Journal of biological and health sciences

Universidad de Sonora

ISSN: 1665-1456

Original Article

Effects of a Leucaena leucocephala leaf extract on xanthine oxidase activity and serum oxypurine levels in mice

Efectos de un extracto de hojas de Leucaena leucocephala en la actividad de xantina oxidasa y en los niveles séricos de oxipurinas en ratones

Flavio Martinez-Morales1 , Juan R. Zapata-Morales2 , Juan F. López-Rodríguez3 , Othir G. Galicia-Cruz1 , Mario A. Isiordia-Espinoza4 and Othoniel H. Aragon-Martinez1*

1 Departamento de Farmacología, Facultad de Medicina, Universidad Autónoma de San Luis Potosí, San Luis Potosí, México.

2 Departamento de Farmacia, División de Ciencias Naturales y Exactas, Universidad de Guanajuato, Guanajuato, México.

3 Bioterio, Facultad de Medicina, Universidad Autónoma de San Luis Potosí, San Luis Potosí, México.

4 Instituto de Investigación en Ciencias Médicas, Departamento de Clínicas, División de Ciencias Biomédicas, Centro Universitario de los Altos, Universidad de Guadalajara. Tepatitlán de Morelos, Jalisco, México.


There is a need for novel alternatives to the medical use of allopurinol. In this sense, the present study obtained a leaf extract from L. leucocephala, and its chemical composition, inhibitory action against xanthine oxidase (XO) in vitro, inhi- bitory interaction between the extract and allopurinol, and the inhibitory action on XO in vivo using mice treated with potassium oxonate and hypoxanthine, were determined. Polyphenol and flavonoid compounds were found in the leaf extract. For the leaf extract, the IC50 and maximal values were 334.60 µg/mL and 46.4 % for the inhibition of XO. The 3:1 ratio combination of allopurinol and extract showed IC50 and waDRI values of 1.35 µg/mL, 1.13 (allopurinol) and 1015.72 (extract) to inhibit XO, resulting in a synergistic inte- raction against XO in vitro. This combination also enhanced the therapeutic success in the mouse model compared with allopurinol administered alone. The present study presents the first evidence for the use of an allopurinol and L. leuco- cephala extract combination at a 3:1 ratio, as a substitute for the administration of allopurinol alone.

Keywords: allopurinol; Chou-Talalay theory; synergism; hy- poxanthine


Hay una necesidad de nuevas alternativas al uso medicinal de alopurinol. En este sentido, el presente estudio obtuvo un extracto de hojas de L. leucocephala al cual se le determinó su composición química, acción inhibitoria contra xantina oxidasa (XO) in vitro, interacción inhibitoria entre el extracto y alopurinol, y acción inhibitoria contra XO in vivo empleando ratones bajo administración de hipoxantina y oxonato de potasio. Compuestos polifenoles y flavonoides fueron detec- tados y cuantificados en el extracto de hojas. Para el extracto solo, su valor IC50 y máximo de inhibición contra XO fueron de 334.60 µg/mL y 46.4 %. La combinación a una proporción 3:1 de alopurinol y extracto obtuvo valores IC50 y waDRI de 1.35 µg/mL, 1.13 (alopurinol) y 1015.72 (extracto) para inhibir a XO, resultando en una interacción sinérgica contra

XO in vitro. Donde esta misma combinación incrementó el éxito terapéutico en el modelo murino al compararse con la administración de alopurinol solo. Nuestro estudio presenta la primera evidencia para el uso de una combinación de alopurinol y extracto de L. leucocephala a una proporción 3:1 como un sustituto para la administración de alopurinol solo. Palabras clave: alopurinol; teoría de Chou-Talalay; sinergis- mo; hipoxantina


Uric acid (UA) is the end product of purine metabolism in humans. In the degradation pathway, xanthine oxidase (XO) transforms hypoxanthine to xanthine, which is then degra- ded into UA (Mehmood et al., 2020). The abnormal and excess accumulation of UA in the blood, known as hyperuricemia, is a risk factor for diabetes, cardiovascular complications, metabolic syndrome, and chronic kidney disease (Chen et al., 2019). Hyperuricemia can be caused by the overproduction or reduced excretion of UA (Mehmood et al., 2020; Kim et al., 2021a). Circulating UA is filtered by the glomeruli, and appro- ximately 90 % of the filtered material is usually reabsorbed into the renal proximal tubules via transporter molecules (Oh et al., 2019; Kim et al., 2021a). As kidneys are responsible for approximately 70 % of daily UA excretion (Mehmood et al., 2020), XO and renal urate transporters are important targets for the regulation of hyperuricemia in patients (Oh et al., 2019).

