMSC-Exo were round or oval membrane vesicles with diameters of about 40-100 nm. Western blot results showed that HUCMSC-Exo expressed specific marker proteins CD63 and CD81. CCK-8 dates showed that the cell viability of ARPE-19 cells was significantly decreased with increasing CoCl2 concentration, and the concentration of 400 µmol/L CoCl2 was chosen to be the optimal concentration for oxidative damage. MMP was increased in exosomes intervention group (25, 50 or 100 µg/mL), and the dates were statistically different from 0 µg/mL exosome intervention group (P < 0.05). The activities of mitochondrial complexes I, IV, and V in exosomes intervention groups (100 µg/mL) were higher than those in 0 µg/mL exosome intervention group. In 50 µg/mL and 100 µg/mL exosome intervention group, ATP synthesis was significantly different from the 0 µg/mL exosome intervention group (P < 0.05). CONCLUSION: HUCMSC-Exo had a certain protective effect on ARPE-19 cells induced by CoCl2 in vitro. The protective mechanism of HUCMSC-Exo on oxidative damage ARPE-19 cells might be through saving its aerobic metabolic function, restoring cell ATP synthesis, and improving the ability of cells to repair damage and deal with the hypoxic environment.

Peripheral blood mononuclear cell mitochondrial enzyme activity is associated with parity and lactation performance in early lactation Holstein dairy cows.

Mitochondria are central to metabolism and are the primary energy producers for all biosynthesis, including lactation. The objectives of this study were to determine if high- and low-producing dairy cows exhibit differences in peripheral blood mononuclear cell mitochondrial enzyme activities of citrate synthase, complex I, complex IV, and complex V during early lactation and, thus, to determine whether those differences were related to differences in lactation performance in the dairy cow. Fifty-six Holstein cows were assigned to 1 of 4 groups: (1) primiparous high, (2) primiparous low, (3) multiparous high, or (4) multiparous low. Primiparous and multiparous cows were analyzed separately. Then, cows were divided into high or low production groups for each production parameter [peak milk, average milk, energy-corrected milk (ECM), fat-corrected milk (FCM), milk lactose, milk fat, milk protein, total solids (TS), solids-not-fat, feed efficiency, and somatic cell count (SCC)]. For all data analysis, production parameters are expressed as yields (kg/d) and SCC (103 cells/mL). High and low production groups were defined by their respective mean production parameters for the 56 cows, with below average cows defined as low and above average cows defined as high. Whole blood samples were collected at one time point, approximately 70 d in milk at 0800 h, and processed for crude mitochondrial extracts from peripheral blood mononuclear cells to determine the activity rates of mitochondrial enzymes. Milk samples were collected 9 times (3 d, 3 times per d) during the week of blood collection and analyzed for major components (fat, protein, lactose, TS, and SCC). Multiparous cows had lower citrate synthase activity than primiparous cows across all production parameters. High-producing cows had greater complex I activity for peak milk, milk yield, ECM, FCM, milk fat, TS, and feed efficiency, and greater complex V activity for ECM, FCM, milk lactose, milk fat, and TS across parities. These findings imply that the most influential respiratory chain enzymes on the level of milk production are those responsible for electron transport chain initialization and ATP production.

Preparation of low-molecular-weight chondroitin sulfates by complex enzyme hydrolysis and their antioxidant activities.

Chondroitin sulfate (CS) has attracted widespread attention because of its numerous pharmacological activities. Low-molecular-weight chondroitin sulfates (LMWCSs) derived from the degradation of CS are reported to have better biological properties than whole CS. In this study, to obtain LMWCSs with high antioxidant activity, we depolymerized CS using complex enzymes, namely, chondroitinase ABC I (ChSase ABC I) and ChSase ABC II. The conditions of the complex enzyme hydrolysis (CEH) were optimized, and the structures and antioxidant activities of CS and LMWCSs were investigated. The results showed that the CEH conditions enhanced the antioxidant activities of the products as compared to CS. The basic structures of the LMWCSs and sulfate groups were well preserved after hydrolysis. Therefore, CEH provides an efficient and safe strategy to obtain LMWCSs, which can be used in antioxidant drugs, healthy foods, and cosmetics.

