Analytical Assessment of the Quality of Dietary Supplements and Cosmetic Products Containing Xanthohumol by Thin-Layer Chromatography Along with the Estimation of Its Antioxidant Potential

Abstract

Xanthohumol, a prenylated chalcone in the flavonoid group, naturally occurs in many plants and exhibits antioxidant, anticancer, anti-inflammatory, antibacterial, and antiviral effects. The growing interest in xanthohumol due to its potential therapeutic properties has led to the increase in the pool of products available on the market. The novelty of this study is the proposal of a rapid and cost-effective procedure useful for performing quality control on products containing xanthohumol in the form of dietary supplements and cosmetics as well as testing their stability. For this purpose, the thin-layer chromatography method with densitometric detection was used, which was validated in accordance with ICH (International Conference on Harmonization) guidelines. The mobile phase was toluene, 1,4-dioxane, and glacial acetic acid (37:10:1.5 v/v/v), and TLC silica gel 60 F₂₅₄ plates were used as the stationary phase. The validation process assessed linearity, with a correlation coefficient (r) of 0.9987. The calculated LOD (limit of detection) and LOQ (limit of quantification) values were 3.82 and 11.57 ng/spot, respectively. Accuracy was evaluated by determining percentage recovery at three concentration levels (80, 100, and 120%), with an average recovery of 100% and RSD below 1%, confirming good accuracy. Precision was indicated by an RSD of less than 2.20%. The average content of xanthohumol in dietary supplements ranged from about 8 to 29% of the content declared by the manufacturers. The stability tests showed that XN decomposes most slowly in water (t_0.5 = 10.86 h) compared with acidic (t_0.5 = 10.80 h) and alkaline solutions (t_0.5 = 7.39 h), as well as in the presence of an oxidizing agent (t_0.5 = 18.38 h), at all tested temperatures, which is confirmed by the calculated kinetic parameters. In the tests of antioxidant capacity, xanthohumol shows significantly higher radical scavenging capacity than vitamin C in the entire range of analyzed concentrations (0.03–2.40 mmol/L).

1. Introduction

Xanthohumol (2′,4,4′-trihydroxy-6′-methoxy-3′-prenylchalcone; XN) is a prenylated chalcone from the flavonoid group, which belongs to polyphenolic compounds commonly found in fruits, vegetables, and certain beverages such as coffee, tea, beer, and wine [1]. From a chemical perspective, a distinctive structural element of XN is the flavonoid backbone with two trans-configured aromatic rings, A and B, connected by a three-carbon chain and an unsaturated carbonyl moiety (Figure 1) [2].

In nature, XN is found in the female inflorescences of hops (*Humulus lupulus* L.), constituting 0.1 to 1% of the dry mass of hop cones. The primary method of obtaining this compound is through extraction from female hop inflorescences. The developed chemical synthesis of XN is complex and is characterized by low yield [3]. In the diet, the primary source of XN is beer, where its concentration can reach 0.96 mg/L, and in dark beer, it can reach up to 3.5 mg/L due to the use of colored malt [4].

Due to the presence of the α, β-unsaturated carbonyl group, xanthohumol exhibits a wide spectrum of biological activities, including antioxidant, anticancer, anti-inflammatory, antibacterial, and antiviral effects [5]. Over the past decade, numerous researchers have studied these properties, especially in terms of antioxidant activity [6]. Oxidative stress, defined as an imbalance between the presence of reactive oxygen species (ROS) and the body’s compensatory mechanisms for their removal, is a significant factor in the etiology of many diseases, including cardiovascular diseases [7]. The antioxidant activity of XN and other prenylated flavonoids is attributed to their polyphenolic structure, which allows them to chelate metals, scavenge singlet oxygen, and neutralize ROS [8,9]. Additionally, XN increases the activity of glutathione and superoxide dismutase (SOD), which catalyzes the breakdown of superoxide anions during anticancer treatment with cisplatin [10]. There are various methods available for determining the antioxidant activity of compounds, and the differences lie in the use of different mechanisms [11]. Zang et al. investigated the antioxidant capacity of XN using the TEAC (Trolox equivalent antioxidant capacity) through ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) scavenging test, DPPH (1,1-diphenyl-2-picrylhydrazyl radical), and FRAP (ferric reducing antioxidant power) assays. The TEAC of XN was 0.32 ± 0.09 μmol/L in the ABTS and 0.27 ± 0.04 μmol/L in the FRAP tests. However, XN did not show a significant scavenging effect in the DPPH radical reaction system [6]. Yamaguchi et al. demonstrated that XN possesses very strong antioxidant properties. The use of the ORAC (oxygen radical absorbance capacity) method, which measures the ability of a compound to neutralize ROS, confirmed that XN exhibits higher antioxidant capacities than vitamins C and E. Furthermore, studies on singlet oxygen absorbance capacity (SOAC) showed that XN is significantly more effective in this regard than vitamin E [12].

