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Biotecnia

versión On-line ISSN 1665-1456

Biotecnia vol.25 no.3 Hermosillo sep./dic. 2023  Epub 27-Mayo-2024

https://doi.org/10.18633/biotecnia.v25i3.2153 

Artículos

Comprehensive characterization of the overlooked residue generated during roselle calyxes brewing with potential use as functional ingredient

Caracterización integral del residuo generado durante la decocción de cálices de jamaica con uso potencial como ingrediente funcional

Jesús Eduardo Serna-Tenorio1 

Ana María Sotelo-González2 

Rosalía Reynoso-Camacho2 

Miriam Aracely Anaya-Loyola1 

Iza Fernanda Pérez-Ramírez* 

1Facultad de Ciencias Naturales, Universidad Autónoma de Querétaro, Avenida de las Ciencias s/n, Juriquilla 76230, Querétaro.

2Facultad de Química, Universidad Autónoma de Querétaro, Cerro de las Campanas s/n, Las Campanas, Querétaro 76010, Querétaro.


Abstract

A significant quantity Hibiscus sabdariffa L. calyxes are generated as by-products during the decoction process commonly used for roselle beverage preparation. We conducted an extensive characterization of polyphenols, organic acids, and antioxidant potential in roselle calyxes, decoction, and their by-product. Roselle calyxes were found to be a rich source of diverse polyphenols, including delphinidin sambubioside, caffeoylquinic acids, hibiscus acid, and citric acid as major components. Importantly, we extracted a significant proportion of these bioactive compounds during the decoction process, resulting in a polyphenol-rich beverage. The used calyces (decoction by-product) retained from 23 % to 140 % of the extractable polyphenols and organic acids found in roselle calyxes. Additionally, due to the leaching of hydrophobic components like soluble dietary fiber and extractable polyphenols and organic acids, the roselle by-products were enriched with non-extractable constituents attached to dietary fiber (126 % - 272 %). Therefore, roselle calyxes and their decoction by-products emerge as promising sources of polyphenols with the potential for use in dietary supplements, alongside the commonly consumed roselle decoction.

Keywords: Hibiscus sabdariffa L.; by-product; bioactive compounds; antioxidant capacity; nutraceutical value

Resumen

Durante el proceso de decocción comúnmente utilizado en la preparación de la bebida de jamaica (Hibiscus sabdariffa L.), se genera una cantidad significativa de cálices de esta planta como subproducto. En este estudio, realizamos una exhaustiva caracterización de polifenoles, ácidos orgánicos y el potencial antioxidante en los cálices de jamaica, la decocción y su subproducto. Los cálices de jamaica son una fuente rica en diversos polifenoles, incluyendo delfinidina sambubiosido, ácidos cafeoilquínicos, ácido hibisco y ácido cítrico como principales componentes. De manera importante, una proporción significativa de estos compuestos bioactivos se extrajo durante el proceso de decocción, lo que resultó en una bebida rica en polifenoles. Los cálices utilizados (subproducto de la decocción) retuvieron entre el 23 % y el 140 % de los polifenoles y ácidos orgánicos extraíbles totales de la jamaica. Además, dicho subproducto fue enriquecido con componentes no extraíbles unidos a la fibra dietaria (126 % - 272 %) debido a la lixiviación de componentes hidrofóbicos, tales como fibra dietaria soluble y polifenoles y ácidos orgánicos extraíbles. Por lo tanto, los cálices de jamaica y sus subproductos de decocción emergen como fuentes prometedoras de polifenoles con potencial para su uso en suplementos dietéticos, junto con la comúnmente consumida decocción de jamaica.

Palabras clave: Hibiscus sabdariffa L.; subproducto; compuestos bioactivos; capacidad antioxidante; valor nutracéutico

Introduction

Hibiscus sabdariffa L, commonly known as roselle, is cultivated in numerous tropic and subtropic countries. The calyxes are the main part consumed of this plant, which are consumed fresh, pickled, or dried depending on the local gastronomy. Nevertheless, a common use of roselle calyxes is for the elaboration of infusions or decoctions, leading to a refreshing deep red colored soft drink with a soothing taste (Jamrozik et al., 2022) rich in anthocyanins and non-pigmented flavonoids, phenolic acids, and organic acids (Sapian et al., 2023). While roselle calyxes have been recognized for their nutraceutical value, a substantial environmental issue arises during their preparation. The significant amount of herb material used for infusions or decoctions is often discarded, contributing to environmental waste. These wastes, as recent studies suggest (Debnath et al., 2021), may contain valuable compounds.

Sáyago-Ayerdi et al. (2014) reported that roselle residues are rich in dietary fiber with considerable antioxidant capacity. Interestingly, the polyphenols found in roselle decoction by-product were found to be highly bioaccesible in the gastrointestinal tract (Mercado-Mercado et al., 2015). In a previous study carried out by our research group, we demonstrated that the decoction process increased the porosity in used roselle calyxes (Amaya-Cruz et al., 2018).

Therefore, in our study we hypothesized that the modifications of the chemical structure and composition during roselle decoction leads to the leaching of extractable polyphenols and organic acids, which are found as free components within the food matrix; whereas the non-extractable polyphenols and organic acids, which are bound to the dietary fiber of the food matrix can either be released and found as extractable components, or can be concentrated and found in higher amount. Interestingly, these non-extractable components are mainly found in fruit, vegetable, and herbs by-products (Ding et al., 2020) and have been reported to exert numerous health beneficial effects.

In this regard, we previously demonstrated that roselle decoction by-product exert similar anti-obesity, hypoglycemic, and hypolipidemic effects than roselle calyx (Amaya-Cruz et al., 2019), which could be associated with the high extractable polyphenol content in roselle calyx, and the high non-extractable polyphenol content in roselle by-product. Even though in our study a qualitative polyphenol profile was carried out, we recognized that the quantification of the potential bioactive compounds is essential. Therefore, in our study, we aimed not only to identify, but also quantify, the constituents of the extractable and non-extractable fractions of roselle calyx and its decoction by-product and to determine their impact on their antioxidant capacity, as well as the polyphenol and organic acid composition of roselle decoction through high-resolution mass spectrometry.

