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In vitro inhibition of xanthine oxidase by hydroalcoholic extracts of Corynaea crassa Hook. F
1 *Faculty of Chemical Sciences "Salvador Allende". University of Guayaquil. Guayaquil, Ecuador.
2 Department of Pharmacy. Institute of Pharmacy and Food. University of Havana. Havana, Cuba.
* Correspondence: [email protected]; [email protected] Tel.: (+593 990225506,ALB)
Corynaea crassa is a hemiparasitic plant native to America, used for the treatment of erectile dysfunction and has been shown to have antimicrobial properties. However, it lacks other biological studies to justify its use in traditional medicine. The objective was to examine the in vitro inhibitory activity of xanthine oxidase in hydroalcoholic extracts of the species from Ecuador and Peru. The extracts obtained by maceration and the Allopurinol used as the reference drug, at concentrations of 10, 30, 40, 50 and 60 µg/mL, were tested to measure the degree of in vitro inhibition of xanthine oxidase employing spectrophotometric determination at 295nm, which is associated with the formation of uric acid. The two extracts showed significant inhibitory activity on xanthine oxidase in a concentration-dependent manner, with the highest percentages being observed at the highest concentrations, being higher for the extract from the Ecuadorian species with enzyme inhibition percentages comparable to Allopurinol. Median inhibitory concentration (IC50) values of 15.35µg/mL and 17.42µg/mL were observed for the extracts from Ecuador and Peru, respectively, although the activity was more notable for the reference drug, which was shown to be an IC50 of 12.21 µg/mL. The results concluded the basis for the potential use of C. crassa in the treatment of hyperuricemia.
Keywords: xanthine oxidase, hydroalcoholic extracts, Allopurinol
The species Corynaea crassa Hook. F (Balanophoraceae) is a hemiparasitic plant native to America, found in various countries, including Bolivia, Colombia, Costa Rica, Ecuador, Mexico, Panama, Peru and Venezuela. In Ecuador, it is located in the provinces of Azuay, Carchi, Chimborazo, Cotopaxi, Imbabura, Loja, Morona Santiago, Napo, Pichincha, Sucumbios, Tungurahua and Zamora Chinchipe1.
In previous studies, the antioxidant activity of hydroalcoholic extracts of the species was evaluated, with similar or superior results to the tested reference substances (vitamin C and Trolox)2. Other studies demonstrated the anti-inflammatory effect of the aqueous and hydroalcoholic extracts of the plant from Ecuador and Peru3 and the erectogenic effect on induced sexual dysfunction in rodents4.
Phytochemical studies revealed by liquid chromatography-mass spectrometry the presence of catechin, quercetin, and a flavanone glycoside)2. Other investigations by gas chromatography-mass spectrometry allowed the detection of terpenoids and fatty acids as significant compounds3. On the other hand, Malca et al. 5, reported sterols, triterpenoids, flavonoids, tannins and anthocyanins.
In Ecuador, C. crassa is little used due to the population's ignorance. Because it develops in the Andean forests and lacks sufficient scientific studies to support said ancestral knowledge, the present investigation was carried out to examine the in vitro inhibitory activity of xanthine oxidase in hydroalcoholic extracts of the species from Ecuador and Peru.
Figure 1 . Corynaea es un género monotípico de plantas parásitas, perteneciente a la familia Balanophoraceae. Su única especie: Corynaea crassa Hook.f., es originaria de América.
MATERIALS AND METHODS
The species Corynae crassa was collected in August 2018, coming from Ecuador (Yanachoca reserve in the north of Pichincha Province (00° 05′S, 78° 33′E, 3700 m elevation)) and Peru (province of La Libertad, department of Santiago de Chuco, Agasmarca (08°07′53″S, 78°03′23″E, 2900 m elevation). One specimen from each collection was identified in the herbarium of the Faculty of Natural Sciences of the University of Guayaquil. It was deposited under the coupon specimen of 13,115 and 13,116, respectively. The whole plant was worked with. The hydroalcoholic extracts were obtained by maceration from 20 g of the dry drug in 100 mL of the hydroalcoholic mixture at 80%. Subsequently, they were concentrated to dryness in a rotary evaporator (Buchi REF 120) at a temperature of 50 °C and redissolved in dimethylsulfoxide (Sigma-Aldrich) for in vitro analysis.
Xanthine oxidase inhibition assay
The inhibitory activity of xanthine oxidase (XO) was assayed spectrophotometrically under aerobic conditions, according to the procedure described by Nguyen et al.6. The assay mixture consisted of 50 mL of test solution, 35 mL of 70 mM phosphate buffer (Sigma, Aldrich) (pH=7.5), and 30 mL of xanthine oxidase enzyme solution (Sigma, Aldrich) (0.01 units / mL in 70 mM phosphate buffer, pH=7.5), was prepared immediately before use. After preincubation at 25 °C for 15 min, the reaction was started by adding 60 mL of substrate solution (150 mM xanthine (Sigma, Aldrich) in the same buffer). The assay mix was incubated at 25°C for 30 min. The reaction was stopped by adding 25 mL of 1N HCL, and the absorbance at 290 nm was measured in a spectrophotometer (RAY LEIGH UV-1601 of Chinese origin). The enzyme solution was added to the assay mix after adding 1N HCL, and a blank was prepared in the same way. One XO unit is defined as the enzyme needed to produce 1 mol of uric acid/min at 25 °C.