Clinically, allopurinol is commonly used to inhibit XO activity and thus reduce serum UA levels (Chen et al., 2019; Oh et al., 2019). Depending on the concentration, allopurinol may cause serious side effects, such as a reaction involving eosinophilia and systemic symptoms, toxic epidermal necrolysis, Stevens-Johnson syndrome, allopurinol hyper- sensitivity syndrome, and gastrointestinal toxicity (Stamp and Barclay, 2018; Oh et al., 2019). Consequently, there is a clear need for novel therapies for regulating serum UA levels or, alternatively, the use of allopurinol at lower doses. Plant- derived extracts have potential use as effective and natural

*Author for correspondence: Othoniel H. Aragon-Martinez e-mail:

Received: September 14, 2023

Accepted: March 1, 2024

Published: April 8, 2024

Volume XXVI


DOI: 10.18633/biotecnia.v26.2155

antihyperuricemic therapies (Kim et al., 2021a). Combination studies are common methods for identifying synergy among drugs to facilitate the reduction of doses while maintaining a similar level of therapeutic efficacy to that obtained from the use of the drug on an individual basis (Martinez-Morales et al., 2022).

Leucaena leucocephala is a leguminous tree native to southern Mexico and northern Central America. Previous studies on the use of different parts of the tree have found antioxidant, anti-inflammatory, and anticancer properties (Chung et al., 2017). To date, the effects of L. leucocephala extracts on in vitro XO activity or serum UA levels in either hu- mans or animal models have not been evaluated, although quercetin, which is one of the major flavonoids found in the leaf extracts obtained from this plant, inhibits the generation of UA and the superoxide radicals catalyzed by XO in vitro (Xu et al., 2018). Moreover, to our knowledge, a combination approach has never been applied to a combination of L. leu- cocephala extract and allopurinol.

The present study aimed to identify the in vitro syner- gistic inhibitory action exerted on XO, by a mixture of allo- purinol and L. leucocephala leaf extract and, subsequently, evaluate its effects on the serum levels of UA, xanthine and hypoxanthine in an experimental model of hyperuricemia recognized in mice (Liang et al., 2018).



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 SigmaAldrich (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 ace- tonitrile were of liquid chromatography grade (Mallinckrodt Baker Inc., Mexico City, Mexico). Deionized water (MontRial, San Luis Potosi, Mexico) was used for the aqueous solutions.

Collection and preparation of plant material

L. leucocephala (Lam.) de Wit samples were collected from Santa María Tonameca, Oaxaca de Juárez, Mexico, in Novem- ber 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 Univer- sidad 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 tem- perature (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.

Identification and content of polyphenols and flavonoids in the extract

The total polyphenol content in leaf extracts was determined using a modified version of the Folin-Ciocalteu method (Mus- ci 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) alu- minum 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 interpo- lation 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 condi- tions 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 compo- sition 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 in- terpolating 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 cinna- moyl 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.

Evaluation of the inhibitory effect on XO

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 concentra- tions 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 per- centage 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.

Interaction assessment of the XO inhibitors

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 allo-

purinol 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 isobolo- gram (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.

Drug administration and animal model

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 ad- ministered 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 admi- nistration, 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 fur- ther analysis.

Determination of serum UA, xanthine, hypoxanthine, and creatinine levels

LC analysis was performed as described previously (Tsikas et al., 2004; Pleskacova et al., 2017) with some mo- difications. 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 inter- polating their response into calibration curves performed with the pure compounds. The results were expressed as µmol of compound/L of serum sample.

Absorbance and chromatographic measurement

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 sys-

tem consisting of a quaternary pump with degasser, a stan- dard 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 chro- matographic 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).

Data analysis

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 determi- ned, 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 isobo- lograms were conducted using Compusyn software (Com- boSyn 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.


Chemical composition of the leaf extract

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, respecti- vely, while the phenolic compound gallic acid was abundant in the leaf extract, and a nonidentified compound (Peak 1)

Table 1. Chemical profile of the leaf extract.

Tabla 1. Perfil químico del extracto de hojas.

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. Representative chromatogram and spectra records for the compounds (G, Q, R, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11) eluted from an L. leucocephala leaf extract. The absorbance (mAU) on the Y-axis, the wavelength (nm) on the X-axis, and the time (min) on the Z-axis are plotted in each 3D spectrogram. G, gallic acid; R, rutin; Q, quercetin.