Material and methods

Roselle calyx, decoction, and by-product

Dried roselle (Hibiscus sabdariffa L.) calyxes were obtained from local producers from Guerrero, Mexico. Calyxes were disinfected with 1 % Nobac citrus 373 solution for 10 min by immersion, drained, and then dried at 45 ºC for 48 h in a forced circulation oven (BF 400, Binder, Tuttlingen, Germany). Subsequently, 100 g of dried and disinfected roselle calyxes were subjected to a decoction process with 1 L of boiling water for 15 min as reported by Amaya-Cruz et al. (2019). The used calyces (by-product) were separated from the liquid (decoction) by decantation. The roselle by-product was dried as previously described, then ground to a size particle < 420 µm. The roselle calyx, decoction, and by-product were stored at -20 ºC until they were further analyzed. Roselle decoction was thawed at 4 ºC for 24 h and subsequently brought to room temperature before undergoing chemical analyses, while roselle calyx and by-product were directly analyzed. The decoction process was performed in triplicate. Within each decoction replicate, the by-product and the calyx were both analyzed in triplicate for each assay.

Polyphenols extraction from roselle calyx and by-product

The roselle decoction was directly utilized to determine the polyphenol composition. The roselle calyx and by-product were subjected to a sequential polyphenol extraction with organic solvents as reported by Hassan et al. (2011). Briefly, 500 mg of each sample were extracted with 5 mL of 50:50 (v/v) methanol:water, adjusted to pH 2 using hydrochloric acid. The extraction was performed at room temperature for 60 min under continuous stirring. Then, samples were centrifuged (1500 x g for 10 min). Supernatants were recovered and the residue was further extracted with 5 mL of 70:30 (v/v) acetone:water as previously described. The combination of these supernatants formed the extractable polyphenol (EPP) fraction, utilized to quantify extractable polyphenols, flavonoids, anthocyanins, and proanthocyanidins. The residues were dried at 45 º C for 25 h, forming the non-extractable polyphenol (NEPP) fraction utilized to quantify hydrolysable polyphenols (HPP) and non-extractable proanthocyanidins (NEPA).

Total and extractable phenolic compounds

The polyphenol content was determined as reported by Singleton et al. (1999) with minor modifications. Briefly, 10 µL of each sample were mixed with 25 µL of 1 N Folin Ciocalteu, 125 µL of a 20 % (w/v) aqueous solution of sodium carbonate, and 40 µL of distilled water. The reactions were incubated for 30 min under darkness. Then, absorbances were measured at 765 nm. The results were expressed as mg of gallic acid equivalents/mL of roselle decoction or mg of gallic acid equivalents/g of roselle calyx or by-product.

Total and extractable flavonoid content

The flavonoid content was determined as reported by Oomah et al. (2005) with minor modifications. Briefly, 100 µL of each sample were mixed with 20 µL of a 1 mg/mL methanolic solution of 2-aminoehtyldiphenylborate, and 130 µL of distilled water. Then, absorbances were immediately measured at 404 nm. The results were expressed as mg of rutin equivalents/mL of roselle decoction or mg of rutin equivalents/g of roselle calyx or by-product.

Total and extractable anthocyanin content

The anthocyanin content was determined as described by Giusti and Wrolstad (2001) with minor modifications. Briefly, 50 µL of each sample were mixed with 175 µL of each buffer: 0.25 M potassium chloride at pH 1 or 0.40 M sodium acetate at pH 4.5. Then, absorbances were immediately measured at 510 and 700 nm. The results were expressed as mg of cyanidin 3-O-glycoside equivalents/mL of roselle decoction or mg of cyanidin 3-O-glycoside equivalents/g of roselle calyx or by-product.

Extractable proanthocyanidins

The extractable proanthocyanidins (EPA) content was determined as reported by Zurita et al. (2012). Briefly, 500 µL of each sample were mixed with 4.5 mL of a 95:5 (v/v) buthanol:hydrochloric acid solution containing 0.07 g/L of iron chloride. The reactions were incubated in a boiling water bath for 1 h. Then, samples were centrifuged at 1500 x g for 10 min and supernatants were recovered. Absorbances were measured at 450 and 550 nm. The results were expressed as mg of proanthocyanidin equivalents/mL of roselle decoction or mg of proanthocyanidin equivalents/g of roselle calyx or by-product.

Hydrolysable polyphenols content

A sequential alkaline and acid hydrolysis was carried out as reported by Quatrin et al. (2019). The NEPP residues were incubated with 5 mL of 10 M sodium hydroxide and 12 mL of distilled water under continuous stirring for 16 h. Then, their pH was adjusted to 2 - 3 with 6 M hydrochloric acid. Samples were centrifuged at 2000 x g for 10 min and supernatants were recovered. Afterwards, the residues were re-extracted with 5 mL of distilled water as previously described. The supernatants were mixed to quantify polyphenols as described. Results were expressed as mg of gallic acid equivalents/mL of roselle decoction or mg of gallic acid equivalents/g of roselle calyx or by-product, reflecting the alkaline HPP content. Then, the residue obtained during the alkaline hydrolyses was subjected to acid hydrolysis with 2.5 mL of hydrochloric acid. Samples were incubated at 85 ºC for 30 min. Then, their pH was adjusted to 2 - 3 with 10 M sodium hydroxide. Afterwards, the residues were re-extracted with 5 mL of distilled water as previously described. The supernatants were mixed to quantify polyphenols as described. The results were expressed as mg of gallic acid equivalents/mL of roselle decoction or mg of gallic acid equivalents/g of roselle calyx or by-product, reflecting the acid HPP content.

Non-extractable proanthocyanidins content

The NEPA content was determined as reported by Zurita et al. (2012) in the NEPP residue as previously described. The results were expressed as mg of proanthocyanidin equivalents/mL of roselle decoction or mg of proanthocyanidin equivalents/g of roselle calyx or by-product.

Polyphenol profile by UPLC-ESI-QToF MS

The roselle decoction was filtered using a syringe filter with PTFE membrane (13 mm, 0.2 µm). The EPP and HPP extracts obtained from the roselle calyx and by-product were vacuum-dried using a Speedvac system (Savant, Thermo Fisher Scientific, MA, USA), re-suspended in 200 µL of mobile phase, and subsequently filtered. The polyphenol profile was assessed in an Ultra-Performance Liquid Chromatograph coupled to a high-resolution Quadrupole/Time-of-Flight Mass Spectrometer (UPLC-QToF MSE, Vion, Waters Co., MA, USA). The autosampler was maintained at 4 ºC. The chromatographic separation was carried out with 1 µL of each sample injected into a C18 column (2.1 x 100 mm, 1.7 µm, BEH Acquity, Waters Co.) at 35 ºC. The mobile phase was constituted by (a) water with 1 % formic acid and (B) acetonitrile with 1 % formic acid using the gradient conditions throughout 17 min as reported by Reynoso-Camacho et al. (2021). An atmospheric pressure electrospray ionization (ESI) source was used at negative and positive mode at 120 ºC using nitrogen as desolvation gas (800 L/h at 450 ºC) and as cone gas (50 L/h). The mass spectra were acquired at 100-1200 m/z at low (6 V) and high (15-45 V) collision energy. Data were mass corrected during acquisition using a 50 pg/mL leucine enkephalin solution at 10 µL/min.

