Quantitative analysis of Abees and A195 extractsDetermination of the nutritional value

In our study we investigated the nutritional importance of two Egyptian sweet potato cultivars (Abees and A195). Voucher specimens of the studied cultivars (no. 17-9-24-F and 18-9-24-F) were deposited in the Herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Cairo University. We determined the content of vitamins, minerals, lipids, proteins, carbohydrates, moisture, and the ash value in these two cultivars. Roots of A195 and Abees were obtained from the Potato and Vegetatively Propagated Vegetables Dep., Horticulture Research Institute, Agricultural Research Center, Egypt. The roots were authenticated by professor Dr. Mahmoud Mohamed Samy (Research Head) at Horticulture Research Institute, Cairo University, Egypt. The proximate analysis, estimation of moisture, ash, proteins, lipids, carbohydrates, minerals, vitamin C along with β carotene contents were carried out according to AOAC procedures.

Proximate analysis

Moisture, ash, protein, lipid, carbohydrate and β- carotene contents were estimated according to (AOAC) procedures. The moisture level was estimated via the oven method. The dry ash method was used for determination of Ash content, and Kjeldahl method for total protein content. Lipid Content was estimated using chloroform–methanol extraction method. Carbohydrate Content was estimated by calculating the difference (dry extract − (ash + lipids + proteins)). β-carotene was determined by UV Spectrophotometric method [15, 16].

Determination of mineral content

Determination of (K) by flame emission, (Fe) by the sulphonated bathophenanthroline colorimetric method, (P) via molybdophosphovanadate method and (Mn, Zn) using atomic absorption spectroscopy method [17, 18].

Determination of vitamin C

Estimation of vitamin C content was carried out using UV/Vis spectrophotometric methods at temperature 24 °C and humidity 38%rH for 2 min by extraction of 5 g of each cultivar with 100 ml oxalic acid -EDTA solution in a blender. The extract is filtered and subjected to centrifugation. By transferring 5-ml aliquot to calibrated flask (25 ml) and the colour was developed. The blue solution is filtered after 15 min, and its absorbance is determined at 760 nm. Determination of ascorbic acid is done using the calibration curve [19].

HPLC standardization of Abees and A 195 extracts

Abees and A195 ethanolic extracts were prepared as mentioned in Elgabry et al. (two kilograms of dried powdered Abees and A 195 cultivars were extracted with (6 L) of ethanol (85%) at room temperature for 10 days with frequent shaking. The filtrate was evaporated to dryness using a rotary evaporator (Buchi, Germany), to yield 100 g of each extract) [20]. Quantitative determination of chlorogenic acid (CA) in Abees and A195 ethanolic extracts was done using HPLC [21]. The analysis was performed using Waters 2690 Alliance HPLC system equipped with a Waters 996 photodiode array detector. Fifty µL of each sweet potato extract solution in methanol (50 mg/ml) or chlorogenic acid solution in methanol (40, 60, 80, 100, 120 and 140) µg/mL were injected into a Inertsil 18 °C (5 µm, 4.6 × 250 mm). The mobile phase was 0.1% Orthophosphoric acid (A): Methanol (B). Gradient elution was used with a flow rate (1 mL/min) as follows: 0–50 min (95% A: 5% B), 50–55 min (50% A: 50% B), 55–75 min (30% A: 70% B), 75–76 min (10% A: 90% B), and 76–80 min (5% A: 95% B). The absorbance was detected at 280 nm. The assay was performed three replicates for the samples and for each chlorogenic acid standard solution (40, 60, 80, 100, 120 and 140) µg/mL. Standard calibration curve of chlorogenic acid (40–140) µg/mL was used for the results expression and the concentration was calculated as mg chlorogenic acid per gram of sweet potato extract.