XO inhibitory activity was expressed as the percentage inhibition of XO in the above assay system, calculated as (1 – B/A) x 100, where A and B are the activities of the enzyme without and with test material, respectively. The extracts (from samples from Ecuador and Peru) and the Allopurinol (Sigma) used as a positive control were prepared at concentrations of 10, 30, 40, 50 and 60 µg/mL.
The results were expressed as mean/standard deviation (SD), and the experiments were carried out in triplicates. A one-way analysis of variance (ANOVA) was used to determine the differences between groups, followed by the Duncan test with p ≤ 0.05. The mean inhibitory concentration (IC50) of the reference drug and the extracts was calculated from the mean values of the data of three determinations. The Statgraphics statistical program Plus version 5.1 was used to process the experimental data.
The inhibitory activity of xanthine oxidase (XO) was demonstrated by the hydroalcoholic extracts of C. crassa, with enzyme inhibition percentages more significant than 50% from a concentration of 30 µg/mL. The results of this study demonstrated that the active extracts inhibited XO in a dose-dependent manner. The results of the in vitro XO inhibitory activity of test samples and control are shown in table 1.
Table 1. In vitro xanthine oxidase inhibitory activity of C. crassa extract
Xanthine oxidase (XO) inhibitors are generally used for the treatment of gout and hyperuricemia since they can hinder the enzymatic reactions involved in the synthesis of uric acid, reduce the formation of uric acid and relieve the symptoms of said disease, a very painful medical condition caused by high levels of uric acid. Scientists have recently attempted to find new safe XO inhibitors from various plants7,8.
In the Balanophoraceae family, especially in the Balanophora genus, many phytochemicals have been found with a hyperuricemic effect7,9. However, in the genus Corynaea of the same family, the inhibitory activity of the enzyme xanthine oxidase has not been studied, which allowed this research to be carried out on C. crassa.
Five concentrations of hydroalcoholic extracts of species from Ecuador and Peru were tested, and Allopurinol as a reference drug, which prevents the formation of uric acid and is the mainstay of prophylactic treatment for hyperuricemia in patients undergoing chemotherapy10,11.
XO is an important enzyme that converts xanthine and hypoxanthine to uric acid; thus, an increased rate of XO activity leads to excessive uric acid production11. In the present investigation, the reference drug and the two tested extracts (Ecuador and Peru) were able to reduce the activity of the said enzyme with mean inhibitory concentration values of 12.21, 15.35 and 17.42 µg/mL, respectively. The extract from the Ecuadorian sample showed enzyme inhibition percentages comparable to Allopurinol at all concentrations tested (10, 30, 40, 50 and 60 µg/mL). An analysis of the results concerning the literature reports9 demonstrated the excellent capacity of the extracts to inhibit xanthine oxidase, compared to the species Balanophora laxiflora (Balanophoraceae), where at a concentration of 10 µg/mL of the crude extract of the plant, an inhibition of 31.7% was obtained, lower than the experience carried out where the evaluated extracts registered values of 40.01% (Ecuador) and 38.77% (Peru). In addition, the IC50 values (the concentration required to inhibit uric acid formation by 50%) of crude extract was 28.2 µg/mL, while for the extracts under study, the IC50 was 15.35 and 17.42 µg/mL.
Comparison with other investigations of the species Balanophora subcupularis P.C. Tam (IC50= 48.41 μg/mL), also confirms the high property of C. crassa extracts as inhibitors of the enzyme xanthine oxidase. In contrast, Balanophora tobiracola Makino (Balanophoraceae) showed higher activity (IC50=11.87 μg/mL)7.
Natural products have been taken as an ideal source of bioactive compounds with specific pharmacological activities. Inhibitors of xanthine oxidase have been identified and isolated in many plant extracts12,13. Several bioactive compounds, including polyphenols, saponins, terpenoids, phenylethane glycosides, and alkaloids, among other compounds, are effective inhibitors of XO13,14.
Previous studies with hydroalcoholic extracts of C. crassa demonstrated the presence of a variety of metabolites, including catechin flavonoids, quercetin glycoside, and a flavanone glycoside2, terpenoids (triterpenoids and sesquiterpenoids), saponins, tannins, among others compounds3 that could contribute to the inhibition of xanthine oxidase
The results of this research provide scientific evidence for the first time on the inhibitory capacity of C. crassa xanthine, and the species may be considered a potential resource in the treatment of hyperuricemia and gout.
Author Contributions: AJLB proposed the concept of this study and analyzed the results and wrote the initial draft; YIGG. Investigation, writing-review and editing.
Funding: This research received no external funding.
Acknowledgments: Special thanks to Ph.D. Migdalia Miranda Martínez who, although she is not with us, will continue to be the guide and teacher in all our investigations, thank you for transmitting that great wisdom.
Conflicts of Interest: The authors declare no conflict of interest.
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Received: 8 October 2022 / Accepted: 3 December 2022 / Published:15 February 2023
Citation: Lopez Barrera A J, Gutiérrez Gaitén Y I. In vitro inhibition of xanthine oxidase by hydroalcoholic extracts of Corynaea crassa Hook. F. Revis Bionatura 2023;8 (1) 5. http://dx.doi.org/10.21931/RB/2023.08.01.5