Properties of a Non-canonical Complex Formed Between a Tepary Bean (Phaseolus acutifolius) Protease Inhibitor and α-Chymotrypsin.

Protease inhibitors are crucial for the control of proteolytic activity in different physiological processes. However, some inhibitors do not show canonical enzyme recognition of the enzyme under certain conditions. In this work, we present evidence that indicates the formation of an active complex between the protease bovine α-chymotrypsin and the Tepary bean protease inhibitor (TBPI). The composition of the active chymotrypsin-TBPI complex (AC) was confirmed by three different methods: size-exclusion chromatography, polyacrylamide gel electrophoresis (PAGE), and mass spectrometry. The kinetic parameters for the AC were similar to those of the enzyme alone, indicating that TBPI binding does not produce any large changes in chymotrypsin. The molecular model proposed here postulates that TBPI binds outside the active cleft of the protease, but near enough to hinder the binding of high molecular weight substrates into the active site. This model was experimentally supported by the inhibitory effect on casein as a substrate, and the unaltered protease activity when a small synthetic substrate was used. We also found that the formation of this complex provided the enzyme with extra stability in denaturing conditions or in the presence of a reducing agent. The chymotrypsin-TBPI complex exhibited higher stability, indicating that autolysis can be partially prevented. When the enzyme was first inactivated followed by the addition of the inhibitor, the activity of the protease was restored. We described a possible mechanism where a plant protease inhibitor binds outside the active site of the enzyme while increasing its stability.

The effects of relative gain and age on peripheral blood mononuclear cell mitochondrial enzyme activity in preweaned Holstein and Jersey calves.

Mitochondria are central to metabolism, nutrition, and health, but many factors can influence their efficiency. The objectives of this study were to determine if the mitochondrial enzyme activities of citrate synthase, complex I, complex IV, and complex V from peripheral blood mononuclear cells in Holstein and Jersey dairy calves were affected by age or relative gain as a percent of initial weight. Twenty-three Holstein and 23 Jersey heifer calves were enrolled between 3 and 6 d of age and whole blood samples were collected via jugular venipuncture at 1, 2, and 8 wk of age. Crude mitochondrial extracts were obtained from the peripheral blood mononuclear cell fraction at each time point and subsequently assayed for enzymatic activity. Age-dependent changes in activity were observed in complex V for both breeds. In Jersey calves complex IV and citrate synthase activity differed with age. Complex I activity was greater for high relative gain Jerseys and tended to be greater for high relative gain Holstein calves. Holstein calves had greater incidence of scours compared with Jersey calves, and in both breeds scouring calves exhibited greater complex V activity compared with those without scours. These data suggest that age and immune challenge in the form of scours affect mitochondrial complex V activity. Additionally, complex I activity may serve as a marker for calf growth potential because it was influenced by relative gain and not age.

Study on content of polysaccharide during processing of Polygonatum sibiricum by RSM and PMP-HPLC characteristic map technology / 中草药