The commonly used techniques for the XN assay are chromatography and spectrometry, which provide high accuracy, precision, and the ability to detect and quantify even low concentrations of this compound in various matrices. Reversed-phase high-performance liquid chromatography (RP-HPLC) with isocratic elution was employed for the determination of XN and curcumin in rat plasma. The developed method exhibited linearity with an r² value of 0.9996 over the concentration range of 50–250 ng/mL, and the lowest limits of detection (LODs) and quantification (LOQ) were 8.49 ng/mL and 25.73 ng/mL, respectively [13]. Another quantitative method used to determine six prenylflavonoids, including XN and isoxanthohumol, in hops and beer is tandem mass spectrometry HPLC-APCI-MS/MS. Isoxanthohumol, formed from the isomerization of XN during the brewing process, was the most frequently occurring flavonoid in hopped beers, with concentrations ranging from 0.04 to 3.44 mg/L [14]. In the subsequent study, a method based on solid-phase extraction and LC–ESI-MS/MS was developed. The method was optimized to prevent the degradation of selected analytes, such as isoxanthohumol, XN, and 8-prenylnaringenin, throughout the analytical process, as well as to minimize the urine matrix effect. It was applied for the quantitative determination of prenylflavonoids in 10 human urine samples after the consumption of a single dose of beer [15]. Quantitative determination of 20 selected phenolic compounds (including XN) in six different types of craft beers in worts and raw and spent products was performed using a validated HPLC–MS/MS method. The extractions were performed in water to promote the sustainable utilization of brewing byproducts [16]. In the next study, UHPLC-MS/MS was developed and validated for the simultaneous determination of XN, isoxanthohumol, 6-prenylnaringenin, and 8-prenylnaringenin in human serum. The LOQs were 0.50 ng/mL for XN and 1.0 ng/mL for 6-PN, 8-PN, and IX [17]. The analysis of hop flowers and commercially available dietary supplements in the form of capsules, as well as various types of beers, was carried out using RP-HPLC with a diode array detector (DAD) at 370 nm. The linear range was between 0.05 and 20 mg/L, the LOD was 0.016 mg/L, and the LOQ was 0.049 mg/L [18]. Another material from which XN was isolated and determined by HPLC-DAD was yeast after the brewing process. The LOD and LOQ were determined to be 0.05 µg/mL and 0.15 µg/mL, respectively [19]. HPLC with UV spectrophotometric detection was also used to determine XN in beer samples. The calibration curve was established in the concentration range of 0.01–10 mg/L, with LOD and LOQ values of 0.003 mg/L and 0.01 mg/L, respectively [20].

One of the variants of liquid chromatography is thin-layer chromatography (TLC). It is a basic analytical technique for the identification of organic compounds, the advantages of which over HPLC include its capability to analyze multiple samples simultaneously, accommodate diluted samples, shorter analysis duration, and lower costs [21,22]. The analyzed XN samples, including extracts from powdered hop pellets from different crops and varieties, as well as beer enriched with XN, were determined using high-performance thin-layer chromatography (HPTLC) with densitometric detection. Standard and extracted samples were applied onto silica gel plates and separated with a mixture of toluene, dioxane, and acetic acid (77:20:3, v/v/v) as the mobile phase. Next, plates were quantitatively evaluated densiotmetrically at 368 nm. A linear correlation, peak area vs. XN concentration within the range of 7.7–77.0 ng/spot, was established with a correlation coefficient of 0.997. The LOD and LOQ were determined as 2 and 5 ng/spot, respectively [23].

Cancer is one of the most serious diseases of the 21st century, characterized by high morbidity and mortality. There is a growing need to improve cancer treatment methods, also by searching for new alternative approaches for prevention and treatment. Numerous studies have demonstrated that XN has an inhibitory effect on cell survival, growth, proliferation, angiogenesis, and metastasis in various types of cancers [24]. Its chemopreventive properties include protecting DNA from damage and oxidative stress, which helps defend against mutations. XN inhibits tumor angiogenesis by blocking the signaling pathways, as well as reducing the production of angiogenic factors. It also acts pro-apoptotically, enhancing the effectiveness of chemo- and radiotherapy treatments. Additionally, XN inhibits cancer cell invasion and regulates the expression of proteins involved in cell adhesion [4]. Many patients, especially the elderly, suffer from rheumatoid or autoimmune diseases, which are characterized by the presence of inflammatory markers in the body. Commonly used nonsteroidal anti-inflammatory drugs (NSAIDs) have many side effects that complicate treatment. Therefore, alternative compounds and herbals with fewer side effects are sought. One of them is XN, which blocks the production of nitric oxide, reduces the expression of the lipopolysaccharide receptor, and inhibits the activation of various inflammatory factors [3].

In recent years, thanks to the widespread availability of antibiotics, the treatment of bacterial infections has become increasingly difficult due to the increasing resistance of bacterial strains to antibiotics. The discovery of new antimicrobial agents remains crucial. Xanthohumol has a strong synergistic effect with antibiotics such as tobramycin, ciprofloxacin, and polymyxin B sulfate. It has been shown that the minimum inhibitory concentration (MIC) for such use is significantly lower compared with antibiotics alone, both for Gram-negative and Gram-positive bacteria [25]. Studies have shown that XN is a potent bacteriostatic agent against Staphylococcus aureus and Streptococcus mutans strains. It also has high antimicrobial potential against Clostridioides difficile, as evidenced by a significant reduction in the number of C. difficile bacteria in stool samples after two days of treatment [26]. XN also exhibits broad-spectrum antiviral properties that inhibit, among others, human cytomegalovirus (CMV), herpes simplex virus (HSV), human immunodeficiency virus (HIV), hepatitis C virus (HCV), porcine reproductive and respiratory syndrome virus (PRRSV), and coronavirus (SARS-CoV-2). Due to its antioxidant properties, it can also reduce tissue damage caused by viruses [27]. XN shows potential as a hypopigmenting agent by inhibiting melanogenesis in B16 melanoma cells. Hop extracts containing XN and lupulones show potent activity against acne pathogens. XN also improves skin texture and firmness by inhibiting elastase and stimulating the biosynthesis of collagen, elastin, and fibrillin, making it a promising anti-aging ingredient. Its use in dermatology may range from photoprotection to the treatment of atopic or contact dermatitis, pigmentation disorders, infections, aging, and skin cancer [3]. Safety studies have demonstrated that XN, at doses comparable with its content in commercial products (24 mg/day), is safe and well tolerated [28].

The growing interest in XN due to its potential medicinal properties has contributed to the increase in the number of products available on the market as dietary supplements and cosmetics. However, taking into account our previous studies on the quality control of dietary supplements [29,30], they indicate the need to develop analytical procedures that allow for a reliable and rapid assessment of the available products. This trend highlights the need for increased vigilance and awareness to prevent further threats to public health from poor-quality products intended to improve health.