Figura 1. Cromatograma y espectrograma representativos de los compuestos eluidos del extracto de hojas de L. leuco-

cephala. La absorbancia (mAU) en el eje Y, la longitud de onda (nm) en el eje X y el tiempo (min) en el eje Z son trazados en cada espectrograma 3D. G, ácido gálico; R, rutina; Q, quercetina.

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.

Activity of XO inhibitors and their combinations in vitro Table 2 shows our IC50, MIE, r, waCI and waDRI values for leaf extract and compounds, as well as the interpretation of possible interactions. Isobolograms confirmed interactions between different allopurinol combinations with either quercetin or leaf extract (Fig. 2).

Effects of allopurinol, leaf extract, and the allopurinol and leaf extract combination in mice

UA and creatinine serum values remained unchanged in all tested groups. Mice in Groups 3, 5, and 6 presented high hy- poxanthine 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).

Mouse serum chromatographic analysis revealed peaks with λmax values of 230, 250, 270, and 290 nm, which matched those of creatinine, hypoxanthine, xanthine, and uric acid

standards, respectively (Fig. 4).


The present study shows, for the first time, that a 3:1 combi- nation of allopurinol and L. leucocephala leaf extract (equiva- lent to 2.2: 0.7 mg/kg dose) administered to hyperuricemia- induced mice exerted a similar inhibitory effect on the XO enzyme and increased the probability of therapeutic success to that produced by allopurinol administered alone (2.5 or 2.2 mg/kg). This combination enabled significant lowering of the individual doses of both allopurinol and leaf extract required to achieve similar effects. Such effects, which were markedly noticed in the extracts, also displayed synergistic action aga- inst XO. On the other hand, leaf extract alone (729.2 mg/kg) did not achieve any inhibitory effect on XO, coinciding with observations in the in vitro experiments.

L. leucocephala leaf extracts possess a great diversity of bioactive compounds, such as tannins, vitamin E, ascorbic acid, carotenes, xanthophylls, alkaloids, and phenolics,

Table 2. Results obtained from the extract, pure compounds, and their combinations in the XO inhibition assay.

Tabla 2. Resultados obtenidos del extracto, compuestos puros y sus combinaciones en el ensayo de la inhibición de XO.


IC50 value

r value

MIE value (%)

waCI value

Type of interaction

waDRI (A / H or Q)






















AH 1:3





Slight antagonism


AH 1:1





Nearly additive

0.97 / 301.69

AH 3:1





Slight synergism

1.13 / 1015.72

AH 5:1





Nearly additive

1.08 / 1643.50

AH 10:1





Slight antagonism


AH 30:1





Slight antagonism


AQ 1:3





Very strong antagonism


AQ 1:1





Strong antagonism


AQ 3:1





Strong antagonism


AQ 5:1







AQ 10:1





Nearly additive

0.98 / 104.29

AQ 30:1





Nearly additive

0.93 / 293.34

Every value includes the mean (N = 5). *P value of < 0.0001 versus A. A, allopurinol; H, leaf extract; MIE, maximal inhibitory effect; na, not applicable; Q, quercetin.

Figure 2. Isobolograms for the 30% (blue), 50% (red), and 90% (green) levels of XO inhibition induced by the 1:3 (a), 1:1 (b), 3:1 (c), 5:1 (d), 10:1 (e), and 30:1 (f ) combinations of allopurinol and quercetin or leaf extract. For the assays with allopurinol and quercetin: A, quercetin; B, allopurinol. For the assays with allopu- rinol and extract: A, allopurinol; B, leaf extract.

Figura 2. Isobologramas de la inhibición al nivel del 30%, 50% y 90% de XO inducida por las combinaciones 1:3 (a), 1:1 (b), 3:1 (c), 5:1 (d), 10:1 (e), and 30:1 (f ) de alopurinol con quercetina o extracto de hojas. Para los ensayos con alopurinol y quercetina: A, quercetina; B, alopurinol. Para los ensayos con alopurinol y extracto: A, alopurinol; B, extracto.

Figure 3. Serum levels (µmol/L) of uric acid (a), hypoxanthine (b), xanthine (c), and creatinine (d) in mice in groups 1, 2, 3, 4, 5, and 6. **P value of < 0.0001 versus groups 1, 2, and 4; *P value of < 0.05 versus groups 1 and 2 in graph b. *P value of < 0.05 versus groups 1, 2, and 4; and $P value of < 0.05 versus group 2 in graph c.

Figura 3. Niveles séricos (µmol/L) de ácido úrico (a), hipoxantina (b), xantina (c) y creatinina (d) en los ratones de los grupos 1, 2, 3, 4, 5 y 6.

