Small angle neutron scattering (SANS) was used to study a series of concentrated protein solutions containing horse heart cytochrome C ranging from 0.6 to 24 g/dL at two ionic strengths. Since the solutions were manifestly nonideal throughout the whole concentration range, a contrast variation method applicable at finite Q (magnitude of the scattering vector) was applied to extract three basic properties of the protein in mixed D 2O/H 2O solutions at a concentration of 12 g/dL.

These properties are the dry volume, the hydration, and the amount of H/D exchange of the protein in D 2O‐containing solvents. These three basic parameters were then used as an input to extract the protein charge at all other concentrations. A generalized one‐component macroion theory (GOCM) was used to compute the interparticle structure factor for a given protein charge, Z p. The Z p ’s so obtained are in good agreement with a separate titration experiment which determines the.

(or biochemical energetics or bioenergetics, as it is also called) is the field of biochemistry concerned with the transformation and use of energy by living cells. Academic Press, Inc., New. Segel IH: Biochemical Calculations. 2nd ed., Wiley-Interscience, New York. Stumpf PK: ATP. Get Instant Access to eBook Biochemical Calculations Segel Pdf PDF. New York Wiley 1976 441 p. Biochemical calculations irwin h segel john.

Results cis-2-methoxycinnamic acid (43.06%) and cinnamaldehyde (42.37%) were found to be the two major components of the CC-EO identified by gas chromatography–mass spectrometry (GC-MS). The inhibitory activities of CC-EO and its major constituents were further evaluated against mushroom tyrosinase. The results showed that CC-EO and cinnamaldehyde exhibited anti-tyrosinase activities with IC 50 values of 6.16 ± 0.04 mg/mL and 4.04 ± 0.08 mg/mL, respectively. However, cis-2-methoxycinnamic acid did not show any anti-tyrosinase activity. The inhibition kinetics were analyzed by Lineweaver-Burk plots and second replots, which revealed that CC-EO and cinnamaldehyde were mixed-type inhibitors.

The inhibition constants (Ki) for CC-EO and cinnamaldehyde were calculated to be 4.71 ± 0.09 mg/mL and 2.38 ± 0.09 mg/mL, respectively. Essential oils The essential oil, which was obtained by steam distillation from the stem bark of C. Cassia Presl, was purchased from Yangsen Biotech, Inc. (Taipei, Taiwan). The extraction procedure is according to previous study with slight modifications (Choi et al.

Cassia stem bark was place into vessel and extracted by steam distillation for 4 hours. The vapors were cooled by a closed cooling system and the liquid were collected in a container. The oils floated towards the top while the water settled below and the essential oils were obtained by simply removing the oils which were separated. Enzymatic assay of tyrosinase Tyrosinase inhibitory activities of the CC-EO and its major constituents were determined by the tyrosinase-dependent L-3,4-dihydroxyphenylalanine (L-DOPA) oxidation assay according to a slight modification of the method of Kubo and Kinst-Hori (). The substrate solution (0.84 mL of 0.89 mM L-DOPA in 16 mM sodium phosphate buffer, pH 6.8) was incubated at 25°C for 10 min. Following incubation, 0.03 mL of each sample solution and 0.03 mL of mushroom tyrosinase (1000 units/mL; T7755, Sigma, one unit = ΔOD 280 of 0.001 per min at pH 6.5 at 25°C in 3 mL reaction mixture containing L-tyrosine) were added. The assay mixture in a total volume of 0.9 mL was immediately monitored for the formation of dopachrome by measuring the linear increase in optical density at 475 nm.

Kojic acid, which is known to inhibit tyrosinase (Chen et al. ), was used as a positive control.

The inhibitory percentage of tyrosinase was calculated as follows:% inhibition = (1-B/A) × 100, where A = ΔOD 475/min without tested sample and B = ΔOD 475/min with tested sample. The 50% inhibition (IC 50) of tyrosinase activity was calculated as the concentrations of the tested sample that inhibited 50% of tyrosinase activity under experimental conditions.