Determination of total phenolic content

Folin–Ciocalteu procedures were used for determination of total phenolic content in triplicate manner [22]. In brief, deionized water was used for Folin–Ciocalteu reagent dilution to ten-folds, (5 mg) of each extract was in (1 ml) MeOH/water (95% v/v) then Na2CO3 (7.5%) solution has been prepared. In tiny test tubes, combine (100) µl of standard, sample or 95% methanol blank with 200 µl 10% Folin–Ciocalteu reagent and carefully vortexed. Add 800 µl Na2CO3 (700 mM) in the tubes and keeping them for incubation 2 h at room temperature. After incubation time, transfer 200 µl standard, sample or blank to 96-well microplate, the absorbance of each well was measured at 765 nm. Total phenolic content values were determined from a calibration curve prepared with a series of gallic acid standards using the equation (Y = 0.004x + 0.1452) and expressed as µg gallic acid equivalent (GA)/5 mg of dried plant extract.

Determination of total flavonoid content

Total flavonoid content determination in Abees and A195 extracts was applied using aluminum chloride colorimetric method taken from [23, 24]. Dissolved 5 mg of the extracts in 1 ml absolute methanol to prepare the test samples. In small test tubes mix (500 µl) of each sample with (250 µl) AlCl3 (10%) and 250 µl distilled, incubate the test tubes at room temperature 10 min. Quercetin standard calibration curve was prepared using different concentrations of quercetin standard. UV–Vis spectrophotometer was used at 420 nm, the mixtures absorbance was measured versus blank [25]. The concentration of total flavonoid content was calculated from the calibration plot (Y = 0.0083x + 0.1221, R2 = 0.9978) and expressed as µg quercetin equivalent (QE)/5 mg of dried plant extract. All the determinations were carried out in triplicate.

Anti-skin aging activities of Abees and A195 extractsMaterials used in the in vivo study

The colorimetric assay kits for superoxide dismutase (SOD, cat. no. SD 25 21), catalase (CAT, cat. no. CA 26 17), and malondialdehyde (MDA, cat. no. MD 25 29) were all acquired from Bio diagnostic Co. (Cairo, Egypt). The.

Materials used in preparation of topical formulas

Beeswax and Cetyl alcohol were obtained from Morgan Co. Cairo, Egypt. Propylene glycol and Sodium lauryl sulphate were purchased from El-Nasr Pharmaceutical Chemicals Co., Cairo, Egypt. Distilled de-ionized water was used.

Preparation of Abees and A195 topical creams

To evaluate the efficacy of the topical use of Abees and A195 extracts, an oil-in-water cream was formulated [26, 27]. In brief, 15% w/w cetyl alcohol and 1% w/w beeswax were combined to form the lipidic phase. Conversely, 2% w/w sodium lauryl sulphate and 10% w/w propylene glycol were dissolved in distilled water to form the aqueous phase. Both phases were heated in a water bath to a temperature of 70 ± 5 °C. Afterward, the aqueous phase was gradually added to the molten lipidic phase with constant agitation until complete homogeneity was attained. Before reaching room temperature, the dried residue of sweet potato ethanolic extract was included and agitated to create creams with concentrations of 3% and 5% (w/w).

AnimalsSample size calculation

Study sample size was determined using the G*Power software (version 3.1.9.7). Based on the estimates obtained from our previous experiments, we hypothesized a considerably large effect size (Cohen’s f = 0.75) when comparing the seven study groups as regards oxidative stress markers (SOD and MDA). In a one-way ANOVA study, a total sample size of 42 (6 rats per group) achieves 94% power to detect meaning differences.

Experimental design

Female C57BL/6 mice, weighing 22 ± 3 g and aged ~ 8 weeks, were obtained from the laboratory animal facility of the National Research Center, Cairo, Egypt. They were kept in a controlled environment with a temperature of 20 ± 2 °C and a 12-h day/night cycle. They had unlimited access to conventional grain-based diet and water. The study protocol received approval from the Research Ethics Committee of the Faculty of Pharmacy at Cairo University, Egypt (serial number. ID: 3308) and was conducted according to the ethical code of animal experiments outlined in the “Principles of Laboratory Animal Care” (NIH publication no. 85-23, revised 1985).