Objective: To investigate the best extracting conditions of Polygonatum sibiricum polysaccharides by single and compound enzymatic methods, and to study on the change content of polysaccharide in processing of P. sibiricum by steamed and shined for nine times, which provide theoretical basis for processing of P. sibiricum and other Chinese herbal medicines. Methods: Response surface method (RSM) was used to optimize the extraction of polysaccharides in P. sibiricum. PMP precolumn derivatization and HPLC characteristic map technology were used to identify and analyze the changes of polysaccharide of P. sibiricum being steamed and shined for nine times and establish polysaccharide characteristic map of P. sibiricum by ancient processing and distillate. The effect of different enzymes on the extraction efficacy of P. sibiricum polysaccharides was also compared. Results: The extracting effect of cellulase in single enzyme was the best. Moreover, cellulose and pectinase were also the most significant factor for extracting polysaccharides from P. sibiricum in four complex enzymes. The best extracting conditions for polysaccharide obtaining by RSM as follows: Cellulase temperature was 51 ℃, cellulase dosage was 2%, and the total polysaccharide content was (675.34 ± 0.10) mg/g when the pH value of enzymolysis was 5.40. PMP-HPLC was used to detect four processed P. sibiricum, which contained mannose, ribose, rhamnose, glucose, and galactose. Glucose had a high proportion in P. odoratum, P. cyrtonema, and P. sibiricum polysaccharide, and P. filipes polysaccharide mainly composed of mannose. With the increase of processed times, the content of each monosaccharide increased first, then decreased, then increased and then tended to be stable. The content of mannose and galactose varied greatly. The content of monosaccharides in the distillate increased first and then stabilized, and the content changes of mannose and glucose were obvious. Conclusion: The extraction efficiency and purity of P. sibiricum polysaccharides can be improved by single and compound enzymatic extraction with cellulase, and the extraction yield of polysaccharide in P. sibiricum could be affected by steamed and shined for nine times. PMP-HPLC characteristic map technology can be used to determine the content of the polysaccharide in processing of P. sibiricum accurately and reproducibly, which can be used for the quality evaluation of P. sibiricum and other Chinese herbal medicines.

Preparation and characterization of the spherical nanosized cellulose by the enzymatic hydrolysis of pulp fibers.

In this work, the pulp fibers were enzymolyzed to prepare the nanosized cellulose (NC). The as-prepared samples were characterized by optical microscopy, electron microscopy, and Raman spectra. The experimental results indicated that enzymatic hydrolysis of pulp fibers could produce the spherical NC with a mean particle size of about 30nm, which had the excellent monodispersity and uniformity. When the concentration of complex enzymes was 20u/mL (cellulase: xylanase=9: 1), the yield of NC was 13.6%. The single cellulase was used, even if the enzyme concentration reached up to 200u/mL, only a mixture of strip and granular flocculation were obtained. The positive synergistic effect between xylanase and cellulase could be due to the enzymolysis of hemicellulose located on the cellulose microfibers to be favorable of cutting and splitting of the microfibers by the endoglucanase in cellulase.

Fermentation and complex enzyme hydrolysis enhance total phenolics and antioxidant activity of aqueous solution from rice bran pretreated by steaming with α-amylase.

In this study, rice bran was successively steamed with α-amylase, fermented with lactic acid bacteria, and hydrolyzed with complex enzymes. The changes in phenolic profiles and antioxidant activities of the corresponding aqueous solutions from three stages were investigated. Compared to the first stage, fermentation and complex enzyme hydrolysis significantly increased the total phenolics, total flavonoids, total FRAP and ORAC values by 59.2%, 56.6%, 73.6% and 45.4%, respectively. Twelve individual phenolics present in free or soluble conjugate forms were also analyzed during the processing. Ferulic acid was released in the highest amount among different phenolics followed by protocatechuic acid. Moreover, a major proportion of phenolics existed as soluble conjugates. The results showed that fermentation and complex enzyme hydrolysis enhanced total phenolics and antioxidant activities of aqueous solution from rice bran pretreated by steaming with α-amylase. This research could provide basis for the processing of rice bran beverage rich in phenolics.

Complex enzyme hydrolysis releases antioxidative phenolics from rice bran.

In this study, phenolic profiles and antioxidant activity of rice bran were analyzed following successive treatment by gelatinization, liquefaction and complex enzyme hydrolysis. Compared with gelatinization alone, liquefaction slightly increased the total amount of phenolics and antioxidant activity as measured by ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC) assays. Complex enzyme hydrolysis significantly increased the total phenolics, flavonoids, FRAP and ORAC by 46.24%, 79.13%, 159.14% and 41.98%, respectively, compared to gelatinization alone. Furthermore, ten individual phenolics present in free or soluble conjugate forms were also analyzed following enzymatic processing. Ferulic acid experienced the largest release, followed by protocatechuic acid and then quercetin. Interestingly, a major proportion of phenolics existed as soluble conjugates, rather than free form. Overall, complex enzyme hydrolysis releases phenolics, thus increasing the antioxidant activity of rice bran extract. This study provides useful information for processing rice bran into functional beverage rich in phenolics.