Kinetic analysis The reaction mixture consisted of 0.3 mL of L-DOPA (0.75-3 mM) as a substrate, 0.54 mL of 25 mM sodium phosphate buffer (pH 6.8), 0.03 mL of mushroom tyrosinase (1000 units/ml) in 25 mM sodium phosphate buffer (pH 6.8), and 0.03 mL of each sample solution (0–12.50 mg/mL for CC-EO; 0–6.25 mg/mL for trans-cinnamaldehyde) in a total volume of 0.9 mL was assayed at 25°C, as described above. The inhibitory kinetics of each sample with tyrosinase were analyzed using Lineweaver-Burk plots. The reciprocal equation for a rapid equilibrium approach from the mixed-type noncompetitive inhibition can be expressed as eq 1 (Segel ). The K i for CC-EO and cinnamaldehyde were calculated from the slope replots ( eq 2). The αK i for CC-EO and cinnamaldehyde were calculated from the 1/ v axis intercept replots ( eq 3). Eq 1, 1 v = K s V max 1 + I K i 1 S + 1 V max 1 + I α K i; eq 2, Slope = K s V max + K s V max K i I and eq 3, 1 v axis intercept = 1 V max α Κ i I + 1 V max, where K s is the dissociation constant of substrate (S) from enzyme-substrate complex (ES), K i is the dissociation constant of inhibitor (I) from enzyme-inhibitor complex (EI), and αK i is the dissociation constant of inhibitor from enzyme-substrate-inhibitor complex (ESI). GC-MS analysis GC-MS analyses were carried out on a GCMS-QP-2010 plus Gas chromatograph Mass Spectrometer (Shimadzu, Japan) and GCMS-solution software (v.

2.50 SU3, Shimadzu, Japan). Compounds were separated on a Forte ID-BPX5 cross-linked 5% phenyl - 95% methyl polysiloxane (30 m × 0.25 mm i.d., film thickness 0.25 μ m) capillary column (SGE, AU). The column was maintained at 50°C for 5 min after injection then programmed at 5°C/min to 150°C, then programmed at 10°C/min to 300°C. The injection volume was 1.0 μl of pure essential oil, with a split ratio of 1:100. Helium was used as the carrier gas at a constant flow-rate of 1.0 ml/min. Injector, transfer line and ion-source temperatures were 250, 230 and 250°C, respectively.

MS detection was performed with electron impact mode at 70 eV ionization energy and 60 μ A ionization current, by operating in the full-scan acquisition mode in the 40–350 amu range. Compounds were identified by comparing the retention times and retention indices of the chromatographic peaks with those of authentic reference standards run under the same conditions.

Peak enrichment on co-injection with authentic reference compounds was also carried out. The comparison of the MS fragmentation pattern with those of pure compounds and mass spectrum database search was performed by using the National Institute of Standards and Technology (NIST) MS spectral database (version 2005). Statistical analysis All the assays to determine enzyme activity (i.e., the tyrosinase inhibitory effect of CC-EO, trans-cinnamaldehyde, cis-2-methoxycinnamic acid and kojic acid and the enzyme kinetics) were conducted at least three times with three different sample preparations. All data were expressed as mean ± standard deviation (S.D.). Analysis of variance (ANOVA) was performed using SPSS (SPSS Inc., U.S.A.).

A one-way ANOVA and Scheffe test were used to determine the difference of means, and p. Chemical composition of CC-EO The chemical composition of CC-EO was analyzed using GC-MS. The 16 constituent compounds identified, along with the retention times and Kovats indices, are listed in Table. Our results showed that the two major constituents of CC-EO were cis-2-methoxycinnamic acid (43.06%) and cinnamaldehyde (42.37%) and that the minor compounds were o-methoxycinnamaldehyde (5.11%), 1,2-dimethoxy-4-(3-methoxy-1-propenyl) benzene (2.05%), cinnamyl acetate (1.83%) and other compounds (1.25~0.16%) in the present study.

Effect of CC-EO, trans-cinnamaldehyde and cis-2-methoxycinnamic acid on the activity of mushroom tyrosinase The effect of CC-EO, trans-cinnamaldehyde and cis-2-methoxycinnamic acid on the oxidation of L-DOPA catalyzed by mushroom tyrosinase as well as that of kojic acid which is a well-known tyrosinase inhibitor were studied. As shown in Figure, it was found that CC-EO and trans-cinnamaldehyde had potent inhibitory effects on the L-DOPA oxidase activity of mushroom tyrosinase in a dose-dependent manner. The results showed that trans-cinnamaldehyde had higher tyrosinase inhibitory activity than CC-EO. Based on the half-inhibition concentration (IC 50) (Table ), trans-cinnamaldehyde is approximately 1.5 times more effective than CC-EO.

However, cis-2-methoxycinnamic acid had no inhibitory effects on the mushroom tyrosinase in this study (data not shown). Then, we had compared the tyrosinase inhibitory activity of the CC-EO with a well-known tyrosinase inhibitor, kojic acid. The IC 50 value of the tyrosinase inhibitory activity of kojic acid was determined to be 0.22 mg/mL which was found to be significantly more pronounced than those of CC-EO (6.16 mg/mL) and trans-cinnamaldehyde (4.04 mg/mL).

