Hesperetin

Animal behavior analysis was performed by using Morris Water Maze (MWM) and Y-maze tests by using a video tracking software (SMART Panlab, Harvard Apparatus, Holliston, MA, USA). Behavioral analysis was conducted daily (after 1 h of drug/chemical administration) according to the plan of experiment. To evaluate the memory and learning performance, MWM was conducted as described previously [29 (link),30 (link)] with some small modifications. After receiving 2 consecutive days of training, latency (sec) was measured to assess the time taken to reach the hidden platform for 6 consecutive days. On the following day, we performed the probe test for the evaluation of memory consolidation by removing the platform and allowing the animal to swim freely for 1 min. The crossing numbers over the previously hidden platform and the time spent in the specific target quadrant were measured.
Next, for Y-maze analysis, we placed each mouse in the middle of the device to freely move for 3–8 min in the maze. Entries into the arms were recorded digitally. Spontaneous alternation behavior is defined as the [successive triplet sets (consecutive entries into three different arms)/total number of arm entries-2] × 100. Improved memory and cognitive function were reflected by a higher % of spontaneous alternation behavior.

The hesperetin content was quantified using a reverse phase HPLC method employing a Perkin Elmer^® Series 200 HPLC equipped with a UV detector (Perkinelmer España S L, Madrid, Spain), detecting the molecule’s absorbance at 288 nm. The analysis utilized a Teknokroma^® Brisa “LC2″ C18 column (150 × 4.6 mm, 5.0 μm) (Teknokroma, Barcelona, Spain) and a mobile phase consisting of a methanol: water MilliQ mixture in a 70:30 proportion, respectively. The flow rate was maintained at 1 mL/min and the sample injection volume was 20 µL. Hesperetin was eluted after 3.5 min and the method was linear in the concentration range of 0.24–62.7 µg/mL with a correlation coefficient of 0.9999. The limits of detection (LOD, Equation (1)) and quantification (LOQ, Equation (2)) were estimated using the calibration curve procedure, as follows: $$LOD=\frac{3.3·SD}{S}$$
$$LOQ=\frac{10·SD}{S}$$
where SD and S are the standard deviation of the y-intercept and the slope of the linear calibration curve, respectively.
Under these conditions, the limit of quantification was 0.918 µg/mL and the limit of detection was 0.303 µg/mL.

Cisd2KO mice were generated as previously described [25 (link)]. All mice used in this study are males with pure or congenic C57BL/6 backgrounds. All mice were bred and housed in a specific pathogen-free facility at a constant room temperature (20–22 °C) with a 12 h light and 12 h dark cycle (7 a.m–7 p.m.). For the anti-aging study, the mice were fed ad libitum with AIN-93G (TestDiet, St. Louis, MO, USA) diet mixed with hesperetin (0.07% [w/w]; Sigma-Aldrich, H4125; 100 mg/kg/day) or mixed with vehicle (3.04% propylene glycol [w/w]; Sigma-Aldrich, 16033). To evaluate the protective effect of hesperetin on UVB-induced skin damage, the mice were treated with hesperetin (30 mg/kg/day) or vehicle by a feeding tube. For the UVB treatment, the UVB apparatus consisted of four UVB lamps (G4T5E, SANKYO DENKI, Hiratsuka, Kanagawa, Japan); the spectral wavelength range of the UVB lamps was 280–360 nm, and peak light source intensity was 306 nm. Mice under anesthesia were placed individually in a plastic box with UVB lamps; the fluence of UVB on the mouse dorsal surface was 349 mJ/cm² for 75 s [31 (link), 32 (link)]. Mice were treated with hesperetin or vehicle for 7 days before UVB irradiation followed by UVB irradiation for 5 consecutive days as hesperetin or vehicle treatment continued. The dorsal skins were dissected two days after the final UVB treatment. After each specific treatment, the mice were sacrificed by CO₂ inhalation, which is a humane method of euthanasia. The animal protocol was approved by the Institutional Animal Care and Use Committee (No. 1040103) of National Yang Ming Chiao Tung University. The animal protocols were designed to follow the associated guidelines and the 3R principles (Replacement, Reduction and Refinement) in accordance with the “Animal Protection Act” of Taiwan.