The aim of the undertaken research was to develop a procedure for determining XN in various dosage forms (commercially available dietary supplements and cosmetic products). To achieve these goals, TLC with densitometric detection was optimized and validated. Furthermore, the developed procedure was used to study the stability of XN in variable environmental and temperature conditions. Tests based on various mechanisms were also carried out to determine its antioxidant potential.

2. Materials and Methods

2.1. Chemicals and Reagents

Xanthohumol (CAS 6754-58-1) was obtained from PhytoLab GmbH & Co. (Dutendorfen, Germany), and L-ascorbic acid (CAS 50-81-7; AA) was purchased from Sigma-Aldrich (St. Louis, MA, USA).

All tested dietary supplements and cosmetics were obtained at local pharmacies in Poland. The following products containing XN were selected for testing: dietary supplement 1 (40 mg/2 mL), dietary supplement 2 (200 mg/3 caps.), dietary supplement 3 (no data/mL), and cosmetic 1 (no data/amp.) and cosmetic 2 (no data/vial.). All tested products were within their expiration date.

1,4-dioxane was purchased from POCH (Gliwice, Poland). Methanol for LC-MS was purchased from Fluka (Buchs, Switzerland); water for HPLC, toluene and glacial acetic acid were obtained from Witko (Łódź, Poland); iron (III) and iron (II) chlorides, phosphate buffer, sulfuric (VI) acid, potassium hexacyanoferrate (III), trichloroacetic acid, ammonium phosphate (V), ethanol, hydrochloric acid, sodium molybdate (VI), sodium hydroxide, and 30% hydrogen peroxide were obtained from CHEMPUR (Piekary Śląskie, Poland). Dimethyl sulfoxide (DMSO) was obtained from Merck KGaA (Darmstadt, Germany), and ferrozine was obtained from Chemat (Konin, Poland). All chemicals were an analytical grade.

All qualitative and quantitative TLC densitometric determinations were performed using Camag instrumental equipment (Muttenz, Switzerland), including: a semi-automatic sample applicator for applying the probes on the Linomat V plate, a 100 µL glass syringe (Hamilton, Bonaduz, Switzerland), a TLC Scanner 3 densitometer with winCATS software, and a UV lamp (254 and 366 nm). A vertical glass chromatographic chamber with the dimensions of 20 × 10 cm was obtained from Sigma-Aldrich (Laramie, WY, USA). TLC silica gel 60F₂₅₄ plates with the dimensions of 20 × 20 cm (No. 1.05554.0001) were purchased from Merck (Darmstadt, Germany).

Spectrophotometric analysis of XN and AA was performed using a Cary 100 UV–Vis spectrophotometer (Agilent, Santa Clara, CA, USA) and HELLMA Optic GmbH quartz cuvettes (Jena, Germany).

Other instrumental equipment used during experiments included a WPA 120C1 analytical balance (Radwag, Radom, Poland) and an EcoCell BMT dryer (Brno, Czech Republic).

2.2. Standard and Sample Solutions

The XN standard solution was prepared by weighing approximately 1.0 mg of XN standard substance into a 10.0 mL volumetric flask, which was then filled with methanol. These prepared solutions were stored in a refrigerator at 2–8 °C, out of sunlight. To obtain lower concentrations of XN for testing, the standard solution prepared in this way was diluted in an appropriate proportion with methanol.

Extracts of the tested products were prepared by weighing approximately 200 mg of each; then dissolving in 10.0 mL of methanol, vortexed for different times: 10, 30, 45, 60 and 90 min; and then centrifuging at 3000 g for 10 min. The obtained solutions (the supernatant) were subjected to further analysis and stored in a refrigerator at a constant temperature of 2–8 °C, protected from sunlight. Each extraction was performed three times. To prepare XN solutions with lower concentrations, the obtained solutions were diluted with methanol.

2.3. Chromatographic Conditions

TLC silica gel 60 F₂₅₄ plates were cut to obtain an appropriate dimension. The test samples were sprayed using a semi-automatic applicator (with 100 psi air pressure) using a 100 µL glass syringe with a constant sample application rate of 200 nL/s. The first application position X was 10.0 mm (distance from each side), application position Y was 10.0 mm (distance from lower edge), and distance between tracks (measured between the centers of the spots) was 20 mm. The application volume for the calibration curve ranged from 1.0 to 35.0 µL of a 0.005 µg/µL standard XN solution.

The plates with applied samples were dried for 10 min at room temperature (25 ± 2 °C) without exposure to light and then developed at a distance of 10 cm in a chromatographic chamber, previously saturated with the mobile phase for 15 min. The mobile phase contained toluene, 1,4-dioxane, and glacial acetic acid at a volume ratio of 37:10:1.5 v/v/v. The developing time was about 30 min. The procedure was applied for both the reference substance and preparation samples. The XN standard was applied to each of the tested chromatographic plates on the first position. Each sample was analyzed in triplicate.

The standard substance XN was in the first position in each of the analyses performed, and each of the tested samples were analyzed in triplicate.

The TLC 3 scanner was used to perform densitometric detection. A D2&W lamp emitting a continuous spectrum in the UV–Vis range was used for measurements. The scanning parameters were as follows: scanning speed 20 mm/s and slit dimensions 4.00 × 0.45 mm. Absorption spectra were recorded in the range of 200–800 nm, based on which the analytical wavelength of λ = 368 nm was selected and used for next measurements.

As a result of scanning, the following data were obtained: retention factors (Rfs), peak areas [mm²], peak areas in percent [%], and absorption spectra of the separated substances.