**Valor P de < 0.0001 comparado con los grupos 1, 2 y 4; *Valor P de < 0.05 comparado con los grupos 1 y 2 en el gráfico b. *Valor P de < 0.05 comparado con los grupos 1, 2 y 4; $Valor P de < 0.05 comparado con el grupo 2 en el gráfico c.

mainly flavonoids (Chew et al., 2011; Xu et al., 2018). Fla- vonoid glycosides conjugated to either arabinose and rhamnose or glucuronide, galactose, and glucose have been isolated from L. leucocephala leaves, with quercetin, quercetin-3-O-α-rhamnopyranoside, and myricetin-3-O-α- rhamnopyranoside described as the major flavonoids (Xu et al., 2018). Other flavonoids present in low quantities include quercetin-3-O-α-arabinofuranose, naringenin, geraldone, 7,3-dihydroxy-4-methoxyflavone, apigenin, chrysoeriol, diosmetin, kaempferol, luteolin, 3,4,7-trihydroxyflavone, juglanin, kaempferol-3-O-α-rhamnopyranoside, (+) taxifolin, and myricetin (Xu et al., 2018). Coinciding with the foregoing, analysis of the L. leucocephala leaf extract administered in the present study showed that it contains polyphenol com- pounds, a large proportion of which were flavonoids, with quercetin in low quantities, while a substantial percentage of other flavones or flavanols were also identified (Table 1 and Fig. 1).

For the first time, the present study reported the presen- ce of gallic acid and rutin in an L. leucocephala leaf extract. The differences in the chemical composition and abundance of compounds in extracts obtained from the same plant species, may be due to its adaptation to the ecological en- vironment and the extraction method used, among other

factors (Cui et al., 2015; Córdova-Guerrero et al., 2016). Other compounds that can be measured with the chromatographic

method, such as ascorbic acid (λmax = 250 nm), catechin (λmax= 279 nm), methyl gallate (λmax= 275 nm), caffeic acid (λmax= 327, 295, 243, and 217 nm), syringic acid (λmax= 217 nm), p-coumaric acid (λmax= 285/305 nm), sinapic acid (λmax= 303 or 311 nm), ferulic acid (λmax= 285/300 nm), myricetin (λmax= 255/375), apigenin (λmax= 269/340 nm), and kaempferol (λmax= 268/369 nm) were not detected in our extracts (Barthelmebs

et al., 2000; Galano et al., 2011; Li et al., 2016; Tosovic, 2017;

Zhang et al., 2018; Kim et al., 2021b).

The L. leucocephala leaf extract presented a lower efficiency and potency in inhibiting the XO enzyme than allopurinol (Table 2). This finding cannot be compared with previous studies, as, to our knowledge, the present study is the first report on such effects. However, in line with our fin- dings, the pure flavonoid rutin exhibited a higher IC50 value than allopurinol during in vitro XO inhibition assays (Malik et al., 2019). In vitro evaluations also found that quercetin was equally or less efficient in inhibiting XO activity compared to leaf extract and allopurinol, respectively, displaying an intermediate potency compared to that observed between leaf extract and allopurinol (Table 2). The literature reports contradictory results for the in vitro effects of quercetin aga- inst the XO enzyme, including reports of either a greater or

Figure 4. Representative chromatograms obtained from a serum sample of mice pertaining to the group administered with oxonate, allopurinol, leaf extract, and hypoxanthine. Furthermore, the spectrum record for each peak is shown. The absorbance (mAU) represented on the Y-axis, the wavelength (nm) represented on the X-axis, and the time (min) represen- ted on the Z-axis are plotted in each 3D spectrogram.

Figura 4. Cromatogramas representativos obtenidos de una muestra sérica de ratón perteneciente al grupo con admi- nistración de oxonato, alopurinol, extracto de hojas e hipoxantina. Además, El espectro de cada pico es mostrado. La absorbancia (mAU) en el eje Y, la longitud de onda (nm) en el eje X y el tiempo (min) en el eje Z son trazados en cada espectrograma 3D.

lesser inhibition of XO than that achieved with allopurinol (Mohos et al., 2019).