The release and skin permeability assessments of hesperetin were conducted on the selected emulgel using a Franz vertical cell (effective diffusion area: 0.785 cm²) with a sample size of n = 3. The assembled cell was placed in a thermostatic bath at 37 ± 1 °C and maintained under constant agitation. For this purpose, the ‘infinite dose’ condition was used to determine the release and skin permeation coefficients. Briefly, the experiment consisted of adding 500 mg of the test formulation to the donor chamber and a 50% (v/v) hydroalcohol mixture to the receptor chamber. In the release tests, a cellulose acetate membrane separated the chambers, while the human epidermis was used for the skin permeability tests. The human epidermis was abdominal female skin, which was obtained from patients after they signed informed consent for inclusion before participating in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethical Committee of the University of Valencia (protocol number: H 1540295606992, approved date: 8 November 2018). The epidermal membranes were obtained via heat separation and immersed in water at 60 °C for 60 s [30 (link)] or frozen and stored after fatty tissue removal.
Samples of 200 µL were collected from the receptor chamber, and an equal volume was replenished at different time intervals for up to 24 h. Simultaneously, the accumulated hesperetin in the skin was quantified after methanol extraction. To achieve this, after the 24-h period, the epidermis was cut, placed into a glass vial containing methanol, and sonicated for 2 min to ensure the complete extraction of hesperetin. The resulting dispersion underwent centrifugation at 3000 rpm for 10 min. Subsequently, the supernatant was filtered, and the hesperetin content was measured using HPLC.
The in vitro release kinetics of hesperetin from the emulgel were modeled mathematically. For this purpose, the data were fitted to zero-order (Equation (3)), first-order (Equation (4)) and Higuchi (Equation (5)) equations.
$$Q_t=K_{0}t$$
$$Q_t=Q_0·e^{-k1t}$$
$$\frac{Q_t}{Q_{\infty }}=K_h·t^{0.5}$$
where t is time, Q_t is the amount of hesperetin released at time t, Q₀ is the initial amount of hesperetin, Q_∞ is the amount of hesperetin released at time ∞, and K₀, K₁, and K_h are the hesperetin release rate constants of each of the kinetics.
To determine the mechanism of hesperetin release, Korsmeyer–Peppas (Equation (6)) and Peppas–Sahlin (Equation (7)) mathematical models were employed and fitted to the experimental data using Sigmaplot 10.0^® (Systat Software, Inc., San Jose, CA, USA).
$$\mathrm{Korsmeyer}-\mathrm{Peppas}: \frac{Q_t}{Q_{\mathrm{\infty }}}=K·t^n$$
$$\mathrm{Peppas}-\mathrm{Sahlin}: \frac{Q_t}{Q_{\mathrm{\infty }}}=K_1{t}^n{+K}_2{t}^{2n}$$
where Q_t/Q_∞ represents the fraction released of the active ingredient, K, K₁, and K₂ are the diffusion constants, t is the time in which the active substance is released, and n is the exponent that characterizes the diffusion process. So for n < 0.5, the release is Fickian, between 0.5 < n < 1.0, it indicates that an anomalous process has occurred, while for n = 1, the release obeys zero-order kinetics [31 (link)].
Additionally, for the skin permeation studies, as a representative of the diffusion process in the experimental conditions, the Scheuplein equation was used, which relates the quantities (Q, mg/cm²) permeated with time (t, hours) (Equation (8)): $$Q_{\left(t\right)}=A·P·L·C·⌊D·\frac{t}{L^2}-\frac{1}{6}-\frac{2}{{\pi }^2}·\sum _{n=1}^{\infty }\frac{{\left(-1\right)}^n}{n^2}·Exp\left(\frac{-D·n^2·{\pi }^2·t}{L^2}\right)⌋$$
where A is the useful diffusion area (cm²), P is the distribution coefficient of the drug between the skin and the donor vehicle, L is the thickness of the membrane (cm), C is the drug concentration in the donor solution (mg/mL), D is the diffusion coefficient in the membrane (cm²/h), and n is a value integer between one and infinity.
Equation (8) has been simplified to Equation (9) and represents the linear section of the percutaneous absorption process: $$Q_{\left(t\right)}=A·P·L·C·⌊D·\frac{t}{L^2}-\frac{1}{6}⌋$$
The accumulated hesperetin quantities against time were fitted to Equation (9). The values of P and D were obtained as the primary parameters of the fit. From these values, the latency time (t_L, h), the permeability coefficient (K_p, cm/h), and the flow (J, µg/cm²·h) were calculated: $$t_L=\frac{1}{6·D}$$
$$K_p=\frac{P·D}{L}$$
$$J=K_p·C$$