2.4. Statistical Analysis

For each dietary supplement and cosmetic included in the study plan, the extraction procedure and analysis were performed in triplicate. The obtained experimental data were expressed as the mean value ± standard deviation and analyzed using an ANOVA one-way analysis of variance, with significant differences between means being set at p < 0.05. Statistical analyses were conducted by software STATISTICA version 12.

2.5. Validation of the Method

The applied procedure was validated by determining the parameters: linearity, limit of detection (LOD), limit of quantification (LOQ), precision, repeatability, and accuracy [31].

Linearity: XN solutions at a concentration of 0.005 µg/µL were prepared by diluting the standard solution in methanol, then applied to chromatographic plates in volumes ranging from 1.0 to 35.0 µL and analyzed as described above. The peak areas were recorded, and a calibration curve was established by plotting the peak area [mm²] versus the amount of XN in the volume applied to the spot [µg/spot].

LOD = (3.3 × Sb)/a

(1)

LOQ = (10 × Sb)/a

(2)

where: Sb—standard deviation of the response; a—the slop of calibration curve.

The precision of the densitometer was examined by scanning the same XN band (volume 15 µL, concentration 0.005 µg/µL) 10 times and then calculating the RSD [%].

Repeatability (intra-day precision) was checked by applying 5 spots of XN standard solution to the TLC plate and calculating the RSD [%].

Inter-day precision was determined by applying 5 spots of a standard XN solution to a chromatographic plate, 7 days apart, and calculating the RSD [%].

The accuracy of the method was established by determining the recovery at three different concentration levels. For this purpose, known amounts of standard XN (corresponding to concentration levels of 80, 100, and 120%, respectively) were added to the sample solution, and the total amount of XN was determined as described above, and then the % recovery was calculated for each concentration level.

2.6. Stability Testing

A 0.02% methanolic solution of XN standard was prepared, which was mixed at a 1:1 (v/v) ratio with 0.2 mol/L HCl, 0.2 mol/L NaOH, 6% H₂O₂ solution (to obtain final solutions for tests with a concentration of 0.1 mol/L HCl, 0.1 mol/L NaOH, and 3% H₂O₂) and distilled water in tightly closed 2.0 mL vials. The obtained solutions were incubated in a thermoblock at temperatures of 25, 60, and 90 °C in the time range of 0 to 5 h, taking samples at an interval of 1 h, and they were then subjected to chromatographic analysis under previously described conditions. Each determination was repeated three times.

2.7. Antioxidant Activity

The antioxidant activity and reducing and chelating power properties of XN samples were evaluated using four various methods, described below. The mentioned activity was assessed in relation to the reference substance, which was a vitamin C solution.

2.7.1. DPPH Assay

To 3 mL of DPPH solution at a concentration of 0.1 mmol/L, 40 µL of methanolic solutions of AA or XN (in concentrations from 0.03 to 2.40 mmol/L) were added, which were then vigorously mixed and then incubated for 20 min in a dark place (temp. 22 °C). Then, at a wavelength of =517 nm, absorbance values were measured for the tested solutions. DPPH solution was used as a reference solution. The method of scavenging DPPH free radicals is characterized by high effectiveness and short duration [32]. After mixing the reactants, the color of the solution changes from dark purple (color of DPPH ethanol solution) to yellow (color of the mixture after adding the antioxidant). This is the result of the DPPH reduction reaction, which occurs under the influence of the antioxidant (HA). The reaction can be schematically described by the following equation:

$$\left(\mathrm{D}\mathrm{P}\mathrm{P}\mathrm{H}\right)+\left(\mathrm{H}\mathrm{A}\right)\to {\mathrm{D}\mathrm{P}\mathrm{P}\mathrm{H}}_2+\left(\mathrm{A}\right)$$

(3)

The formation of the reduced product (DPPH₂) causes a decrease in absorbance. The antioxidant activity of a compound in terms of its ability to scavenge free radicals can be calculated based on the equation:

$$\%\mathrm{R}\mathrm{S}\mathrm{A}={\displaystyle \frac{\mathrm{A}\mathrm{b}\mathrm{s} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}-\mathrm{A}\mathrm{b}\mathrm{s} \mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}{\mathrm{A}\mathrm{b}\mathrm{s} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}}\times 100$$

(4)

where: RSA—radical scavenging activity; Abs control—the absorbance of the control solution; Abs sample—the absorbance of the sample solution.

2.7.2. Reduction of Iron (III) Ions

Each sample was prepared by combining 140 µL of either AA or XN solution (in methanol) with 140 µL of phosphate buffer at pH = 6.6 and 250 µL of 1% potassium hexacyanoferrate (III) solution. The resulting mixtures were incubated at 50 °C for 20 min, followed by a cooling period. Then, 250 µL of 10% TCA solution and 3 mL of 0.1% iron (III) chloride solution were introduced into each sample. The absorbance was then recorded at 700 nm to evaluate the solutions. The antioxidant activity of the tested compound was assessed based on its ability to reduce hexacyanoferrate (III) to hexacyanoferrate (II), according to the following formula:

$${\mathrm{F}\mathrm{e}\left(\mathrm{C}\mathrm{N}\right)}_6^{3-}+\mathrm{A}\mathrm{H}\to {\mathrm{F}\mathrm{e}\left(\mathrm{C}\mathrm{N}\right)}_6^{4-}+\mathrm{A}+{\mathrm{H}}^{+}$$

$${\mathrm{F}\mathrm{e}\left(\mathrm{C}\mathrm{N}\right)}_6^{4-}+{\mathrm{F}\mathrm{e}}^{3+}\to {\mathrm{F}\mathrm{e}}_4{\left[\mathrm{F}\mathrm{e}\right(\mathrm{C}\mathrm{N})}_6^{4-}{]}_3+\mathrm{A}+{\mathrm{H}}^{+}$$

The introduction of iron (III) ions into the reaction medium results in the formation of iron (III) hexacyanoferrate (II), a dark blue compound commonly known as “Prussian blue”. Antioxidant compounds change the color of the reagent mixture from yellow to various shades of green and ultimately to dark blue. Depending on the degree of antioxidant activity of the compound, the color intensity of the resulting solution varies [33].