To our knowledge, there are no previous reports of a combination study using allopurinol with L. leucocephala leaf extract and either pure rutin or quercetin. As observed in Table 2 and the isobolograms presented in Fig. 2, only the combination of allopurinol and leaf extract in a 3:1 ratio presented a slight synergistic interaction, as defined by the waCI value, which was observed at three of the four levels of effect tested (75, 50, and 30 %, with the exception of 90

%). This interaction was determined using the Chou-Talalay method, presenting an acceptable conformity with the theory applied. The synergistic interaction of the 3:1 ratio combination enabled a respective minor and major reduc- tion of the allopurinol and extract doses required (waDRI > 1). Other allopurinol and extract combinations presented a nearly additive or slight antagonism, wherein both types of interactions did not perform better than the synergistic inte- raction observed, a finding supported by the IC50, waCI, and waDRI values. Consequently, the 3:1 combination of allopuri- nol and leaf extract was selected to be administered in mice. Moreover, the allopurinol and pure quercetin combinations did not present superior performance to, or any advantage over, the allopurinol and leaf extract combination.

The administration of potassium oxonate in BALB/c mice did not result in a significantly high level of serum UA by Day 3 (Fig. 3). This situation was produced by the different urate excretion by kidneys of the present strain rodent, compared to the mice used in the original study and by the low absorp- tion of potassium oxonate from the peritoneal fluid into the systemic blood circulation (Bobulescu and Moe, 2012; Lemos Lima et al., 2015; Wishart et al., 2018; Al Shoyaib et al., 2020).

The administration of an oral hypoxanthine load in ani- mals (268 mg/kg) subjected to potassium oxonate injection, allied to the use of the LC measurement of oxypurines in serum samples (Fig. 4), enabled the adequate interpretation of XO inhibition in vivo. The measurement of compounds involved in the purine catabolism pathway is desirable to effectively interpret UA generation (Aragon-Martinez et al., 2014). In the present study, the hypoxanthine dose admi- nistered was lower than the doses administered previously in mouse models (500 to 600 mg/kg) in studies that also applied the coadministration of potassium oxonate (Yong et al., 2016; Liang et al., 2018; Yong et al., 2018). The administe- red dose was limited by the saturation of the hypoxanthine suspension and the volume of administration, as it was close to the maximum volume considered good practice in mouse models (Diehl et al., 2001). We applied the XO inhibitor before the hypoxanthine load to enable the action of the inhibitor against the enzyme, which is the inverse of the administration regime applied in previous reports (Yong et al., 2016; Liang et al., 2018; Yong et al., 2018). As observed in Fig. 3, the adminis- tration of allopurinol partially inhibited the conversion of the hypoxanthine load to xanthine, and completely inhibited the degradation of the resulting xanthine into UA in mice (Zhang et al., 2021). It is important to note that there was a basal pre-

sence of serum UA in all the mouse groups tested, including the allopurinol group, as we selected an allopurinol dose that did not cause hypouricemia, a disorder associated with several inflammatory and degenerative diseases, as well as an increased risk of declining kidney function via a reduced renal antioxidant capacity (Park et al., 2020).

The activity found for the combined therapy can be ex- plained by the synergistic interaction of allopurinol with the components found in the extract, an interaction determined in our in vitro studies (Table 2). Moreover, the application of this interaction strengthened the evidence for the change observed in the serum hypoxanthine levels of mice, under the administration of the allopurinol and leaf extract com- bination compared with allopurinol alone (2.2 mg/kg) (Fig. 3). Due to the link between the P value and the Type I error, the combination of L. leucocephala extract and allopurinol reduced the probability of treatment failure from 28.9, which corresponds to the administration of allopurinol alone, to <

1.8 % (Gao, 2020). It is clear that the present reduction in the allopurinol dose, does not have clinical relevance, but the improved probability of therapeutic success by the use of the combination has a very important impact in real clinical settings.

Finally, the inefficacy of the in vivo use of the extract alo- ne is a consequence of its limited efficiency and potency in inhibiting XO, which was observed in our in vitro experiments (Table 2). The present study evaluated the interactions bet- ween a drug and a natural extract, to ascertain the possibility of reducing the dose required of the drug while continuing to inhibit XO both in vitro and in vivo. We employed the Chou-Talalay method for drug combination analyses, since it has physical-chemical bearings, mathematically verifiable equations, and theories. However, there are numerous mo- dels, approaches, hypotheses, and theories that can be used instead of the current experimental design (Chou, 2006), and therefore different outcomes can be obtained. It is clear that further studies are required to obtain additional evidence to support our study, including long-term preclinical and clinical studies. The present study represents a new develo- pment for the use of synergistic combinations to inhibit XO, an important target for regulating hyperuricemia in patients.