Polyphenol identification was carried out by comparison of the experimental mass with the theorical mass according to their molecular formula, with a mass error <10 ppm cut-off and by the analysis of the fragmentation pattern. For compounds for which a standard was available, quantification was carried out using its corresponding calibration curve. In cases where a commercial standard was unavailable, quantification was conducted using the most similar standard available. The results were expressed as mg/mL of roselle decoction or mg/g of roselle calyx or by-product.

Antioxidant capacity

The antioxidant capacity was determined in the roselle decoction, as well as in the EPP and HPP fractions of the roselle calyx and by-product, using three widely recognized methods. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay was performed following the procedure outlined by Fukumoto and Mazza (2000). The 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging assay was conducted according to the method described by Re et al. (1999). The ferric reducing antioxidant power (FRAP) assay was carried out as reported by Firuzi et al. (2005). The results were expressed as mmol of Trolox equivalents/mL of roselle decoction or mmol of Trolox equivalents/g of roselle calyx or by-product.

Statistical analysis

Results are presented as mean values ± standard deviation. Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by means comparison by Tukey’s test (p < 0.05), using the JMP software (v14.0).

Results and discussion

Traditional medicine commonly advocates the preparation of orally suitable beverages, primarily infusions and decoctions, to extract bioactive compounds from various botanical sources (Jaiswal et al., 2016). These processes rely on hot water extraction of hydrophilic components such as polyphenols and organic acids found in roselle calyxes. In a beverage like roselle decoction, polyphenols are typically measured as a whole entity, either via spectrophotometry or mass spectrometry techniques. However, in complex matrices like roselle calyxes and by-products, it is crucial to independently analyze the content and composition of extractable and non-extractable components. The former refers to components soluble in the extraction solvents, while the latter can correspond to polymeric constituents or monomeric components bound to macromolecules (Báez-García et al., 2023).

In this study, we conducted a comprehensive analysis of the polyphenols, organic acids, and antioxidant potential of roselle calyxes, decoction, and its by-product. We quantified the total, extractable, and non-extractable polyphenols using spectrophotometric techniques (Table 1). Subsequently, we employed a high-resolution UPLC-QToF MSE system for an exhaustive identification of antioxidant compounds, with a primary focus on polyphenols and organic acids (Tables 2 - 4), which are major constituents of roselle (Sapian et al., 2023). Finally, the antioxidant capacity of each fraction was assessed by three spectrophotometric techniques that assess different mechanisms (Table 5).

Table 1 Total, extractable, and non-extractable polyphenol content of roselle decoction, calyx, and by-product.  

Tabla 1. Contenido de polifenoles totales, extraíbles y no extraíbles de decocción, calix y subproducto de jamaica. 

Polyphenols Decoction Calyx By-product
Polyphenols
Total content (mg GAE/mL) 1.11 ± 0.05
Extractable fraction (mg GAE/g) 25.05 ± 0.98 a 14.82 ± 0.23 b
Acid hydrolysable fraction (mg GAE/g) 5.71 ± 0.20 b 13.14 ± 1.10 a
Alkaline hydrolysable fraction (mg GAE/g) 8.13 ± 0.59 b 10.61 ± 0.95 a
Flavonoids
Total content (mg RE/mL) 0.22 ± 0.02
Extractable fraction (mg RE/g) 10.87 ± 0.41 a 7.91 ± 0.10 b
Anthocyanins
Total content (mg C3GE/mL) 0.13 ± 0.00
Extractable fraction (mg C3GE/g) 2.24 ± 0.07 a 1.29 ± 0.10 b
Proanthocyanidins
Total content (mg CE/mL) 0.57 ± 0.03
Extractable fraction (mg CA/g) 1.54 ± 0.14 a 0.87 ± 0.03 b
Non-extractable fraction (mg PA/g) 2.41 ± 0.10 a 2.60 ± 0.02 a

Different letters indicate significant (p < 0.05) differences. GAE: gallic acid equivalents; RE: rutin equivalents; C3GE: cyanidin 3-O-glucoside equivalents; CE: (+)-catechin equivalents; PA: proanthocyanidin equivalents.

Letras diferentes indican diferencia significativa (p < 0.05). GAE: equivalentes de ácido gálico; RE: equivalentes de rutina; C3GE: equivalentes de cianidina 3-O-glucósido; CE: equivalentes de (+)-catequina; PA: equivalentes de proantocianidina.

Table 2 Polyphenol profile of roselle decoction by UPLC-ESI-QToF MSE.  

