The 2 N Folin-Ciocalteu reagent, allopurinol, quercetin, xanthine, hypoxanthine, XO, rutin, dimethyl sulfoxide (DMSO), carboxymethylcellulose (CMC), potassium oxonate, UA, ascorbic acid, gallic acid, and creatinine were obtained from Sigma‒Aldrich (St. Louis, MO, USA). Sodium carbonate, acetic acid, hydrochloric acid, disodium hydrogen phosphate, potassium dihydrogen phosphate, aluminum chloride, and potassium acetate were of reagent grade. Ethanol and acetonitrile were of liquid chromatography grade (Mallinckrodt Baker Inc., Mexico City, Mexico). Deionized water (MontRial, San Luis Potosi, Mexico) was used for the aqueous solutions.

L. leucocephala (Lam.) de Wit samples were collected from Santa María Tonameca, Oaxaca de Juárez, Mexico, in November 2019 and authenticated by Eleazar Carranza González, who is affiliated with the Herbarium Isidro Palacios of the Instituto de Investigación de Zonas Desérticas of the Universidad Autónoma de San Luis Potosi, with a voucher specimen then deposited in the herbarium. Subsequently, the leaf samples were air-dried without exposure to the sun. Two hundred eighty-two grams of the powdered leaf sample was macerated in 1 liter of ethanol:water 96:4 v/v at room temperature (22 °C) for 12 d. The resulting solution was filtered, and the solvent was removed under reduced pressure at 40 °C. Finally, the plant extract was stored at 0 °C and protected from exposure to light.

The total polyphenol content in leaf extracts was determined using a modified version of the Folin-Ciocalteu method (Musci and Yao, 2017), wherein 30 µL of leaf extract, representing 0.0112 mg dry weight, or gallic acid standard solution was mixed with 150 µL of Folin-Ciocalteu reagent diluted to 1:10 with deionized water. After 5 min at room temperature, 120 µL of a 10 % (w/v) sodium carbonate solution was added to the mixture and incubated at room temperature for 30 min. The mixture was then centrifuged at 3000 × g for 5 min at 10 °C, and 200 µL of the supernatant was placed into one well of a 96-well plate (Costar^® 3595, Corning Incorporated, NY, USA), and the absorbance measured at 765 nm against a blank sample (ethanol). Polyphenol concentrations were obtained by interpolating the absorbance values of the extract samples into gallic acid calibration curves. Using the data of volume and grams of the extract used, the results are expressed as mg gallic acid equivalents/g of extract.

The total flavonoid content in extracts was quantified by a colorimetric technique (Farasat et al., 2014), wherein 40 µL of leaf extract, representing 0.0150 mg dry weight, or rutin standard solution were mixed with 40 µL of a 10 % (w/v) aluminum chloride solution, 40 µL of a 1 M potassium acetate solution, and 360 µL deionized water. After 30 min at room temperature, 200 µL of the mixture was placed into one well of a 96-well plate, and the absorbance was recorded at 415 nm against blanks (ethanol). Considering the data of interpolation into rutin calibration curves and volume and grams of the extract used, the flavonoid concentration is expressed as mg rutin equivalents/g of extract.

A liquid chromatographic (LC) analysis was performed as described previously (Seal, 2016; Cefali et al., 2019) for the identification of flavanols and flavones and the quantification of gallic acid, quercetin, and rutin in the extract samples. The analysis was optimized for quercetin and rutin, as these two flavonoids are described as the main compounds found in biologically relevant extracts obtained from natural sources (Cefali et al., 2019). For chromatographic separation, the column compartment was maintained at 24 °C, while the mobile phase consisted of 1 % acetic acid in an acetonitrile gradient. The separation and column regeneration conditions applied were 80 % mobile phase plus 20 % acetonitrile for 2 min, followed by a change to 10 % mobile phase and 90 % acetonitrile for 8 min and a second change to 80 % mobile phase plus 20 % acetonitrile for 5 min, with the final composition then maintained for 5 min. The analysis was performed using an injection volume of 20 µL of the sample dissolved in ethanol at a 1 mL/min rate, with monitoring conducted at 257 nm, a run time of 20 min, and drawing spectra of 200 to 400 nm. Gallic acid, rutin, and quercetin were identified by means of their retention times and spectra records, while their concentration in the plant extract was obtained by interpolating their responses into calibration curves performed for each pure compound, with the results expressed as µg compound/g of leaf extract. The other flavanols and flavones were identified using the spectral data for their B-ring cinnamoyl and A-ring benzoyl systems, which are observed as two major UV absorption peaks (Band I and Band II) in this class of compounds (Mabry et al., 1970). The percentage abundance of flavanols and flavones in the extract was calculated using the area values of their peaks and all peaks eluted in the chromatogram.