2.7.3. Phosphomolybdenum Method

To 200 µL of either AA or XN solution (dissolved in methanol), 660 µL of a mixture containing 0.6 mol/L sulfuric acid, 4 mmol/L ammonium heptamolybdate, and 28 mmol/L ammonium phosphate (in equal volumes, 1:1:1) was added. The resulting mixture was incubated at 95 °C for 90 min. Following incubation, the solutions were cooled to room temperature, and absorbance was measured at a wavelength of 695 nm.

The phosphomolybdenum method relies on the reduction of Mo (VI) to Mo (V) and the formation of a colored complex with phosphate present in the reaction medium. The intensity of the color produced, which shifts from green to blue, correlates with the level of antioxidant activity present. A deeper color signifies higher antioxidant potency [34]. The total antioxidant capacity was estimated as the equivalent of AA by using following equation:

Antioxidant activity [%] = [(Absorbance control − Absorbance sample)/Absorbance control] × 100

(5)

2.7.4. Chelation of Iron Ions

For each 1 mL of AA or XN solution (dissolved in methanol) within a concentration range of 0.03–2.4 mmol/L, 80 µL of 20 mmol/L iron (II) chloride solution was added. Various amounts of ferrozine solution with a concentration of 2.9 mg/mL were added to the solutions in order to achieve an equal concentration of AA (or XN) and ferrozine in the final mixtures. The samples were mixed thoroughly and incubated at room temperature in the dark for 10 min. After incubation, the absorbance was recorded at 562 nm in the presence of the solvent alone as the reference solution. Additionally, Na₂EDTA solution was used as a positive control.

Compounds with low iron-chelating capacity cause a purple coloration, which diminishes as chelating strength increases. Consequently, stronger antioxidants lead to lighter-colored solutions, resulting in lower absorbance values [35]. The percentage of iron ion chelation was calculated using the following equation:

$$\mathrm{C}\mathrm{R}\left(\%\right)=\left(1-{\displaystyle \frac{\mathrm{A} \mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}{\mathrm{A} \mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k}}}\right)\times 100$$

(6)

where: CR(%)—chelating rate (the degree of chelation, expressed as a percentage); A sample—the absorbance of the tested compound solution; and A blank—the absorbance of the solvent.

The experiments were carried out in triplicate, and the results are given as the arithmetic mean. The data in all the experiments were analyzed statistically.

3. Results and Discussion

3.1. Validation Method

Based on the available literature [23], the XN analysis conditions were adopted and validated in our laboratory conditions in accordance with ICH guidelines in terms of linearity, sensitivity, precision, and accuracy. The obtained values of individual parameters are collected in

Table 1. Statistical parameters of the validation method.

Parameter	Statistical Evaluation
Linearity	0.01–0.100 µg/spot a = 57,264.35; b = 7.70; Sa = 1120.14; Sb = 66.268; Se = 100.12; r = 0.9987; r2 = 0.9973; F = 2613.51
LOD [µg/spot]	3.82 ng/spot
LOQ [µg/spot]	11.57 ng/spot
Recovery	80%	$\overline{x}=$ 99.23 SD = 0.39 S$\overline{x}$ = 0.23 RSD% = 0.39
	100%	$\overline{x}=$ 100.18 SD = 0.18 S$\overline{x}$ = 0.11 RSD% = 0.18
	120%	$\overline{x}=$ 101.20 SD = 0.73 S$\overline{x}$ = 0.42 RSD% = 0.72
Precision	intra-day	$\overline{x}=$ 3499.00 SD = 34.27 S$\overline{x}$ = 13.99 RSD% = 0.98
	inter-day	$\overline{x}=$ 3377.47 SD = 74.09 S$\overline{x}$ = 30.25 RSD% = 2.19

[31].

Silica gel 60F₂₅₄ TLC chromatography plates as a stationary phase and a mobile phase that consisted of toluene, 1,4-dioxane, and glacial acetic acid at a volume ratio of 37:10:1.5 (v/v/v) were chosen. Densitometric detection was performed at the analytical wavelength of 368 nm, and the obtained retardation factor Rf for XN was 0.50 (Figure 2).

The precision of the densitometer was examined by scanning the same XN band (volume 15 µL, concentration 0.005 µg/µL) 10 times. The calculated RSD values of about 2% indicate good repeatability of the obtained results.

Analyzing the obtained validation parameters, it can be stated that the proposed method shows a rectilinear dependence of the measured value (r = 0.9987) expressed as surface area [mm²] on the content of the determined substance in the range from 0.01 to 0.100 µg of XN per spot (Figure 3a). To confirm the validity of the calibration curve equation, a residual analysis was performed to identify potential outliers. If the regression model fits the data well, the residuals should show a random distribution around zero. Analyzing the obtained results, the distribution of residuals is random, and the dependence of raw residual values on concentration is characterized by a weak correlation (r = −0.8 × 10⁻⁶) (Figure 3b).

The calculated LOD and LOQ values (appropriately 3.82 and 11.57 ng/spot) indicate high sensitivity, while the RSD value, less than 1.0% for intra-day and about 2% for inter-day precision, indicates a high precision of the developed method. Based on the obtained peak areas, the percentage of XN recovery was calculated. At three levels of concentration (80, 100, and 120%) it was close to 100% and also indicates a high accuracy of the applied method (Table 1).

Based on the conducted tests and the obtained results, it was confirmed that the validated method is precise and accurate.