Tabla 2. Perfil de polifenoles de decocción de jamaica por UPLC-ESI-QToF MSE

Compound Rt (min) Molecular formula Expected mass (Da) Observed mass (Da) Error (ppm) Adduct Fragments Content (mg/mL)
Anthocyanins
Delphinidin sambubioside 3.59 C26H29O16 597.1456 597.1436 -3.2458 [M]+ 303.05903 10.95 ± 0.02
Delphinidin hexoside 3.68 C21H21O12 465.1033 465.1071 8.1315 [M]+ 303.08267 0.02 ± 0.00
Flavonols
Kaempferol aldo pentosyl-hexoside 4.20 C26H28O15 580.1428 580.1432 0.7137 [M-H]- 284.03284 0.19 ± 0.01
Quercetin aldo pentosyl-rutinoside 6.81 C32H38O20 742.1956 742.1957 0.0777 [M-H]- 300.02781, 178.99754, 151.00338 0.01 ± 0.00
Myricetin hexoside 7.00 C21H20O13 480.0904 480.0912 1.7001 [M-H]- 316.02261, 178.99803, 151.00300 0.04 ± 0.01
Kaempferol rhamnosyl-hexoside-rhamnoside 8.07 C33H40O19 740.2164 740.2165 0.1643 [M-H]- 284.03273 0.01 ± 0.00
Quercetin rutinoside* 8.70 C27H30O16 610.1534 610.1532 -0.2614 [M-H]- 300.02711, 178.99815, 151.00322, 107.01334 0.11 ± 0.00
Quercetin hexoside 9.14 C21H20O12 464.0955 464.0959 0.9470 [M-H]- 300.02711, 178.99800, 151.00321, 107.01259 0.08 ± 0.01
Kaempferol hexoside-rhamnoside 10.74 C27H30O15 594.1585 594.1581 -0.6093 [M-H]- 285.0397 0.02 ± 0.00
Myricetin 11.07 C15H10O8 318.0376 318.0378 0.7452 [M-H]- 179.03454, 151.00331, 137.02392 0.01 ± 0.01
Quercetin* 11.40 C15H10O7 302.0427 302.0427 0.1508 [M-H]- 178.99784, 151.00334, 107.01267 0.02 ± 0.02
Hydroxybenzoic acids
Gallic acid* 3.01 C7H6O5 170.0215 170.0213 -1.3736 [M-H]- 125.02403 0.01 ± 0.00
Dihydroxybenzoic acid hexoside 3.20 C13H16O9 316.0794 316.0795 0.1541 [M-H]- 153.01887, 136.99077, 109.02902 1.51 ± 0.25
Vanillic acid* 3.24 C8H8O4 168.0423 168.0420 -1.8337 [M-H]- 136.99077, 109.02902 0.03 ± 0.00
Galloylquinic acid isomer I 3.28 C14H16O10 344.0743 344.0748 1.1862 [M-H]- 191.05583, 169.05030 0.01 ± 0.00
3,4-Dihydroxybenzoic acid* 3.37 C7H6O4 154.0266 154.0266 0.0096 [M-H]- 137.02418, 109.02925 0.04 ± 0.00
Hydroxybenzoic acid isomer I 3.96 C7H6O3 138.0317 138.0317 0.1860 [M-H]- No fragments 0.34 ± 0.02
Dihydroxybenzoic acid isomer II 4.10 C7H6O4 154.0266 154.0266 -0.1822 [M-H]- 136.86270, 109.02951 0.02 ± 0.00
Methylgallic acid 4.47 C8H8O5 184.0372 184.0372 0.1824 [M-H]- 169.01423, 139.04017, 125.02445 0.03 ± 0.02
Hydroxybenzoic acid isomer II 8.91 C7H6O3 138.0317 138.0316 -0.3855 [M-H]- No fragments 0.06 ± 0.00
Hydroxycinnamic acids
Caffeoylquinic acid isomer I 3.46 C16H18O9 354.0951 354.0953 0.6817 [M-H]- 191.05586, 179.03485, 135.04501 3.90 ± 0.19
Coumaroylquinic acid isomer I 3.98 C16H18O8 338.1002 338.1007 1.4770 [M-H]- 191.05603, 163.04009, 119.05021 0.63 ± 0.02
Caffeic acid hexoside 4.01 C15H18O9 342.0951 342.0956 1.6240 [M-H]- 178.99889, 135.01643 0.04 ± 0.00
Caffeoylquinic acid isomer II 4.11 C16H18O9 354.0951 354.0956 1.5046 [M-H]- 191.05586, 179.05521, 135.04492 1.26 ± 0.17
Chlorogenic acid* 4.28 C16H18O9 354.0951 354.0958 1.9330 [M-H]- 191.03346, 179.03518, 135.04523 2.41 ± 0.02
Ferulic acid hexoside 4.71 C16H20O9 356.1107 356.1112 1.4254 [M-H]- 193.05060, 178.02642, 135.04506 0.01 ± 0.00
Caffeic acid* 4.72 C9H8O4 180.0423 180.0424 0.8791 [M-H]- 135.04525 0.07 ± 0.00
Feruloylquinic acid isomer I 4.89 C17H20O9 368.1107 368.1116 2.3544 [M-H]- 192.99567, 134.03622 0.12 ± 0.00
Coumaroylquinic acid isomer II 5.31 C16H18O8 338.1002 338.1008 1.9346 [M-H]- 191.03403, 163.04014, 119.05039 0.78 ± 0.01
Feruloylquinic acid isomer II 6.28 C17H20O9 368.1107 368.1114 1.8727 [M-H]- 192.97484, 179.10743, 134.03476 0.07 ± 0.00
p-Coumaric acid* 6.37 C9H8O3 164.0473 164.0474 0.2458 [M-H]- 119.05004 0.01 ± 0.00
Feruloylquinic acid isomer III 7.23 C17H20O9 368.1107 368.1112 1.3841 [M-H]- 191.05562, 179.03476, 135.04494 0.10 ± 0.00
Organic acids
Citric acid* 1.38 C6H8O7 192.0270 192.0265 -2.7462 [M-H]- 111.00859 2.80 ± 0.06
Hibiscus acid 0.61 C6H8O8 208.0219 208.0215 -1.9499 [M-H]- 189.00344, 127.00330 14.08 ± 0.01
Hydroxycitric acid 1.28 C6H8O8 208.0219 208.0216 -1.3813 [M-H]- 189.00371, 127.00347 0.31 ± 0.04
Quinic acid 0.59 C7H12O6 192.0634 192.0630 -1.9364 [M-H]- 127.00344 0.43 ± 0.01

*Identification confirmed with commercial standards.

*Identificación confirmada con estándares comerciales.

Table 3 Extractable polyphenols and organic acid profiles of roselle calyx and by-product by UPLC-ESI-QToF MSE.  

Tabla 3. Perfil de polifenoles y de ácidos orgánicos extraíbles de calix y subproducto de jamaica por UPLC-ESI-QToF MSE