To obtain 80% tyrosinase inhibitory activity, the concentrations needed for CC-EO, trans-cinnamaldehyde, and kojic acid were 51.35, 16.65, and 0.96 mg/mL, respectively. Inhibition type of CC-EO and trans-cinnamaldehyde on the activity of mushroom tyrosinase The kinetic behaviors of CC-EO, on the mushroom tyrosinase for the oxidation of L-DOPA were first studied. Under the experimental conditions, the Michaelis constant ( K m) and maximum velocity ( V max) of the L-DOPA oxidation reaction catalyzed by the tyrosinase (30 units) were 0.80 mM and 0.469 ΔOD 475/min, respectively. The inhibitory kinetics of CC-EO were analyzed using Lineweaver-Burk double reciprocal plots as shown in Figure A. The five lines, obtained from the uninhibited enzyme and from four different concentrations of CC-EO, intersected to the left of the 1/ v axis above the 1/ S axis. Increased concentrations of CC-EO resulted in decreased V max and an increased K m.

These results indicate that CC-EO exhibited mixed-I type inhibition for the oxidation of L-DOPA catalyzed by mushroom tyrosinase. Similar results were obtained with trans-cinnamaldehyde (Figure A), showing that it was also a mixed-I type inhibitor for the enzyme. The dissociation constants for inhibitor binding with the free enzyme and the enzyme-substrate complex, K i and αK i, were obtained from the double-reciprocal plots and the replots of the slope and the vertical intercept versus the concentration of CC-EO (Figure B, C ) or trans-cinnamaldehyde (Figure B, C ), respectively. The obtained values are summarized in Table. Discussion The essential oil extracted by steam distillation from the stem bark of C. Cassia Presl was quantitatively analyzed by GC-MS. Cis-2-Methoxycinnamic acid and cinnamaldehyde were determined as the major compounds of the oil (43.06% and 42.37%, respectively).

For comparison, a previous study reported that cinnamaldehyde (92.2%) was the most plentiful constituent in the C. Cassia essential oil (Giordani et al.

Different extraction processes and assay methods could have contributed to differences in cinnamaldehyde levels of C. Cassia essential oils (Dugoua et al.

Cinnamaldehyde (77.1%) was also found to be the major constituent of volatile oil of the bark of C. Zeylanicum (Marongiu et al. Several studies have demonstrated that cinnamaldehyde has antimicrobial (Ooi et al. ), antimutagenic (Shaughnessy et al.

), antitumorigenic (Ka et al. ) and immunomodulatory activities (Koh et al. ); furthermore, cinnamaldehyde is considered to possess tyrosinase-inhibitory effects with IC 50 values from 0.52~0.97 mM (Lee; Lee et al.; Ngoc et al. Lee () reported that 2-methoxycinnamic acid that had been isolated from Pulsatilla cernua root was a potent noncompetitive inhibitor of mushroom tyrosinase with an IC 50 value of 0.34 mM. In addition, benzaldehyde, one of the flavor compounds characterized in anise oil, showed potent tyrosinase inhibitory activity with an IC 50 of 0.82 mM (Kubo and Kinst-Hori ). Because the CC-EO contained major in cinnamaldehyde and cis-2-methoxycinnamic acid, and trace readings of benzaldehyde, however, there is no report directly evaluating the tyrosinase inhibitory activities of CC-EO. Mushroom tyrosinase has been widely used as a target enzyme in screening and characterizing potential tyrosinase inhibitors.

Because the inhibition mode depends on the structures of both the substrate and inhibitor, L-DOPA has been used as the substrate in this study. Therefore, the activity studied in this paper was the o-diphenolase inhibitory activity of mushroom tyrosinase. The results showed that CC-EO and its major constituent, cinnamaldehyde, showed a dose-dependent anti-tyrosinase effect, however no activity was observed for the other major constituent, cis-2-methoxycinnamic acid. Because CC-EO contains 42.37% of cinnamaldehyde, it is suggested that cinnamaldehyde is responsible for the tyrosinase inhibitory activity of CC-EO.