For the first time, the present study has shown that the use of an allopurinol and L. leucocephala leaf extract combina- tion at a 3:1 ratio, was an effective and simple method for inhibiting XO in vivo, due to the synergistic interaction with an inhibitory effect on XO that is produced between the allopurinol and the compounds found in the extract. This combination produced a minor reduction in the allopurinol dose alongside an important increase in therapeutic success. Nevertheless, further studies are required to obtain data in support of the use of the allopurinol and L. leucocephala extract combination in a clinical setting.


The present study was financially supported by the economic support provided to researchers by the SNI-CONAHCYT. The

authors thank Marco M. González-Chávez for their technical assistance.


The authors declare that they have no conflict of interest.


Al Shoyaib, A., Archie, S. R. and Karamyan, V. T. 2019. Intraperitoneal route of drug administration: Should it be used in experimental animal studies? Pharmaceutical Research. 37: 12. 2745-x

Aragon-Martinez, O. H., Galicia, O., Isiordia-Espinoza, M. A. and Martinez-Morales, F. 2014. A novel method for measuring the ATP-related compounds in human erythrocytes. The Tohoku Journal of Experimental Medicine. 233: 205-214.

Barthelmebs, L., Divies, C. and Cavin, J. F. 2000. Knockout of the p-coumarate decarboxylase gene from Lactobacillus plantarum reveals the existence of two other inducible enzymatic activities involved in phenolic acid metabolism. Applied and Environmental Microbiology. 66: 3368-3375.

Bobulescu, I. A. and Moe, O. W. 2012. Renal transport of uric acid: evolving concepts and uncertainties. Advances in Chronic Kidney Disease. 19: 358-371. ackd.2012.07.009

Cefali, L. C., Ataide, J. A., Fernandes, A. R., Sanchez-Lopez, E., Sousa, I. M. O., Figueiredo, M. C., Ruiz, A. L. T. G., Foglio, M. A., Mazzola, P. G. and Souto, E. B. 2019. Evaluation of in vitro solar protection factor (SPF), antioxidant activity, and cell viability of mixed vegetable extracts from Dirmophandra mollis Benth, Ginkgo biloba L., Ruta graveolens L., and Vitis vinífera L. Plants (Basel). 8: 453. plants8110453

Checkmahomed, L., Padey, B., Pizzorno, A., Terrier, O., Rosa- Calatrava, M., Abed, Y., Baz, M. and Boivin, G. 2020. In vitro combinations of baloxavir acid and other inhibitors against seasonal influenza A viruses. Viruses. 12: 1139. https://doi. org/10.3390/v12101139

Chen, Y., Li, C., Duan, S., Yuan, X., Liang, J. and Hou, S. 2019. Curcumin attenuates potassium oxonate-induced hyperuricemia and kidney inflammation in mice. Biomedicine & Pharmacotherapy. 118: 109195. https://doi. org/10.1016/j.biopha.2019.109195

Chew, Y. L., Chan, E. W., Tan, P. L., Lim, Y. Y., Stanslas, J. and Goh, J. K. 2011. Assessment of phytochemical content, polyphenolic composition, antioxidant and antibacterial activities of Leguminosae medicinal plants in Peninsular Malaysia. BMC Complementary Medicine and Therapies. 11: 12. https://doi. org/10.1186/1472-6882-11-12

Chou, T. C. 2010. Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Research. 70: 440-446. CAN-09-1947

Chou, T. C. 2006. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacological Reviews. 58: 621-681.

Chung, H. H., Chen, M. K., Chang, Y. C., Yang, S. F., Lin, C. C. and Lin, C. W. 2017. Inhibitory effects of Leucaena leucocephala

on the metastasis and invasion of human oral cancer cells. Environmental Toxicology. 32: 1765-1774. https://doi. org/10.1002/tox.22399

Córdova-Guerrero, I., Aragon-Martinez, O. H., Díaz-Rubio, L., Franco-Cabrera, S., Serafín-Higuera, N. A., Pozos-Guillén, A., Soto-Castro, T. A., Martinez-Morales, F. and Isiordia- Espinoza, M. 2016. Antibacterial and antifungal activity of Salvia apiana against clinically important microorganisms. Revista Argentina de Microbiología. 48: 217-221. https://doi. org/10.1016/j.ram.2016.05.007

Cui, H., Zhang, X., Zhou, H., Zhao, C. and Lin, L. 2015. Antimicrobial activity and mechanisms of Salvia sclarea essential oil. Botanical Studies. 56: 16.