Compound Rt (min) Molecular formula Expected mass (Da) Observed mass (Da) Error (ppm) Adduct Fragments Content (mg/g)
Calyx By-product
Anthocyanins
Delphinidin sambubioside 3.59 C26H29O16 597.1456 597.1436 -3.2458 [M]+ 303.05903 1.32 ± 0.14 a 0.35 ± 0.03 b
Delphinidin hexoside 3.68 C21H21O12 465.1033 465.1071 8.1315 [M]+ 303.08267 0.02 ± 0.00 a 0.01 ± 0.00 a
Flavonols
Kaempferol aldo pentosyl-hexoside 4.20 C26H28O15 580.1428 580.1432 0.7137 [M-H]- 284.03284 0.42 ± 0.01 a 0.23 ± 0.04 b
Myricetin rutinoside 6.78 C27H30O17 626.1483 626.1482 -0.1203 [M-H]- 316.02251, 178.99842, 151.00369 0.05 ± 0.01 a 0.02 ± 0.00 b
Quercetin aldo pentosyl-rutinoside 6.81 C32H38O20 742.1956 742.1957 0.0777 [M-H]- 300.02781, 178.99754, 151.00338 0.03 ± 0.01 a 0.01 ± 0.00 a
Myricetin hexoside 7.00 C21H20O13 480.0904 480.0912 1.7001 [M-H]- 316.02261, 178.99803, 151.00300 0.17 ± 0.04 a 0.08 ± 0.00 b
Quercetin rutinoside 8.70 C27H30O16 610.1534 610.1532 -0.2614 [M-H]- 300.02711, 178.99815, 151.00322, 107.01334 0.56 ± 0.12 a 0.33 ± 0.07 b
Quercetin hexoside 9.14 C21H20O12 464.0955 464.0959 0.9470 [M-H]- 300.02711, 178.99800, 151.00321, 107.01259 0.44 ± 0.10 a 0.20 ± 0.05 b
Kaempferol hexoside-rhamnoside 10.74 C27H30O15 594.1585 594.1581 -0.6093 [M-H]- 285.0397 0.07 ± 0.02 a 0.06 ± 0.01 a
Myricetin 11.07 C15H10O8 318.0376 318.0378 0.7452 [M-H]- 179.03454, 151.00331, 137.02392 0.17 ± 0.01 a 0.14 ± 0.02 a
Quercetin* 11.40 C15H10O7 302.0427 302.0427 0.1508 [M-H]- 178.99784, 151.00334, 107.01267 0.49 ± 0.02 0.69 ± 0.08 a
Hydroxybenzoic acids
Gallic acid* 3.01 C7H6O5 170.0215 170.0213 -1.3736 [M-H]- 125.02403 0.44 ± 0.03 a 0.34 ± 0.02 b
Dihydroxybenzoic acid hexoside 3.20 C13H16O9 316.0794 316.0795 0.1541 [M-H]- 153.01887, 136.99077, 109.02902 6.93 ± 0.44 a 4.83 ± 0.27 b
Vanillic acid 3.24 C8H8O4 168.0423 168.0420 -1.8337 [M-H]- 136.99077, 109.02902 0.12 ± 0.01 a 0.10 ± 0.00 a
3,4-Dihydroxybenzoic acid* 3.37 C7H6O4 154.0266 154.0266 0.0096 [M-H]- 137.02418, 109.02925 0.42 ± 0.01 a 0.32 ± 0.00 b
Hydroxybenzoic acid isomer I 3.96 C7H6O3 138.0317 138.0317 0.1860 [M-H]- No fragments 2.43 ± 0.18 a 1.78 ± 0.00 b
Dihydroxybenzoic acid isomer II 4.10 C7H6O4 154.0266 154.0266 -0.1822 [M-H]- 136.86270, 109.02951 0.23 ± 0.01 a 0.11 ± 0.01 b
Methylgallic acid 4.47 C8H8O5 184.0372 184.0372 0.1824 [M-H]- 169.01423, 139.04017, 125.02445 0.27 ± 0.05 a 0.09 ± 0.00 b
Galloylquinic acid isomer II 7.67 C14H16O10 344.0743 344.0746 0.6374 [M-H]- 191.05583, 169.05030 0.81 ± 0.18 a ND
Galloylquinic acid isomer III 7.96 C14H16O10 344.0743 344.0747 0.8987 [M-H]- 190.99818, 169.98481 0.74 ± 0.18 a ND
Hydroxycinnamic acids
Caffeoylquinic acid isomer I 3.46 C16H18O9 354.0951 354.0953 0.6817 [M-H]- 191.05586, 179.03485, 135.04501 15.22 ± 1.29 a 13.74 ± 0.10 a
Coumaroylquinic acid isomer I 3.98 C16H18O8 338.1002 338.1007 1.4770 [M-H]- 191.05603, 163.04009, 119.05021 1.60 ± 0.20 a 1.52 ± 0.01 a
Caffeic acid hexoside 4.01 C15H18O9 342.0951 342.0956 1.6240 [M-H]- 178.99889, 135.01643 0.16 ± 0.02 a 0.10 ± 0.00 b
Chlorogenic acid* 4.28 C16H18O9 354.0951 354.0958 1.9330 [M-H]- 191.03346, 179.03518, 135.04523 10.86 ± 1.27 a 8.69 ± 0.19 a
Caffeic acid* 4.72 C9H8O4 180.0423 180.0424 0.8791 [M-H]- 135.04525 0.59 ± 0.08 a 0.29 ± 0.00 b
Feruloylquinic acid isomer I 4.89 C17H20O9 368.1107 368.1116 2.3544 [M-H]- 192.99567, 134.03622 1.03 ± 0.13 a 1.00 ± 0.02 a
Coumaroylquinic acid isomer II 5.31 C16H18O8 338.1002 338.1008 1.9346 [M-H]- 191.03403, 163.04014, 119.05039 3.24 ± 0.51 a 2.06 ± 0.01 b
Feruloylquinic acid isomer II 6.28 C17H20O9 368.1107 368.1114 1.8727 [M-H]- 192.97484, 179.10743, 134.03476 1.01 ± 0.16 a 0.73 ± 0.02 b
p-Coumaric acid* 6.37 C9H8O3 164.0473 164.0474 0.2458 [M-H]- 119.05004 0.08 ± 0.01 a 0.06 ± 0.00 a
Feruloylquinic acid isomer III 7.23 C17H20O9 368.1107 368.1112 1.3841 [M-H]- 191.05562, 179.03476, 135.04494 1.75 ± 0.28 a 0.86 ± 0.01 b
Ferulic acid* 10.74 C10H10O4 194.0579 194.0572 -3.8475 [M-H]- 178.02718, 134.86543 0.04 ± 0.01 a 0.02 ± 0.00 a
Organic acids
Citric acid* 1.38 C6H8O7 192.0270 192.0265 -2.7462 [M-H]- 111.00859 11.17 ± 0.82 a 11.64 ± 0.88 a
Hibiscus acid 0.61 C6H8O8 208.0219 208.0215 -1.9499 [M-H]- 189.00344, 127.00330 37.57 ± 2.88 a 33.38 ± 2.27 a
Hydroxycitric acid 1.28 C6H8O8 208.0219 208.0216 -1.3813 [M-H]- 189.00371, 127.00347 1.08 ± 0.21 a ND
Quinic acid 0.59 C7H12O6 192.0634 192.0630 -1.9364 [M-H]- 127.00344 1.62 ± 0.68 a 1.00 ± 0.35 b

Different letters indicate significant (p < 0.05) differences. *Identification confirmed with commercial standards. ND: not detected.