Preparation of rat thoracic aortic vascular smooth muscle cells by modified enzyme double digestion / 中国比较医学杂志

Objective This paper reports a modified method for the isolation of rat thoracic aortic vascular smooth muscle cells based on combined enzyme double digestion.Methods An enzyme mixture containing collagenase II, soybean trypsin inhibitor and elastase was prepared and used to remove the aortic tunica intima and tunica adventitia, and then the tunica media was subjected to second digestion using the same enzyme mixture and to isolate vascular smooth muscle cells.Results The isolated VSMCs were cultured in vitro and the growing cells had an elongated spindle-shape, reached confluence within a week, and displaying a typical hill-and-valley pattern.After 1 week, the cells were passaged.Contractile smooth muscle markers(α-SMA, myosin-II and β-tubulin) were highly expressed in the isolated cells.More than 90% of the cells significantly expressed alpha smooth muscle actin and myosin-II.Conclusions Themethod established in this study has advantages of simple and easy to operate, and good reproducibility, with a high activity and purity of the separated cells.It can ensure to obtain a large amount of contraction-type vascular smooth muscle cells within a short time.

Clinical Effect of Four Oral Vitamins Combined with FE Complex Enzyme Rash on Recurrent Oral Ulcer and Serum Inflammatory Factors Levels / 现代生物医学进展

Objective:To explore the clinical effect of four oral vitamins [vitamin E (Vit E) +folic acid (FA)+ vitamin B2 (Vit B2) + vitamin B12 (Vit B12)] combined with FE complex enzyme rash on the recurrent oral ulcer (ROU) and on the serum inflammatory factors levels.Methods:126 cases with ROU in our hospital from January 2014 to February 2016 were selected as research objectives and randomly divided into two groups.FE complex enzyme was provided to the control group,while FE complex enzyme and four Vietnam (Vit E+FA+Vit B2+Vit B12) were given to the observation group.The local efficacy,long-term efficacy,serum inflammatory factor levels before and after treatment as well as the incidence of adverse drug reactions were recorded and compared between two groups.Results:Compared with the control group,the pain index was significantly decreased on 30th treating day in the observation group(P＜0.01),and the average ulcer period was shorten (P＜0.01).After being treated for 6 months,the overall effective rate was 95.2％ in the observation group,which was significantly higher than that of the control group (81.0％,P＜0.05).The serum TNF-α and IL-17 levels on the 30th treating day was significantly lower in both groups after treatment than those before treatment (P＜0.01),but the serum 1L-2 level was significantly increased (P＜0.01),and the improvement of each above inflammatory factors in the observation group were more significant than those of the control group (P＜0.01).No significant difference was found in the incidence of adverse reactions between two groups (P＞0.05).Conclusions:Four oral Vietnam combined with FE complex enzyme could promote the ulcer wound healing,reduce the pain,regulate the body to promote/anti-inflammatory factor balance and improve the long-term efficacy in the treatment of patients with ROU with high safety.

Optimization of total sugar extraction from Cynomorii Herba by using quadratic orthogonal rotation regression combination design / 中草药

Objective: To optimize the extraction processes used for enzymatic hydrolysis of total sugar from Cynomorii Herba (Cynomoriumsongaricum) by employed quadratic orthogonal rotation regression combination design. Methods: Taking the extraction rate of total sugar as index, as well as enzyme ratio, enzyme concentration, extraction temperature, extraction time, and initial pH value as investigation factors to screen out main factors. The quadratic orthogonal rotation regression combination design was applied to optimize the process. And then, to employ response surface analysis method (RSM) for evaluating the impacts on the response values of the single factor effect and interaction effect of each factor level. Results: The optimum conditions were as follows: enzyme concentration was 1% (wt% of Cynomorii Herba powder), initial enzyme solution pH was 5.5, extraction temperature was 55℃, extraction time was 45 min, and pectinase and cellulose was 1∶1 proportion. The extraction yield of total sugar was (17.54 ± 0.35) %. The influence of response values of each factor presented the relationship of the parabolic surface with the opening downward. The results showed that the impact degree on total sugar yield of Cynomorii Herba were extraction temperature, extraction time, and enzyme ratio, respectively. Conclusion: This method is a simple and effective method to increase the extraction yield of total sugar from Cynomorii Herba. Quadratic orthogonal rotation regression combination design has a favourable predictability.