In the next stage of the research, using a previously validated procedure, a qualitative and quantitative analysis of dietary supplements and cosmetic products available on the market was carried out. First, the extraction process of the active substance from the tested products was optimized. The extraction efficiency was checked by changing the shaking time of the weighed substance in methanol. Ultimately, the best results were obtained after 60 min of shaking. The samples were then centrifuged at 3000× g for 10 min, and the supernatant was subjected to further chromatographic analysis. Based on the recorded peak areas corresponding to the XN standard solutions and the tested preparations, the XN content was calculated and then compared with the content declared by the manufacturer on the product leaflet. The obtained results, presented in

Table 2. Evaluation of xanthohumol content in the tested preparations (n = 5).

Preparation	Content Declared by Manufacturers	Determined Content	Statistical Parameters
Dietary supplement 1	40 mg/2 mL	11.960 11.886 12.045 12.086 11.689	$\overline{\mathrm{x}}=$ 11.933 mg/2 mL SD = 0.1569 RSD% = 1.31
Dietary supplement 2	200 mg/3 caps.	15.985 16.319 16.454 15.959 16.224	$\overline{\mathrm{x}}=$ 16.188 mg/3 caps. SD = 0.2138 RSD% = 1.32
Dietary supplement 3	No data/mL	1.762 1.750 1.749 1.787 1.779	$\overline{\mathrm{x}}=$ 1.765 mg/mL SD = 0.0171 RSD% = 0.97
Cosmetic 1	No data/amp.	0.273 0.267	$\overline{\mathrm{x}}=$ 0.27 mg/amp. SD = 0.0026
		0.274	RSD% = 0.97
		0.272
		0.271
Cosmetic 2	No data/vial.	0.141	$\overline{\mathrm{x}}=$ 0.141 mg/vial.
		0.141	SD = 0.0011
		0.142	RSD% = 0.78
		0.139
		0.141

where: $\overline{\mathrm{x}}$—arithmetic mean; SD—standard deviation; RSD%—relative standard deviation.

, indicate a very low degree of compliance between the experimentally determined composition of each of the analyzed products and the value declared by the manufacturer (<29% compliance).

3.2. Stability Analysis

The stability analysis of the test compound involves exposing the probes to numerous factors that could theoretically cause their degradation. The substance may be subjected to various environmental conditions, such as acid and base hydrolysis, oxidation, or thermal degradation.

In the 0.1 mol/L NaOH solution (immediately after preparing the solution), the presence of an additional peak with Rf = 0.20 was recorded (probably originating from degradation product), the area of which decreases during heating, reaching zero after 2 h of incubation (Figure 4). This relationship was observed at all tested temperatures.

To assess the quality of the obtained chromatographic separation for the peaks recorded in the densitograms, the following coefficients were determined: retention factor (k), separation factor (α), and peak resolution (RS). The above-mentioned coefficients allow for the numerical assessment of the separation of individual peaks and determine the possibility of simultaneous quantitative assay of the components corresponding to them.

The following formulas were used to calculate these coefficients:

k = (1 − RF)/RF

(7)

α_n = (k_(n+1))/k_n

(8)

RS = (z₂ − z₁)/(0.5 (w₁ + w₂))

(9)

where: n—peak number; z—the distance between the start line and the peak maximum; and w—width of the peak base.

The calculated parameters (

Table 3. Separation parameters determined for the peaks recorded in the densitograms.

Peak	RF	k	α	RS
A (1)	0.20	4	-	-
XH (2)	0.50	1	0.25	4.36

) confirm good separation of the recorded peaks (RS > 4).

Under the remaining tested environmental and temperature conditions (H₂O, 0.1 mol/L HCl, and 3% H₂O₂), no additional peaks were recorded in the densitograms at all time points. A reduction in the peak area corresponding to XN was recorded, which for the aqueous solution was approximately 25 and 30% at 25 and 60 °C, respectively, and reached a value of approximately 70% at 90 °C after 5 h of incubation. The highest degree of degradation was found in HCl solution with a concentration of 0.1 mol/L, where after 3 h of incubation at 90 °C, no peak corresponding to XN was detected. Similarly, in the environment of 0.1 mol/L NaOH and 3% H₂O₂, after 5 h of incubation at 90 °C, it was found that the tested substance was completely decomposed (

Table 4. Peak areas [mm²] recorded for xanthohumol under various incubation conditions.

Time [h]	H2O (n = 3)
	25 °C	[%]	60 °C	[%]	90 °C	[%]
0	13,375.33	100.00	13,375.33	100.00	13,350.10	100.00
1	13,289.50	99.36	12,473.40	93.26	10,272.43	76.95
2	12,952.22	96.84	11,259.84	84.18	6656.13	49.86
3	11,740.53	87.77	10,778.83	80.59	5712.70	42.79
4	10,589.59	79.17	9854.35	73.66	4564.37	34.19
5	9985.78	74.66	9265.54	69.27	4337.67	32.49
Time [h]	0.1 mol/L HCl (n = 3)
	25 °C	[%]	60 °C	[%]	90 °C	[%]
0	12,989.10	100.00	12,989.10	100.00	6242.23	100.00
1	12,450.30	95.85	11,448.21	88.14	2357.37	37.76
2	11,576.42	89.12	10,807.27	83.20	1982.86	31.77
3	10,871.61	83.70	10,017.53	77.12	1098.00	17.59
4	10,086.50	77.65	9282.37	71.46	0.00	0.00
5	9682.38	74.54	8267.53	63.65	0.00	0.00
Time [h]	0.1 mol/L NaOH (n = 3)
	25 °C	[%]	60 °C	[%]	90 °C	[%]
0	5745.40	100.00	5745.40	100.00	5745.40	100.00
1	5481.73	95.41	4230.37	73.63	3399.25	59.16
2	5037.49	87.68	2807.92	48.87	2349.43	40.89
3	4539.85	79.02	1430.61	24.90	1224.47	21.31
4	4063.51	70.73	1000.84	17.42	698.33	12.15
5	3739.50	65.09	970.52	13.89	0.00	0.00
Time [h]	3% H2O2 (n = 3)
	25 °C	[%]	60 °C	[%]	90 °C	[%]
0	8082.77	100.00	8082.77	100.00	8082.77	100.00
1	7931.11	98.12	7628.47	94.38	5793.67	71.68
2	7485.52	92.61	6108.63	77.56	3225.90	39.91
3	7254.10	89.27	4754.62	58.82	1196.89	14.81
4	7008.09	86.70	4089.96	50.61	563.89	6.98
5	6800.98	84.14	3394.57	41.99	0.00	0.00

).