Letras diferentes indican diferencia significativa (p < 0.05). *Identificación confirmada con estándares comerciales. ND: no detectado.

Table 4 Non-extractable polyphenols and organic acid profiles of roselle calyx and by-product by UPLC-ESI-QToF MSE.  

Tabla 4. Perfil de polifenoles y de ácidos orgánicos no extraíbles de caliz y subproducto de jamaica por UPLC-ESI-QToF MSE

Compound Rt (min) Molecular formula Expected mass (Da) Observed mass (Da) Error (ppm) Adduct Fragments Content (mg/g)
Calyx By-product
ALKALINE HYDROLYSABLE POLYPHENOLS
Hydroxybenzoic acids
3,4-Dihydroxybenzoic acid* 3.37 C7H6O4 154.0266 154.0266 0.0096 [M-H]- 137.02418, 109.02925 0.28 ± 0.02 b 0.62 ± 0.04 a
Hydroxybenzoic acid isomer I 3.96 C7H6O3 138.0317 138.0317 0.1860 [M-H]- No fragments 1.36 ± 0.00 b 2.63 ± 0.15 a
Hydroxycinnamic acids
Ferulic acid* 10.74 C10H10O4 194.0579 194.0572 -3.8475 [M-H]- 178.02718, 134.86543 ND 0.20 ± 0.01 a
Sinapic acid* 11.17 C11H12O5 224.0685 224.0674 -4.9933 [M-H]- 208.03765 0.14 ± 0.01 b 0.68 ± 0.13 a
Organic acids
Quinic acid 0.59 C7H12O6 192.0634 192.0630 -1.9364 [M-H]- 127.00344 0.26 ± 0.02 b 0.72 ± 0.4 a
ACID HYDROLYSABLE POLYPHENOLS
Hydroxybenzoic acids
Dihydroxybenzoic acid hexoside 3.20 C13H16O9 316.0794 316.0795 0.1541 [M-H]- 153.01887, 136.99077, 109.02902 2.09 ± 0.14 b 4.61 ± 0.62 a
Organic acids
Citric acid* 1.38 C6H8O7 192.0270 192.0265 -2.7462 [M-H]- 111.00859 ND 0.37 ± 0.01 a
Hibiscus acid 0.61 C6H8O8 208.0219 208.0215 -1.9499 [M-H]- 189.00344, 127.00330 3.36 ± 0.56 b 4.25 ± 0.27 a
Quinic acid 0.59 C7H12O6 192.0634 192.0630 -1.9364 [M-H]- 127.00344 9.47 ± 0.84 b 12.22 ± 1.7 a

Different letters indicate significant (p < 0.05) differences. *Identification confirmed with commercial standards. ND: not detected.

Letras diferentes indican diferencia significativa (p < 0.05). *Identificación confirmada con estándares comerciales. ND: no detectado.

Table 5 Antioxidant capacity of roselle decoction, calyx, and by-product.  

Tabla 5. Capacidad antioxidante de decocción, calix y subproducto de jamaica. 

Antioxidant capacity Decoction Calyx By-product
ABTS assay
Total content (mmol TE/mL) 49.41 ± 0.66
Extractable fraction (mmol TE/g) 394.60 ± 2.55a 386.96 ± 6.99a
Acid hydrolysable fraction (mmol TE/g) 34.32 ± 1.42a 22.11 ± 1.75b
Alkaline hydrolysable fraction (mmol TE/g) 185.85 ± 1.09a 18.67 ± 0.31b
DPPH assay
Total content (mmol TE/mL) 2.71 ± 0.11
Extractable fraction (mmol TE/g) 364.76 ± 25.10a 382.93 ± 6.27a
Acid hydrolysable fraction (mmol TE/g) 2210.41 ± 47.95b 2400.05 ± 22.76a
Alkaline hydrolysable fraction mmol TE/g) 558.72 ± 8.67b 1886.99 ± 124.78a
FRAP assay
Total content (mmol TE/mL) 1.01 ± 0.09
Extractable fraction (mmol TE/g) 0.23 ± 0.02a 0.17 ± 0.02b
Acid hydrolysable fraction (mmol TE/g) 0.14 ± 0.01a 0.02 ± 0.00b
Alkaline hydrolysable fraction (mmol TE/g) 0.19 ± 0.01a 0.08 ± 0.00b

Different letters indicate significant (p < 0.05) differences. TE: trolox equivalents.

Letras diferentes indican diferencia significativa (p < 0.05). TE: equivalentes de Trolox.

Total polyphenol content of roselle calyx decoction

As shown in Table 1, the roselle decoction exhibits a mean polyphenol content of 111 mg/100 mL, significantly higher than other herb-based beverages (Rothwell et al., 2013). It is worth noting that the proanthocyanidin content in roselle decoction is 4.4 times higher than its anthocyanin content, despite the latter being traditionally associated with its health benefits. Commonly consumed polyphenol-rich beverages, such as Camellia sinensis (tea) infusion and coffee, typically contain a mean total polyphenol content ranging from 62 to 105 mg/100 mL (Rothwell et al., 2013). This positions roselle decoction as a competitive antioxidant beverage.

Total extractable and non-extractable polyphenol content of roselle calyx and its decoction by-product

As the global demand for flavorful herb-based beverages grows, there is a concurrent increase in waste generated from the decoction and infusion processes (Debnath et al., 2021). As shown in Table 1, used roselle calyxes (decoction by-product) displayed a lower content of extractable polyphenols (1.7-fold) compared to roselle calyxes before the decoction process, attributed to the release of these hydrophilic constituents into the decoction (beverage). This trend was observed in most of the extractable polyphenols identified in used roselle calyxes (Table 2). Notably, the antioxidant capacity assessed by ABTS and DPPH scavenging assays remained similar between roselle calyxes and their by-product, while the FRAP capacity decreased significantly (1.4 - fold; Table 5).