The assay was performed following that set out by Nguyen et al. (2004), with some modifications. Stock solutions of XO (0.37 U/mL) and xanthine (1 mM) were prepared in 70 mM phosphate buffer solution (pH 7.5), while stock solutions of allopurinol (1.4 mg/mL), plant extract (9.5 mg/mL), and quercetin (6 mg/mL) were prepared in ethanol:DMSO 96:4 v/v. Quercetin was used as a positive control for the action of flavonoids against XO activity. Subsequently, working standards for allopurinol, quercetin, and plant extract were prepared using ethanol:water 1:1 v/v and ranged from 0.16 to 41.30, 1.20 to 153.75, and 4.07 to 1563.00 µg/mL, respectively (seven to nine points per sample, N= 5). For each preparation tested, the number of standards and the range of concentrations were established in accordance with the guidelines to accurately estimate the concentration level that was able to achieve a 50 % inhibition (IC₅₀) of XO activity (Sebaugh, 2011). The assay mixture comprised 100 µL of test solution, 70 µL of 70 mM phosphate buffer (pH 7.5), and 60 µL of XO stock solution. After preincubation at 37 °C for 30 min, the reaction was initiated by adding 120 µL xanthine stock solution, with the assay mixture then incubated again at 37 °C for 30 min, after which the reaction was stopped by adding 50 µL 1 N HCl. Two hundred microliters of each mixture were placed into a 96-well plate, and the absorbance was read at 290 nm.

A blank sample for each solution tested was prepared in the same way, with the enzyme solution added to the assay mixture after the addition of the 1 N HCl solution. Using the data obtained from the blank samples, the interference-free absorbance was calculated for each experimental sample. Then, the inhibitory effect on XO was expressed as the percentage of XO inhibition, which was calculated using the interference-free absorbance of the assay mixture, both with and without (0 µg/mL) the test material.

For the samples tested, constant ratio combinations were performed (Chou, 2006; Chou, 2010). Once the IC₅₀ value for each sample was obtained via the XO inhibition assay, the combination of allopurinol and plant extract was assessed via the same assay, using six different proportions. For the combinations of allopurinol and leaf extract, concentrations ranging from 0.65 to 20.82, 0.32 to 10.37, 0.66 to 21.29, 0.24 to 7.89, 0.44 to 14.27, and 0.75 to 24.13 µg/mL were used for the 1:3, 1:1, 3:1, 5:1, 10:1, and 30:1 combination, respectively (six points per combination, N= 5). Concentrations ranging from 0.65 to 20.92, 0.32 to 10.62, 0.35 to 11.18, 0.45 to 14.61, 0.42 to 13.42, and 0.28 to 8.94 µg/mL were used for the allopurinol and quercetin combinations at 1:3, 1:1, 3:1, 5:1, 10:1, and 30:1, respectively (six points per combination, N= 5).

Data obtained for the combinations were evaluated using the Chou-Talalay method, which is based on the median-effect equation, which, in turn, is derived from the mass-action law principle (Chou, 2006). The Chou-Talalay theory involves the quantitative definition of synergism, additivity, and antagonism by means of a combination index (CI) value and their visual definition by means of an isobologram (Chou, 2006). Furthermore, the theory includes a dose-reduction index (DRI), which measures decreasing dose folds of each drug (Chou, 2006).