Although pure cis-2-methoxycinnamic acid had no tyrosinase inhibitory activity in our study, Lee () reported that 2-methoxycinnamic acid that had been isolated from Pulsatilla cernua root was a potent noncompetitive inhibitor of mushroom tyrosinase with an IC 50 value of 0.34 mM. The discrepancy of these results may be due to different assay methods or inhibitor purity. The methanolic extracts of C. Cassia or the bark essential oil from the different cinnamon species obtained using different extraction techniques such as supercritical CO 2 fluid extraction have been reported to exert anti-tyrosinase activity (Marongiu et al.; Ngoc et al. However, the extraction methods or plant species mentioned in these studies are different from the present paper. Moreover, the biological activities of plant extracts and essential oils are contributed to their bioactive components which may be affected by seasons, geographical origin, harvest time, agronomic practices and extraction methods (Fiocco et al.

Kojic acid, a fungal secondary metabolic product produced by species of Aspergillus and Penicillium, was shown to inhibit mushroom tyrosinase activity (Chen et al. Kojic acid has been extensively used as a medical agent for the treatment of a number of different skin disorders associated with hyperpigmentation. To reach a similar degree of tyrosinase inhibitory effect, the concentration required for CC-EO or its major constituent, cinnamaldehyde, was significantly higher than that required for kojic acid. Although the anti-tyrosinase abilities of the CC-EO and its major constituent, cinnamaldehyde, were significantly less than that of kojic acid, it was evident that they did have potent tyrosinase inhibitory activity. Kinetic analyses confirmed that both CC-EO and trans-cinnamaldehyde were regarded as mixed-I type inhibitors for the enzyme with L-DOPA as the substrate. This result implies that CC-EO and trans-cinnamaldehyde affected the affinity of the enzyme for L-DOPA but did not bind at the active site (Webb ). Furthermore, the behavior of mixed-I type inhibition indicated that CC-EO and trans-cinnamaldehyde could bind, not only with the free enzyme, but also with the enzyme-substrate complex.

As shown in Table, the determined values showed that the K i value for trans-cinnamaldehyde was approximately 2 times lower than that of CC-EO. In other words, trans-cinnamaldehyde had a more effective binding capacity for the enzyme than CC-EO. Additionally, the value of αK i was almost 4 times as great as K i for the oxidation of L-DOPA, indicating that the affinity of the inhibitor, CC-EO or trans-cinnamaldehyde, for a free enzyme is stronger than that of inhibitor for the enzyme–substrate complex.

() reported that trans-cinnamaldehyde isolated from the bark of C. Cassia exhibited competitive inhibition for L-DOPA oxidation by mushroom tyrosinase, while Lee () reported that cinnamaldehyde isolated from the root of P. Cernua was a noncompetitive tyrosinase inhibitor.

However, different enzyme preparation and assay methods or inhibitor purities could have contributed to these differences in enzyme inhibitory kinetics (Chen et al. Recently, safe tyrosinase inhibitors have become important for their potential applications in improving food quality and preventing pigmentation disorders and other melanin-related human health issues (Fitzpatrick et al. Magicdraw 16 6 Keygen Generator. ; Seo et al. Furthermore, safe tyrosinase inhibitors are important in cosmetics for skin whitening effects. Cassia has been safely used for many years by humans as a flavoring agent and a traditional medicinal herb. According to the United States Food and Drug Administration (USFDA), Cinnamomum spp., including common and cassia cinnamon, are generally recognized as safe (GRAS) when used in amounts commonly found in food (Dugoua et al. Furthermore, cinnamaldehyde and benzaldehyde, which are found in CC-EO, are also GRAS (Kubo and Kinst-Hori ). Thus, it is expected that CC-EO and its major constituent, cinnamaldehyde, may be a safe and viable source of skin-whitening agents.

Conclusions In summary, our findings demonstrate that the CC-EO and its major component, cinnamaldehyde, possess potent inhibitory activity against the diphenolase activity of tyrosinase. A study of the kinetics for the inhibition of mushroom tyrosinase showed that the CC-EO and cinnamaldehyde are mixed-type inhibitors for the enzyme with L-DOPA as the substrate. Both CC-EO and cinnamaldehyde are generally recognized as safe (GRAS).

Therefore, CC-EO and cinnamaldehyde may be suggested as a safe and as a good source of skin-whitening agents for pharmaceutical and cosmetic applications. Electronic supplementary material The online version of this article (doi:10.1186/1999-3110-54-10) contains supplementary material, which is available to authorized users. Competing interests The authors declare that they have no competing interests. Authors’ contributions C-TC, YS and S-TC conceived and designed the experiments, and drafted the manuscript. W-LC carried out the enzymatic assay, kinetic assay and statistical analysis.

J-CH conducted the GC-MS analysis. All authors read and approved the final manuscript.