The release of hydrophilic constituents into the decoction led to the concentration of non-extractable constituents in the roselle decoction by-product, resulting in a higher content of both acid and alkali hydrolysable polyphenols (2.3- and 1.3-fold; Table 1) as comparison to the calyx before the decoction process. These hydrolysable polyphenols include low-molecular-weight polyphenols covalently bound to the polysaccharide constituents of dietary fiber through covalent bonds, hydrogen bonds, and/or hydrophobic interactions (Báez-García et al., 2023).

Identification and quantification of polyphenols and organic acids in roselle calyx, decoction, and its by-product

Delphinidin sambubioside emerged as the major anthocyanin identified in roselle calyxes, decoction, and its by-product. The MSE spectra obtained for this component are depicted in Figure 1, revealing a cation radical ([M]+) with a m/z of 597 and a fragment at m/z 303 corresponding to the delphinidin aglycone after the disaccharide moiety is lost. Although delphinidin hexoside, a minor anthocyanin was also identified, our study did not detect cyanidin derivatives, possibly due to the deep red-purple color of the calyxes used, which is attributed to delphinidin derivatives. For instance, Hinojosa-Gómez et al. (2020) reported both delphinidin- and cyanidin- sambubioside as major anthocyanins in roselle calyxes from red cultivars. It is noteworthy that delphinidin 3-sambubioside extracted from roselle calyxes decreases hepatic steatosis in both in vivo and in vitro studies by decreasing fatty acid lipogenesis and augmenting fatty acid beta-oxidation (Long et al., 2021).

Figure 1 High-resolution mass spectra of delphinidin sambubioside. Upper mass spectra acquired at 6 V (low collision energy) and lower mass spectra acquired at 15-45 V (high collision energy).  

Figura 1. Espectro de masas de alta resolución de delfinidina sambubiosida. El espectro de masas superior fue adquirido a 6 V (baja energía de colisión) y el espectro de masas inferior fue adquirido a 15-45 V (alta energía de colisión). 

Regarding non-pigmented flavonoids, previous research have identified quercetin and kaempferol aglycones and their derivatives in various roselle varieties and plant parts (Da-Costa-Rocha et al., 2014). In our study, these flavonoids were identified as minor components. Some flavones like gossypetin, hibiscitrin, sabdaretin, and hibiscetin, previously reported in roselle flowers (Da-Costa-Rocha et al., 2014), were not detected.

In the context of phenolic acids, the major hydroxybenzoic acid was dihydrobenzoic acid hexoside, appearing at 315 m/z as a deprotonated molecular ion. A fragment at 153 m/z resulted from the loss of the hexoside moiety, while another at 109 m/z stemmed from CO2 detachment (Figure 2). Accordingly, protocatechuic acid has been identified as major component in roselle calyx, with a higher content as compared to other Mediterranean plants (Rababah et al., 2011) and edible flowers (Trinh et al., 2018). Protocatechuic acid (3,4-dihydroxybenzoic acid) and its glucoside derivative are well-known for their antioxidant and anti-inflammatory properties (Kakkar and Bais, 2014); moreover, in silico studies suggest that protocatechuic acid shows a better inhibitory potential of phosphoenolpyruvate carboxikinase, a key enzyme involved in hepatic insulin resistance, than the antidiabetic drug metformin (Mohanty and Bhadra, 2020).

Figure 2 High-resolution mass spectra of dihydroxybenzoic acid hexoside. Upper mass spectra acquired at 6 V (low collision energy) and lower mass spectra acquired at 15-45 V (high collision energy).  

Figura 2. Espectro de masas de alta resolución de ácido dihidroxibenzoico hexósido. El espectro de masas superior fue adquirido a 6 V (baja energía de colisión) y el espectro de masas inferior fue adquirido a 15-45 V (alta energía de colisión). 

The major hydroxycinnamic acids were caffeoylquinic acids, including chlorogenic acid, observed at 353 m/z as a deprotonated molecular ion. Fragments corresponding to quinic acid (191 m/z), caffeic acid (179 m/z), and decarboxylated caffeic acid (135 m/z) were also noted (Figures 3 and 4). Interestingly, these compounds have been reported to contribute to the flavor of roselle (Selli et al., 2021) and are recognized as primary precursors for developing coffee flavor and aroma (Lin et al., 2022). Coumaroulquinic acid was also found as a major phenolic acid, also identified as a deprotonated ion at 337 m/z, with fragments at 191 m/z (quinic acid), 163 m/z (coumaric acid), and 119 m/z (decarboxylated coumaric acid; Figure 5). Accordingly, chlorogenic acids have been identified as the major phenolic acid in roselle extracts, contributing with almost 18 % (Yang et al., 2023), which shows high inhibitory activity against the angiotensin converting enzyme, suggesting an anti-hipertensive potential (Salem et al., 2020). Conversely to our study, coumaroylquinic acids have been identified as minor components in roselle extracts (Yang et al., 2023).

Figure 3 High-resolution mass spectra of caffeoylquinic acid isomer I. Upper mass spectra acquired at 6 V (low collision energy) and lower mass spectra acquired at 15-45 V (high collision energy).  

Figura 3. Espectro de masas de alta resolución de ácido cafeoilquínico isómero I. El espectro de masas superior fue adquirido a 6 V (baja energía de colisión) y el espectro de masas inferior fue adquirido a 15-45 V (alta energía de colisión). 

Figure 4 High-resolution mass spectra of caffeoylquinic acid isomer III (chlorogenic acid). Upper mass spectra acquired at 6 V (low collision energy) and lower mass spectra acquired at 15-45 V (high collision energy).  

Figura 4. Espectro de masas de alta resolución de ácido cafeoilquínico isómero III (ácido clorogénico). El espectro de masas superior fue adquirido a 6 V (baja energía de colisión) y el espectro de masas inferior fue adquirido a 15-45 V (alta energía de colisión). 

Figure 5 High-resolution mass spectra of coumaroylquinic acid isomer II. Upper mass spectra acquired at 6 V (low collision energy) and lower mass spectra acquired at 15-45 V (high collision energy).  

Figura 5. Espectro de masas de alta resolución de ácido cumaroliquínico isómero II. El espectro de masas superior fue adquirido a 6 V (baja energía de colisión) y el espectro de masas inferior fue adquirido a 15-45 V (alta energía de colisión). 