A weighted average CI (waCI) value was calculated for each combination using the following formula: waCI = [CI₉₀ + (2 x CI₇₅) + (3 x CI₅₀) + (4 x CI₃₀)]/10. This formula was designed to increase the relevance of low effect levels, as XO inhibitors should be used at a sufficiently low concentration to achieve the target serum urate level in gout patients (Chou, 2006; Stamp and Barclay, 2018; Checkmahomed et al., 2020). For synergistic or additive interactions only, a weighted average DRI (waDRI) value was determined for each component in the mixture using the formula waDRI = [DRI₉₀ + (2 x DRI₇₅) + (3 x DRI₅₀) + (4 x DRI₃₀)]/10, with low effect levels also of high relevance for this formula.

Once the experiments described above had been completed and their results analyzed, the utility of the leaf extract and its combined use with allopurinol in the animal model was evaluated. All animal procedures were approved by the university’s Institutional Animal Care and Use Committee (approval number: BGFMUASLP-06-19) and conducted in full compliance with international guidelines (National Research Council US, 2011). Male BALB/c mice (16 - 20 g) were kept under standard laboratory conditions (room temperature 21 ± 1 °C with a 12 h dark and light schedule) and housed in acrylic cages with free access to water and a standard diet. All mice were housed under laboratory conditions for one week before the experiment.

Hyperuricemia was induced via potassium oxonate (uricase inhibitor) and hypoxanthine, in accordance with Lemos Lima et al. (2015) and Yong et al. (2016), with some modifications. Hypoxanthine, allopurinol, and leaf extract were suspended in 0.3 % CMC aqueous solution, while potassium oxonate was suspended in 0.5 % CMC aqueous solution. The mice were randomly divided into six experimental groups (N = 5 for each group) and subjected to fasting 2 h prior to the injection of the uricase inhibitor. The inhibitor (280 mg/kg bw, i.p.) was administered once daily to the animals from Groups 2 - 6 for three consecutive days. Vehicle (8.0 mL/kg bw), allopurinol at 2.5 mg/kg bw, leaf extract (729.2 mg/kg bw), allopurinol-extract combination at a ratio of 3:1 (2.2 mg/kg bw allopurinol plus 0.7 mg/kg bw extract), or allopurinol at 2.2 mg/kg bw were administered ten minutes after the application of the inhibitor via gavage once a day for three days to the animals of Groups 2, 3, 4, 5, and 6, respectively.

One hour after the potassium oxonate injection, the hypoxanthine suspension was administered by gavage to the mice in Groups 2 - 6 (268.0 mg/kg bw). Group 1 was administered the vehicle via intraperitoneal and oral routes (8 mL/kg bw) for the same periods of time as the other groups. The allopurinol dose (2.5 mg/kg, Group 3) was selected to produce a uric acid level close to that found in the animals administered the vehicle (Group 1), while the doses of the extract alone (Group 4) or combined with allopurinol (Group 5) were determined on the basis of their IC₅₀ values and in vitro interaction studies. Group 6 was administered the dose of allopurinol used in the foregoing combination but applied without extract. The oral administration of allopurinol and leaf extract was selected, as it is the most preferred route of administration in patients with hyperuricemia.

On the third day, one hour after hypoxanthine administration, mice were anesthetized with a combination of ketamine and xylazine (100 and 20 mg/kg bw, respectively), and their blood was then collected by means of cardiac puncture. Each blood sample was kept at 4 °C for 1 h and then centrifuged at 3000 × g for 10 min at 4 °C. The resultant serum samples were separated and frozen at -70 °C for further analysis.

LC analysis was performed as described previously (Tsikas et al., 2004; Pleskacova et al., 2017) with some modifications. For chromatographic separation, the column compartment was maintained at 32 °C, while the mobile phase consisted of a phosphate buffer solution (100 mmol/L potassium dihydrogen phosphate at pH 6.0) in an acetonitrile gradient. Separation and subsequent column regeneration conditions applied were 100 % phosphate buffer solution for 4 min, followed by a change to 70% phosphate buffer solution and 30 % acetonitrile for 30 s, with this composition then maintained for 2 min, followed by a second change to 100 % phosphate buffer solution for 30 s, with, finally, this composition then maintained for 3 min. The analysis was performed with an injection sample volume of 20 µL and a flow rate of 1 mL/min, wherein monitoring was conducted at 236, 248, 263, and 292 nm for creatinine, hypoxanthine, xanthine, and uric acid, respectively, with a run time of 10 min and drawing spectra of 200 to 400 nm. Each compound was identified by retention time and spectrum record, their concentration in the serum samples was obtained by interpolating their response into calibration curves performed with the pure compounds. The results were expressed as µmol of compound/L of serum sample.