Forty-two female mice were randomly assigned to seven groups, with six mice each:

Normal group, injected with 0.9% NaCl saline 0.3 mL/day SC.

d-Gal group, treated with d-Galactose 1000 mg/kg/day dissolved in 0.3 mL saline SC [27].

Vit E group treated with d-Galactose + topical vitamin E 5% [28, 29].

A195 3% group treated with d-Galactose + topical A195 3% formula.

A195 5% group treated with d-Galactose + topical A195 5% formula.

Abees 3% group treated with d-Galactose + topical Abees 3% formula.

Abees 5% group treated with d-Galactose + topical Abees 5% formula.

The d-Galactose solution (or saline) was injected SC in the mice backs for a total duration of 8 weeks. Starting from the third week, topical treatments were applied daily and continued for the remaining 6 weeks. In order to apply topical treatments, square areas measuring 3 × 3 cm on the backs of mice were consistently shaved of hair, and experimental treatments were uniformly massaged onto the exposed skin surface. Records of body weight for each animal were updated weekly throughout the whole experiment.

Macroscopic assessment of mice skin

24 h following the last topical drug application, a researcher blinded to the experimental design visually examined, photographed, and evaluated the naked skin on the backs of euthanized mice for signs of aging. The adopted grading system spanned from 0, representing intact mouse skin, to 6, indicating severely injured skin [30]. Afterwards, skin tissue samples were collected and either snap frozen in liquid nitrogen or fixed in 4% paraformaldehyde, for later use.

Histopathological assessment

Following fixation and paraffin embedding, tissue sections of 4–6 μm thickness underwent pathological staining with H&E and Masson's trichrome staining. Tissue morphology and collagen expression pattern were observed under a light microscope (Olympus BX-51, Japan; magnification 100 ×). Epidermal (EP) and dermal (DE) thickness (measured in μm), as well as collagen expression area (presented as % relative to the total field area) were analyzed using an image analysis software (ImageJ, v1.53 k).

Assessment of oxidative markers

10% skin tissue homogenizes were prepared for the purpose of detecting the redox status associated with aging and treatment. The antioxidant activity of the enzymes; superoxide dismutase (SOD) and catalase (CAT), and the levels of malondialdehyde (MDA) in the skin were quantified using commercially available kits.

Assessment of the TGF-β/SMAD pathway expression

Quantitative measurement of the protein expression of TGF-β RII (cat. no. ab277719; Elab science Biotechnology, Houston, TX, USA), p-SMAD3 (cat. no. MBS269937; Mybiosource, San Diego, CA, USA) and SMAD7 (cat. no. NBP2-76627; Novus Biologicals, Centennial, CO, USA) was done using corresponding mouse enzyme-linked immunoassay (ELISA) kits. Briefly, skin samples (~ 100 mg in weight) were homogenized and centrifuged at 4 °C to obtain the supernatant. The concentrations of TGF-β RII, p-SMAD3 and SMAD7 in the skin tissue were quantified following the instructions of the providers.

Statistical analysis

Statistical analysis was conducted using the SPSS software (version 26.0; SPSS Inc., Chicago, IL, USA), and the results are presented as mean values ± standard error of means (SEM). A repeated measures ANOVA test was used to examine the effect of different treatments (“between-subjects” variable) on mice body weight (dependent variable) across time (“within-subjects” variables). The normality and sphericity assumptions were evaluated using Shapiro–Wilk and Mauchly’s tests, respectively. Bonferroni correction was then used for pairwise comparisons between the different groups. Analysis of skin macroscopic scores was done using the nonparametric Kruskal–Wallis ANOVA followed by Dunn's-Bonferroni test. Statistical significance of all other data was assessed using one-way ANOVA, followed by Tukey's multiple comparison test. The results were validated as significant at p < 0.05.