Diehl, K. H., Hull, R., Morton, D., Pfister, R., Rabemampianina, Y., Smith, D., Vidal, J. M., van de Vorstenbosch, C. and European Federation of Pharmaceutical Industries Association and European Centre for the Validation of Alternative Methods. 2001. A good practice guide to the administration of substances and removal of blood, including routes and volumes. Journal of Applied Toxicology. 21: 15-23. https://

Farasat, M., Khavari-Nejad, R. A., Nabavi, S. M. and Namjooyan,

F. 2014. Antioxidant activity, total phenolics and flavonoid contents of some edible green seaweeds from northern coasts of the Persian Gulf. Iranian Journal of Pharmaceutical Research. 13: 163-170.

Galano, A., Francisco-Márquez, M. and Alvarez-Idaboy, J. R. 2011. Mechanism and kinetics studies on the antioxidant activity of sinapinic acid. Physical Chemistry Chemical Physics. 13: 11199-11205.

Gao, J. 2020. P-values - a chronic conundrum. BMC Medical Research Methodology. 20: 167. s12874-020-01051-6

Kim, O. K., Yun, J. M., Lee, M., Kim, D. and Lee, J. 2021.

Hypouricemic effects of Chrysanthemum indicum L. and Cornus officinalis on hyperuricemia-induced HepG2 Cells, Renal Cells, and Mice. Plants (Basel). 10: 1668. https://doi. org/10.3390/plants10081668

Kim, S., Chen, J., Cheng, T., Gindulyte, A., He, J., He, S., Li, Q., Shoemaker, B. A., Thiessen, P. A., Yu, B., Zaslavsky, L., Zhang, J. and Bolton, E. E. 2021. PubChem in 2021: new data content and improved web interfaces. Nucleic Acids Research. 49: D1388-D1395.

Lemos Lima, R. de C., Ferrari, F. C., de Souza, M. R., de Sá Pereira, B. M., de Paula, C. A. and Saúde-Guimarães, D. A. 2015. Effects of extracts of leaves from Sparattosperma leucanthum on hyperuricemia and gouty arthritis. Journal of Ethnopharmacology. 161: 194-199. https://doi. org/10.1016/j.jep.2014.11.051

Liang, D., Yong, T., Chen, S., Xie, Y., Chen, D., Zhou, X., Li, D., Li, M., Su, L. and Zuo, D. 2018. Hypouricemic effect of 2,5-dihydroxyacetophenone, a computational screened bioactive compound from Ganoderma applanatum, on hyperuricemic mice. International Journal of Molecular Sciences. 19: 1394.

Li, B., Zhang, W. and Ma, H. 2016. Physicochemical characterization of inclusion complex of catechin and glucosyl-β-cyclodextrin. Tropical Journal of Pharmaceutical Research. 15: 167-172. v15i1.23

Mabry, T. J., Markham, K. R. and Thomas, M. B. 1970. The ultraviolet spectra of flavones and flavonols. En The Systematic Identification of Flavonoids. T.J. Mabry, K.R. Markham y M.B. Thomas (ed.), pp. 41-164. Springer, Berlin.

Malik, N., Dhiman, P. and Khatkar, A. 2019. In silico design and synthesis of targeted rutin derivatives as xanthine oxidase inhibitors. BMC Chemistry. 13: 71. s13065-019-0585-8

Martinez-Morales, F., Zapata-Morales, J. R. and Aragon-Martinez,

O. H. 2022. Evaluation of the antioxidant interaction between butylated hydroxytoluene and quercetin and their utility for beef patties preservation. Biotecnia. 24: 69-78. https://doi. org/10.18633/biotecnia.v24i1.1546

Mehmood, A., Zhao, L., Ishaq, M., Xin, W., Zhao, L., Wang, C., Hossen, I., Zhang, H., Lian, Y. and Xu, M. 2020. Anti- hyperuricemic potential of stevia (Stevia rebaudiana Bertoni) residue extract in hyperuricemic mice. Food & Function. 11: 6387-6406.

Mohos, V., Pánovics, A., Fliszár-Nyúl, E., Schilli, G., Hetényi, C., Mladěnka, P., Needs, P. W., Kroon, P. A., Pethő, G. and Poór,

M. 2019. Inhibitory effects of quercetin and its human and microbial metabolites on xanthine oxidase enzyme. International Journal of Molecular Sciences. 20: 2681.

Musci, M. and Yao, S. 2017. Optimization and validation of Folin- Ciocalteu method for the determination of total polyphenol content of Pu-erh tea. International Journal of Food Sciences and Nutrition. 68: 913-918.


National Research Council US. 2011. Committee for the update of the guide for the care and use of laboratory animals, guide for the care and use of laboratory animals. National Academies Press (US). Washington, D.C.