Then, natural logarithms were calculated from the percentage loss of XN concentration over time to determine the order of its degradation reactions in the tested conditions.

When analyzing the determined parameters of the lnc = f(t) regression curves (

Table 5. Statistical parameters describing the relationship lnc = f(t) for the tested solutions (n = 3).

Time [h]	a	b	Sa	Sb	Se	r	F
H2O
25 °C	−0.0654	4.6519	0.0086	0.0260	0.0359	0.9672	57.99
60 °C	−0.0746	4.6048	0.0037	0.0113	0.0156	0.9951	402.29
90 °C	−0.2351	4.5262	0.0278	0.0843	0.1165	0.9731	71.32
0.1 mol/L HCl
25 °C	−0.0626	4.6148	0.0023	0.0069	0.0096	0.9973	745.51
60 °C	−0.0857	4.5043	0.0051	0.0155	0.0214	0.9930	281.25
90 °C	−0.539	4.4510	0.1066	0.1995	0.2384	0.9630	25.56
0.1 mol/L NaOH
25 °C	−0.0900	4.6333	0.0044	0.0132	0.0183	0.9953	425.25
60 °C	−0.4257	4.6476	0.0297	0.0898	0.1241	0.9904	206.07
90 °C	−0.5240	4.6400	0.0246	0.0602	0.0777	0.9967	454.60
3% H2O2
25 °C	−0.0380	4.6133	0.0024	0.0074	0.0102	0.9919	244.55
60 °C	−0.1863	4.6724	0.0133	0.0404	0.0558	0.9899	195.24
90 °C	−0.6910	4.8240	0.0691	0.1692	0.2184	0.9853	100.12

), it was found that the XN degradation process in all tested conditions follows a linear relationship (first-order reaction kinetic), which is confirmed by the values of the correlation coefficients (r) being close to one, as well as the high F-values.

In the next stage of the research, the kinetic parameters of the XN degradation process were determined, such as:

-

Reaction rate constant

$$\mathrm{k}=2.303\times {\displaystyle \frac{\mathrm{l}\mathrm{o}\mathrm{g}{\mathrm{c}}_1-\mathrm{l}\mathrm{o}\mathrm{g}{\mathrm{c}}_2}{{\mathrm{t}}_2-{\mathrm{t}}_1}}$$

(10)

where: c₁, c₂—concentrations [%] after time t₁, t₂ [h]).

-

Time needed to reduce the concentration of the substance to half of the initial value.

t_0.5 = 0.635/k

(11)

-

And the time at which the degradation reaches 10% of the initial value.

t_0.1 = 0.1053/k

(12)

-

And thermodynamic, such as activation energy E_a.

$$\mathrm{l}\mathrm{n}{\displaystyle \frac{{\mathrm{k}}_2}{{\mathrm{k}}_1}}={\displaystyle \frac{{\mathrm{E}}_{\mathrm{a}}}{\mathrm{R}}}\times {\displaystyle \frac{{\mathrm{T}}_2-{\mathrm{T}}_1}{{\mathrm{T}}_1·{\mathrm{T}}_2}}$$

(13)

where: T—temperature [K]; T₂ > T₁, R—gas constant (8.315 J/molK); k₂ > k₁.

The determined kinetic and thermodynamic parameters differ depending on the environment and temperature used during the degradation processes. With increasing k values and decreasing times t_0.5 and t_0.1, the stability of a compound decreases, i.e., the rate of its degradation in given conditions increases. The highest values of the reaction rate constant were observed for the solution of XN in 3% H₂O₂ at 90 °C (k = 0.6657 h⁻¹), where it is decomposed most rapidly. The slowest degradation occurred for XN in water at 25 °C (k = 0.0585 h⁻¹). The obtained k values in water were lower than the k values obtained for the analyzed acidic and basic solutions, as well as in the presence of an oxidizing agent, at all tested temperatures (

Table 6. Calculated kinetic and thermodynamic parameters of the xanthohumol degradation process.

Temperature [°C]	k [h−1]	t0.5 [h]	t0.1 [h]	Ea [kJ/moL]
H2O
25	0.0585	10.86	1.80	-
60	0.0734	8.65	1.43	80.85
90	0.2249	2.82	0.47	1675.65
0.1 mol/L HCL
25	0.0588	10.80	1.79	-
60	0.0904	7.03	1.17	153.25
90	0.5794	1.09	0.18	2780.23
0.1 mol/L NaOH
25	0.0859	7.39	1.23	-
60	0.3949	1.61	0.27	543.54
90	0.5271	1.20	0.20	432.13
3% H2O2
25	0.0345	18.38	3.05	-
60	0.1736	3.66	0.61	575.73
90	0.6657	0.95	0.16	2011.44

). The determined values of the kinetic parameters indicate that the values of the reaction rate for all analyzed samples increase with the increase in temperature, while the values for parameters t_0.5 and t_0.1 decrease. Both the environment and used temperature affect the given substance, causing its degradation. The energy needed to dissolve the chemical compound may come from the dissolved substance (it can be absorbed or released as heat), or its source may be the reaction environment. The lowest value of the determined activation energy was found for the aqueous environment at 60 °C 80.85 kJ/moL, while the highest was found in the 0.1 mol HCl environment at 90 °C 2780.23 kJ/moL (Table 6).

