Uv visible spectrophotometer

Peroxidase (POD; EC 1.11.1.7) activity of frozen root samples was measured as per the modified method of using guaiacol as substrate (electron donor) (Castillo et al., 1984 (link); Prakash et al., 2016 (link)). The root samples (100 mg) were homogenized using tissue lyzer (EzLyzer, Genetix, Biotech Asia, Pvt. Ltd.) in 300 µl extraction buffer (0.1 M phosphate buffer and 0.1 mM EDTA, pH 7.0). Lysates were centrifuged for 20 min at 15,000 × g at 4°C temperature, and the supernatants were collected and stored for assay. The 3-ml reaction mixture contained 16 mM guaiacol, 2 mM H₂O₂, 50 mM phosphate buffer (pH 6.1), and 0.1 ml enzyme extract. The change in optical density (OD) of the reaction mixture was recorded at 436 nm for a period of 3 min in time scan mode using a UV-Visible spectrophotometer (Thermo Scientific, USA). The result was presented as a change in OD per min/g fresh weight.

The Folin-Ciocalteu method previously detailed by Waterhouse was utilized to quantify total polyphenol content (TPC) [35 (link)]. Essentially, the Folin-Ciocalteu reagent and the extract were allowed to incubate at room temperature. The sodium carbonate solution was used to quench the reaction and sample absorbance was measured immediately using a UV-visible spectrophotometer (Thermo Fisher; Waltham, MA, USA) at 765 nm. Therefore, TPC was calculated as gallic equivalents (GE) using a standard curve prepared under the same conditions.

The specific activity of the GST in tenocytes was measured according to Zhang et al. [64 (link)], using 5 mM GSH and 0.5 mM CDNB as the second substrate in 0.1 M potassium phosphate buffer pH 6.5 at room temperature. The changes in absorbance at 340 nm were monitored for 5 min with the UV/visible spectrophotometer (Thermo-Fisher Scientific, Waltham, MA, USA). The molar extinction coefficient used for CDNB conjugation was 9.6 mM⁻¹ cm⁻¹. Enzymatic activities were calculated after correction for the non-enzymatic reaction.
The CAT activity in horse tenocytes was measured using the CAT activity assay kit according to the manufacturer’s instructions (Elabscience, E-BC-K031-S). The UV/visible spectrophotometer used to measure CAT activity was a JascoV-550 ETC 505S (Portland, OR, USA).

The HMF content was determined based on the UV absorbance of HMF at 284 nm using a UV-Visible spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) [32 (link)]. In order to avoid the interference of other components at this wavelength, the difference between the absorbance of a clear aqueous honey solution and the same solution after the addition of bisulfite was determined. The HMF content was calculated after the subtraction of the background absorbance at 336 nm. In a 50 mL volumetric flask containing 2 mg of honey dissolved in 25 mL of water, 0.5 mL of Carez I solution was added, followed by 0.5 mL of Carez II solution. Water was added to the flask to make up a volume of 50 mL, and the resulting solution was filtered. After discarding the first 5 mL of the filtrate, 5 mL of 0.2% sodium bisulfite solution was added to the test tube. In another test tube, 5 mL of pure water was added as a blank. A UV-Visible spectrophotometer was used to measure the solution’s absorbance at 284 and 336 nm in 10 mm quartz cells within 1 h. The calculation was performed using the formula below: $$\mathrm{HMF} (\mathrm{mg}/\mathrm{kg})=(\mathrm{A}284-\mathrm{A}336) \times 149.7 \times 5 \times \frac{\mathrm{D}}{\mathrm{W}}$$
It should be noted that 149.7 is a constant, A284 is the absorbance at 284 nm, A336 is the absorbance at 336 nm, 5 is the theoretical nominal sample weight, D is the dilution factor (in case dilution is necessary), and W is the weight of honey taken.

Encapsulation efficiency was measured using the method of Hu et al. [21 (link)] with slight modifications. One mL of 0.1 M trisodium citrate buffer (pH 6.0) was added to the curcumin encapsulated cryogel beads and stirred at 40 °C for 90 min to degrade the matrix. After all the matrix of cryogel beads was degraded, 0.8 mL of dimethyl sulfoxide (DMSO) was added to extract curcumin and stirred for 3 h. Next, the curcumin extract mixture was centrifuged at 3500 rpm for 10 min, and the supernatant was collected. The undissolved curcumin remaining in the sediment was dissolved again by adding 0.2 mL of DMSO and vortexed for 1 min. The mixture was centrifuged at 3500 rpm for 10 min, and the supernatant was collected. The total supernatant was used to determine the amount of encapsulated curcumin. The curcumin concentration (encapsulated amount of curcumin) in the cryogel beads was measured at 435 nm by a UV-Visible spectrophotometer (Thermo Scientific, Vantaa, Finland) with a calibration curve (0–10 μg/mL free curcumin). Measurements were performed in triplicate. The encapsulation efficiency was determined by using Equation (1), as shown below:

A single yeast colony harboring FoCYP539A7 and FoCYP655C2 gene was individually inoculated into 10 mL of SD-U (except uracil) medium with 2% dextrose. S. cerevisiae harboring only pESC_URA plasmid without any FoCYP was used as control. The overnight grown cells were inoculated into 50 mL of YPG media with 4% galactose and 2 mM 5-ALA to obtain an OD₆₀₀ of 0.4 and cultured again. The cells were collected, resuspended in 500 mL of fresh galactose media, and cultured for about 2 days with shaking at 150 rpm until reaching an OD₆₀₀ of 2-4. The galactose induced yeast cells were then harvested and the microsomes were isolated as described earlier [49 (link)]. UV absorbance spectra of CO-bound microsomes after sodium dithionate reduction were recorded using a UV-visible spectrophotometer (Thermo Labsystems, NY, USA) scanning between the wavelengths 400 and 500 nm.