A Cytation™ 3 microplate reader controlled by Gen5™ software (Biotek Instruments Inc., Vermont, USA) was used for the absorbance measurements. A 1100 series Agilent LC system consisting of a quaternary pump with degasser, a standard autosampler, a thermostatted column compartment, and a diode array detector (Agilent Technologies, Palo Alto, CA, USA) was used for the LC analyses. Agilent ChemStation software for LC 3D systems was used for data collection, integration, and evaluation of the spectra records. The chromatographic separations described above were carried out using a 150 mm long AcclaimTM 120 C18 column with a 5 µm particle size and a 4.6 mm internal diameter (Thermo Fisher Scientific, CA, USA).

For the combination analyses, the waCI value was interpreted as described previously (Chou, 2006). If the data points fell on the isobologram hypotenuse, an additive effect was determined, while if the data points fell on the lower or upper left of the hypotenuse, synergism or antagonism was determined, respectively (Chou, 2006). A waDRI of > 1 indicated a more favorable dose reduction for the combination applied than that achieved using each compound alone (Chou, 2006). Additionally, the conformity of the data with the mass-action law was evaluated using the linear correlation coefficient (r) of the median-effect plot, where an r value close to 0.9 was considered an acceptable value (Martinez-Morales et al., 2022). The IC₅₀ values, combination analyses, and isobolograms were conducted using Compusyn software (ComboSyn Inc, NJ, USA).

The IC₅₀ values, maximal inhibitory effects (MIE), and serum oxypurine and creatinine levels are presented as the mean or mean plus standard deviation. These results were analyzed using one-way ANOVA with Tukey’s posttest, which was performed using GraphPad Prism 5 software (San Diego, CA, USA). A P value of < 0.05 was considered statistically significant.

Table 1 shows the identification and quantification of the L. leucocephala leaf extract components. Rutin and quercetin comprised 7.39 % and 0.77 % of the total flavonoids, respectively, while the phenolic compound gallic acid was abundant in the leaf extract, and a nonidentified compound (Peak 1) coeluted with it (Fig. 1). Gallic acid, rutin, and quercetin in the extract presented wavelength absorption maxima (λmax) values of 270, 260/350, and 250/370 nm, respectively (Fig. 1). Compounds (peaks) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11 were detected in extracts and exhibited λmax values at 270, 230, 270, 260/350, 260/350, 260/350, 260/350, 260/350, 260/350, 260/340, and 270/330 nm, respectively. Similar to quercetin and rutin, compounds 4 - 11 presented flavanol and flavone bands I and II. Retention times and spectra records for gallic acid, rutin, and quercetin in extracts coincided with those used as standards.

Table 1

Compound	Content in the extract
Total polyphenols	202.05 ± 21.89 mg GAE/ g extract
Total flavonoids	127.17 ± 24.62 mg RE/ g extract
Rutin	9397.4 ± 536.8 µg/ g extract
Quercetin	975.6 ± 79.5 µg/ g extract
Gallic acid	40922.0 ± 2189.6 µg/ g extract
Other flavones or flavanols	43.38 ± 0.82% of eluted peaks
Nonidentified compounds	17.60 ± 0.84% of eluted peaks

Every value includes the mean ± standard deviation (N = 5). GAE, gallic acid equivalents; RE, rutin equivalents.

Figure 1

UA and creatinine serum values remained unchanged in all tested groups. Mice in Groups 3, 5, and 6 presented high hypoxanthine and xanthine serum levels, in contrast with mice in Groups 1, 2, and 4. In terms of serum hypoxanthine levels, leaf extract administration to Group 5 modified the P value in contrast to Group 6, although both groups received the same dose of allopurinol (Fig. 3).

Figure 3

Mouse serum chromatographic analysis revealed peaks with λ_max values of 230, 250, 270, and 290 nm, which matched those of creatinine, hypoxanthine, xanthine, and uric acid standards, respectively (Fig. 4).

Figure 4