Comparing the validation parameters obtained in the TLC procedure using densitometric detection with the parameters presented by other authors (available methods discussed in the Introduction [13,14,15,16,17,18,19,20,21,22,23]), we can state that we received appropriately low LOD and LOQ values in a comparable concentration range. The method is also characterized by good precision and accuracy. Compared with chromatographic (HPLC) and spectrometric methods, which are time-consuming and require higher financial outlays, the procedure we propose is cost-effective and does not require the tedious and time-consuming preparation of high-purity samples as well as high-quality solvents. Moreover, many samples can be determined simultaneously during a single analysis. TLC-densitometry can be an excellent alternative to other available methods for the qualitative and quantitative assessment of preparations containing XN as well as stability studies.

3.3. Antioxidant Activity Testing

The literature data indicate that antioxidants play an important role in maintaining human health, preventing diseases, and also treating them, which results from their ability to reduce oxidative stress. The measurement of the antioxidant activity/capacity of samples is necessary both for quality assurance and, more importantly, to investigate the effectiveness of antioxidants in preventing and treating diseases associated with oxidative stress.

The analysis of the antioxidant capacity of compounds related to the chelation of iron (II) ions allows for the determination of the ability of a substance containing OH groups to complex ions of this metal. The resulting combination prevents the reaction of free iron ions with reactive oxygen species. Studies using the reduction reactions of both iron (III) and molybdenum (VI) ions allow for the determination of whether the oxidation potential of the tested substance is lower/higher than the oxidation potentials of the used ions with oxidizing properties. In turn, the test using the assessment of the efficiency of 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging allows for the assessment of the antioxidant properties of the substance with particular consideration of the strength of free radical binding.

The results of free radical scavenging and the reducing and chelation power of the XN and AA solutions are shown in Figure 5. In the tested samples, the antioxidant activity was found to be concentration-dependent, which led to the general conclusion that the strength of the action potential increased with increasing concentration.

In antioxidant capacity assays, XN showed significantly higher radical scavenging in the entire range of analyzed concentrations. The phosphomolybdate method used helps to determine whether the tested compound can act as an antioxidant. The ability of compounds to participate in a reduction reaction is related to their electron-donating and antioxidant potential. Compounds with antioxidant activity can act as reducing agents, thereby deactivating oxidants. Iron ions are essential for the proper functioning of the human body. However, even a small excess of them in physiological conditions can lead to various irregularities, including reactions that facilitate the synthesis of ROS. Due to its high degree of reactivity, iron is an important pro-oxidant, taking part among others in the lipid peroxidation process. Therefore, the potential of a specified substance to chelate iron is a very important aspect from the clinical point of view [35].

The obtained results show that XN demonstrates a stronger antioxidant effect than ascorbic acid in the entire range of analyzed concentrations. The total antioxidant capacity of this compound was tested at different doses, and it was observed that the antioxidant power increases slightly with concentration, reaching maximum values at a concentration of 0.15 mmol/l, and then it decreases significantly (Figure 6). The data obtained during the analysis of iron ion reduction results confirm these observations.

Then, the antioxidant activity of XN was computed, expressed in its ability to capture free radicals (%RSA; Figure 7). The results indicate a high antioxidant capacity of XN, but no significant changes were observed in the tested concentration range.

The chelating effect on iron ions was determined by measuring the rate of reduction of the color of iron ion complexes. Ferrozine, by forming a complex with free Fe (II) ions, reduces the amount of Fe (II)–ferrozine complexes formed after adding an antioxidant agent. In the tested concentration range (from 0.15 to 2.4 mmol/L), high absorbance values were recorded. The calculated values of the degree of chelation (CR%; Figure 8) indicate the lack of chelating capacity of XN.

To conclude, we report notable information on the basic antioxidant properties of XN, as well as a simple, rapid, and inexpensive procedure for the qualitative and quantitative determination of XN and for stability testing under variable environmental and temperature conditions.

In the conducted studies, significant differences in the content of XN were observed in the products covered by the research plan compared with those declared by the manufacturer. This may be influenced by the stability of the active substance, i.e., the method of storage (too high of an ambient temperature) and reactions with active/additional substances (e.g., acidic environment often used in the case of cosmetics). Considering the fact that the half-life (t_0.5) of XN at 25 °C is about 10 h, it would be recommended to store products containing XN at a reduced temperature. However, such large differences in the marked content of XN in dietary supplements and cosmetic products compared with those declared by the manufacturer may result mainly from the lack of quality control during the production process. Therefore, quality control is already so important at the stage of selecting the raw material for production, as well as at each stage of its production until the final product is obtained.

4. Conclusions

With its multi-faceted biological properties, XN has significant therapeutic potential and may be widely used in dietary supplements and cosmetic products. Its ability to modulate key signaling pathways involved in inflammatory and oncogenic processes positions it as a promising alternative to NSAIDs, offering similar effects with reduced risks of side effects. XN may also enhance the efficacy of antibiotics, which is important in light of the growing issue of antibiotic resistance, and holds potential in the treatment of viral infections. The development of xanthohumol-based products could improve public health, making it a significant and innovative component of dietary supplements and cosmetics. However, the analysis of products available on the market containing XN, as shown in our research, clearly indicates that the content of the active substance significantly differs from that declared by the manufacturers, which is also associated with the lack of the expected biological effects.

The results of the current study represent an important step in understanding the need to control dietary supplements and cosmetics commercially available on the market. As a future perspective, it would be valuable to conduct additional, extended stability studies of XN including, among others, photostability studies.