Decreased mitochondrial bioenergetics and calcium buffering capacity in the basal ganglia correlates with motor deficits in a nonhuman primate model of aging.

Altered mitochondrial function in the basal ganglia has been hypothesized to underlie cellular senescence and promote age-related motor decline. We tested this hypothesis in a nonhuman primate model of human aging. Six young (6-8 years old) and 6 aged (20-25 years old) female Rhesus monkeys (Macaca mulatta) were behaviorally characterized from standardized video records. Additionally, we measured mitochondrial bioenergetics along with calcium buffering capacity in the substantia nigra and putamen (PUT) from both age groups. Our results demonstrate that the aged animals had significantly reduced locomotor activity and movement speed compared with younger animals. Moreover, aged monkeys had significantly reduced ATP synthesis capacity (in substantia nigra and PUT), reduced pyruvate dehydrogenase activity (in PUT), and reduced calcium buffering capacity (in PUT) compared with younger animals. Furthermore, this age-related decline in mitochondrial function in the basal ganglia correlated with decline in motor function. Overall, our results suggest that drug therapies designed to enhance altered mitochondrial function may help improve motor deficits in the elderly.

Extraction optimization, preliminary characterization and immunological activity of polysaccharides from figs.

Complex enzyme extraction (CEE), purification, characterization of fig polysaccharides (FPs) from dried figs were investigated. Orthogonal experiment was used to optimize the concentration of cellulose, pectinase and papain. Response surface methodology (RSM) was employed to optimize extraction conditions. The optimum extraction conditions were: enzyme concentration of 1.5%, 1.5%, 0.5% (wt%) of pectinase, papain, cellulose, ratio of water to raw material 40.3 mL/g, extraction time 54.1 min, temperature 34.15 °C and pH 3.8. Under these conditions, the experimental yield was 7.98 ± 0.17%. Two homogeneous heteropolysaccharides (FPs-1-1, FPs-2-1) were purified by DEAE-Sepharose and Sephadex G-200 chromatography, which were composed of rhamnose, arabinose, xylose, mannose, glucose and galactose with molecular weight of 1.52 × 10(6) and 4.75 × 10(5)Da, respectively. The bioactivity assay showed that FPs-1-1 and FPs-2-1 could more significantly enhance splenic lymphocyte proliferation, phagocytosis and NO production of macrophages, could be explored as potential immunopotentiating agent for use in functional food or medicine.

Effects of maize source and complex enzymes on performance and nutrient utilization of broilers.

The objective of this study was to investigate the effect of maize source and complex enzymes containing amylase, xylanase and protease on performance and nutrient utilization of broilers. The experiment was a 4×3 factorial design with diets containing four source maize samples (M1, M2, M3, and M4) and without or with two kinds of complex enzyme A (Axtra XAP) and B (Avizyme 1502). Nine hundred and sixty day old Arbor Acres broiler chicks were used in the trial (12 treatments with 8 replicate pens of 10 chicks). Birds fed M1 diet had better body weight gain (BWG) and lower feed/gain ratio compared with those fed M3 diet and M4 diet (p<0.05). Apparent ileal crude protein digestibility coefficient of M2 was higher than that of M3 (p<0.05). Apparent metabolisable energy (AME) and nitrogen corrected AME (AMEn) of M1 were significant higher than those of M4 (p<0.05). Supplementation of the basal diets with enzyme A or B improved the BWG by 8.6% (p<0.05) and 4.1% (p>0.05), respectively. The fresh feces output was significantly decreased by the addition of enzyme B (p<0.05). Maize source affects the nutrients digestibility and performance of broilers, and a combination of amylase, xylanase and protease is effective in improving the growth profiles of broilers fed maize-soybean-rapeseed-cotton mixed diets.