Among the organic acids, hibiscus acid highlighted as the major compound, consistent with previous studies (Sapian et al., 2023). This organic acid appeared in its deprotonated form ([M-H]-) at 207 m/z, with fragments at 189 and 127 m/z (Figure 6). Additionally, citric and quinic acids were identified as deprotonated ions at 191 m/z with their representative characteristic fragment at 111 and 127 m/z, respectively (Figures 7 and 8). Importantly, the hibiscus acid and citric acid content, the major organic acids identified in this study, remained unaffected by the decoction process (Table 2). It is noteworthy that most studies carried out with roselle have associated its beneficial health effects to their high content of anthocyanins, phenolic acids, and non-pigmented flavonoids, whereas only few studies have focused on the bioactive potential of organic acids (Izquierdo-Vega et al., 2020). In this regard, Morales-Luna et al. (2018) demonstrated that white roselle calyx extract, with a high organic acid content and no anthocyanins, exert a similar anti-obesogenic effect than red roselle calyx extract with a high anthocyanin content but low organic acid content. Among the composition of organic acids found in roselle, citric acid has been attributed with high antioxidant, anti-inflammatory and anti-coagulant activities, whereas hibiscus acid shows anti-diabetic and anti-hypertensive potential. On the other hand, hydroxycitric acid has been recently proposed as an anti-obesity nutraceutical; however, most studies have been assessed in hydroxycitric acid isolated from Garcinia cambogia (Yang et al., 2020).

The roselle decoction analyzed in this study was found to have hibiscus acid as the major component, accounting for 79.9 % of the identified organic acids, followed by delphinidin sambubioside (Table 2). Moreover, dihydroxybenzoic acid hexoside represented 73.7 % of all hydroxybenzoic acids, while the three caffeoylquinic acid isomers collectively made up to 80.5 % of the hydroxycinnamic acids. These results are consistent with previous studies demonstrating that roselle calyx decoctions are rich in bioactive compounds, known for their numerous health benefits (Da-Costa-Rocha et al., 2014; Ríaz and Chopra, 2018; Bedi et al., 2020; Sapian et al., 2023).

Figure 6 High-resolution mass spectra of citric acid. Upper mass spectra acquired at 6 V (low collision energy) and lower mass spectra acquired at 15-45 V (high collision energy).  

Figura 6. Espectro de masas de alta resolución de ácido cítrico. El espectro de masas superior fue adquirido a 6 V (baja energía de colisión) y el espectro de masas inferior fue adquirido a 15-45 V (alta energía de colisión). 

Figure 7 High-resolution mass spectra of hibiscus acid. Upper mass spectra acquired at 6 V (low collision energy) and lower mass spectra acquired at 15-45 V (high collision energy).  

Figura 7. Espectro de masas de alta resolución de ácido hibiscus. El espectro de masas superior fue adquirido a 6 V (baja energía de colisión) y el espectro de masas inferior fue adquirido a 15-45 V (alta energía de colisión). 

Figure 8 High-resolution mass spectra of quinic acid. Upper mass spectra acquired at 6 V (low collision energy) and lower mass spectra acquired at 15-45 V (high collision energy).  

Figura 8. Espectro de masas de alta resolución de ácido quínico. El espectro de masas superior fue adquirido a 6 V (baja energía de colisión) y el espectro de masas inferior fue adquirido a 15-45 V (alta energía de colisión). 

Interestingly, organic acids were identified in both acid and alkali hydrolysable fractions, with higher amounts in the acid hydrolysis fraction. It is noteworthy that quinic acid has been reported to be obtained after the hydrolysis of caffeoylquinic acids and other phenolic acids esterified with quinic acid (Clifford et al., 2017). Nevertheless, hibiscus acid and quinic acid were found to be increased by 1.3-fold in the roselle decoction by-product compared to the roselle calyxes. The non-extractable fractions of the roselle decoction by-product primarily consisted of hydroxybenzoic acid derivatives, which were enriched by 1.9 to 2.2-fold after the decoction process (Table 4).

Antioxidant capacity of roselle calyx, decoction, and its by-product

The results indicate that the non-extractable fraction of the roselle decoction by-product exhibits significant antioxidant capacity when assessed using the DPPH method, showing a 1.1- to 3.4-fold increase as compared to roselle calyxes before the decoction process (Table 5). However, the antioxidant capacity, as determined by the ABTS and FRAP assays, is notably lower, with reductions of 1.6- to 9.9-fold and 7.0- to 2.4-fold, respectively. These findings suggest that the non-extractable fraction of the roselle decoction by-product contains antioxidant components, such as polyphenols and organic acids, that are effective in donating protons and scavenging the DPPH radical. On the other hand, it appears to have a limited ability to donate electrons, as indicated by the reduced capacity in scavenging the ABTS radical and reducing Fe3+. This variation in antioxidant activity across different assays highlights the diverse mechanisms by which antioxidants can operate and underscores the importance of considering multiple methods for a comprehensive evaluation of antioxidant potential (Danet, 2021).

Notably, this study presents the first quantitative profile of the non-extractable fraction of roselle calyxes and their decoction by-product. Moreover, we demonstrate that hibiscus acid, the major roselle organic acid, is not only found as a free component but can also be found bound to the matrix by glycosidic linkage, as observed in the composition of the acid hydrolysable fraction. Few studies have determined non-polyphenol constituents in the non-extractable fraction. In this regard, Dong et al. (2021) identified eleven organic acids, along with several polyphenols, bound to carrot dietary fiber which were released by alkaline hydrolysis, contributing to its antioxidant capacity. Therefore, further studies must be undertaken to fully characterize the non-extractable fraction of plant sources and wastes.

Conclusions

This study highlights the substantial content of diverse extractable and non-extractable constituents with significant antioxidant potential in the waste generated during roselle decoction. The extraction and purification of these compounds for use as nutraceuticals, or in the creation of innovative value-added dietary supplements or functional foods, could contribute to a circular economy by reclaiming and repurposing an underappreciated waste stream with potential health benefits.

Conflicts of interest

The authors declare no actual or potential conflict of interests, including financial, personal or relationship with other organizations.

Acknowledgments

This research study was supported by Fondo para el Desarrollo del Conocimiento (FONDEC-UAQ) 2022 (Grant no. FQU-2022-08). The authors are grateful to CONAHCYT for the scholarships granted to E.S.-T. and A.M.S-G. We thank the Chemistry Faculty of the Universidad Autónoma de Querétaro for providing facilities for the UPLC-ESI-Q-ToF MS analysis (CONAHCyT project number: INFR-15-255182).

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Received: September 11, 2023; Accepted: November 23, 2023; Published: December 08, 2023

*Author for correspondence: Iza Fernanda Pérez-Ramírez e-mail: iza.perez@uaq.mx.

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