Nguyen, M. T., Awale, S., Tezuka, Y., Tran, Q. L., Watanabe, H. and Kadota, S. 2004. Xanthine oxidase inhibitory activity of vietnamese medicinal plants. Biological and Pharmaceutical Bulletin. 27: 1414-1421. bpb.27.1414

Oh, D. R., Kim, J. R., Choi, C. Y., Choi, C. H., Na, C. S., Kang, B. Y., Kim,

S. J. and Kim, Y. R. 2019. Effects of chondroT on potassium oxonate-induced hyperuricemic mice: downregulation of xanthine oxidase and urate transporter 1. BMC Complementary Medicine and Therapies. 19: 10. https://doi. org/10.1186/s12906-018-2415-2

Park, J. H., Jo, Y. I. and Lee, J. H. 2020. Renal effects of uric acid: hyperuricemia and hypouricemia. The Korean Journal of Internal Medicine. 35: 1291-1304. kjim.2020.410

Pleskacova, A., Brejcha, S., Pacal, L., Kankova, K. and Tomandl,

J. 2017. Simultaneous determination of uric acid, xanthine and hypoxanthine in human plasma and serum by HPLC–

UV: uric acid metabolism tracking. Chromatographia. 80: 529-536.

Seal, T. 2016. Quantitative HPLC analysis of phenolic acids, flavonoids and ascorbic acid in four different solvent extracts of two wild edible leaves, Sonchus arvensis and Oenanthe linearis of North-Eastern region in India. Journal of Applied Pharmaceutical Science. 6: 157-166. https://doi. org/10.7324/JAPS.2016.60225

Sebaugh, J. L. 2011. Guidelines for accurate EC50/IC50 estimation. Pharmaceutical Statistics. 10: 128-134. https://

Stamp, L. K. and Barclay, M. L. 2018. How to prevent allopurinol hypersensitivity reactions? Rheumatology (Oxford). 57: i35-i41.

Tosovic, J. 2017. Spectroscopic features of caffeic acid: theoretical study. Kragujevac Journal of Science. 39: 99-108. https://doi. org/10.5937/KgJSci1739099T

Tsikas, D., Wolf, A. and Frölich, J. C. 2004. Simplified HPLC method for urinary and circulating creatinine. Clinical Chemistry. 50: 201-203.

Wishart, D. S., Feunang, Y. D., Guo, A. C., Lo, E. J., Marcu, A., Grant,

J. R., Sajed, T., Johnson, D., Li, C., Sayeeda, Z., Assempour, N., Iynkkaran, I., Liu, Y., Maciejewski, A., Gale, N., Wilson, A., Chin, L., Cummings, R., Le, D., Pon, A., Knox, C. and Wilson, M. 2018. DrugBank 5.0: a major update to the DrugBank database for 2018. Nucleic Acids Research. 46: D1074-D1082. https://doi. org/10.1093/nar/gkx1037

Xu, Y., Tao, Z., Jin, Y., Yuan, Y., Dong, T. T. X., Tsim, K. W. K. and Zhou, Z. 2018. Flavonoids, a potential new insight of Leucaena leucocephala foliage in ruminant health. Journal of Agricultural and Food Chemistry. 66: 7616-7626. https://doi. org/10.1021/acs.jafc.8b02739

Yong, T., Li, D., Li, M., Liang, D., Diao, X., Deng, C., Chen, S., Xie, Y., Chen, D. and Zuo, D. 2018. Anti-Hyperuricemic effect of 2-hydroxy-4-methoxy-benzophenone-5-sulfonic acid in hyperuricemic mice through XOD. Molecules. 23: 2671.

Yong, T., Zhang, M., Chen, D., Shuai, O., Chen, S., Su, J., Jiao, C., Feng, D. and Xie, Y. 2016. Actions of water extract from Cordyceps militaris in hyperuricemic mice induced by potassium oxonate combined with hypoxanthine. Journal of Ethnopharmacology. 194: 403-411. https://doi. org/10.1016/j.jep.2016.10.001

Zhang, D., Zhao, M., Li, Y., Zhang, D., Yang, Y. and Li, L. 2021. Natural xanthine oxidase inhibitor 5-O-caffeoylshikimic acid ameliorates kidney injury caused by hyperuricemia in mice. Molecules. 26: 7307. molecules26237307

Zhang, L., Liu, Y. and Wang, Y. 2018. Deprotonation mechanism of methyl gallate: UV spectroscopic and computational studies. International Journal of Molecular Sciences. 19: 3111.