Front. Chem. Frontiers in Chemistry Front. Chem. 2296-2646 Frontiers Media S.A. 1661223 10.3389/fchem.2025.1661223 Original Research Rheum officinale Baill.: chemical characterization and in-vitro biological activities Kerroum et al. 10.3389/fchem.2025.1661223 Kerroum Fatima 1 Formal Analysis Validation Project administration Methodology Data curation Supervision Software Conceptualization Resources Writing - review and editing Visualization Writing - original draft Funding acquisition Investigation Atoui Aicha 2 Visualization Data curation Validation Investigation Methodology Conceptualization Funding acquisition Supervision Project administration Resources Software Writing - review and editing Formal Analysis Writing - original draft Khattabi Latifa 1 Funding acquisition Writing - review and editing Formal Analysis Supervision Investigation Writing - original draft Software Project administration Validation Data curation Resources Methodology Conceptualization Visualization Yilmaz Mustafa Abdullah 3 4 Formal Analysis Visualization Resources Funding acquisition Writing - original draft Project administration Investigation Supervision Data curation Methodology Writing - review and editing Validation Conceptualization Software Cakir Oguz 4 5 Funding acquisition Supervision Resources Software Formal Analysis Writing - review and editing Writing - original draft Visualization Data curation Validation Project administration Conceptualization Methodology Investigation Atoki Ayomide Victor 6 * Supervision Writing - review and editing Writing - original draft Formal Analysis Data curation Software Funding acquisition Conceptualization Resources Investigation Validation Visualization Project administration Methodology Messaoudi Mohammed 7 Data curation Methodology Validation Investigation Writing - review and editing Formal Analysis Writing - original draft Conceptualization Resources Supervision Visualization Funding acquisition Project administration Software Biotechnology Research Center-C.R.Bt, Constantine, Algeria Laboratory of the Development and Valorization of Plant Genetic Resource, Faculty of Sciences, Brothers Mentouri University, Constantine, Algeria Department of Analytical Chemistry, Faculty of Pharmacy, Dicle University, Diyarbakir, Türkiye Science and Technology Research and Application Center, Dicle University, Diyarbakir, Türkiye Department of Nutrition and Dietetics, Faculty of Health Sciences, Dicle University, Diyarbakir, Türkiye Department of Biochemistry, Kampala International University, Ishaka, Uganda Laboratory for Research on Bioactive Products and Biomass Valorization, Department of Chemistry, ENS Kouba, Algiers, Algeria Correspondence: Ayomide Victor Atoki, atokiav@kiu.ac.ug 10 12 2025 2025 13 1661223 07 07 2025 15 09 2025 04 11 2025 Copyright © 2025 Kerroum, Atoui, Khattabi, Yilmaz, Cakir, Atoki and Messaoudi. 2025 Kerroum, Atoui, Khattabi, Yilmaz, Cakir, Atoki and Messaoudi https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

Medicinal plants constitute a valuable natural resource of bioactive phytochemicals, which are increasingly studied for their therapeutic potential and broad applications in the pharmaceutical, nutraceutical, and cosmetic fields. Rheum officinale, a medicinal rhubarb species, is appreciated for the presence of biologically active compounds with therapeutic relevance. This work analyses the chemical composition, including the phytochemical profile, and pharmacological activities of Rheum officinale Baill. stems in Algeria. The plant extract was analyzed for its notable antioxidant capacity using various assays, including DPPH, ABTS, β-carotene bleaching, ferric and also cupric reducing power, and metal chelation. The inhibitory potential against cholinesterase and α-amylase was assessed through specific enzymatic assays. LC–ESI-MS/MS assessment highlighted the phytochemical profile within the extract, with quinic acid identified as the major component. Antimicrobial potential against P. aeruginosa, S. aureus, E. coli, E. faecalis, and C. albicans was confirmed via agar diffusion and inhibition zone (C) tests. The extract demonstrated potent antioxidant activity, with radical scavenging IC50 values less potent than reference antioxidants such as BHT and α-tocopherol (IC50 = 0.42 ± 1.43 μg/mL). Total phenol and flavonoid content were quantified using Folin-Ciocalteu and AlCl3 methods, yielding high values (373.10 ± 0.055 mg GAE/g and 38.012 ± 0.05 mg QE/g, respectively). Enzyme inhibition assays demonstrated significant activity against key enzymes related to Alzheimer’s disease (IC50: 28.14 ± 2.22; 73.71 ± 1.48 μg/m) and diabetes (IC50: 36.21 ± 0.56 μg/m). The extract also exhibited antimicrobial effects. Given its bioactive potential, Rheum officinale presents promising opportunities for therapeutic product development, supporting the pharmaceutical industry.

 Rheum officinale antimicrobial activity enzyme inhibition antioxidant phytochemical profile The authors declare that no financial support was received for the research and/or publication of this article. section-in-acceptance Chemical Biology Introduction

The discovery and development of novel pharmaceuticals are significantly influenced by natural products, particularly those originating from plants (Krajewska et al., 2024; Yener et al., 2020). Natural products are valuable sources of leads for pharmaceutical research due to their extensive biological activities and structural diversity (Demir et al., 2024; Oncu et al., 2024; Yilmaz et al., 2024). Consequently, traditional medicine, which has been practiced for centuries, is founded on plants that possess potent medicinal properties, including antioxidant, enzyme inhibitory, and antimicrobial activity (Findik et al., 2024).

The Rheum genus includes approximately 60 species of robust herbaceous species (Xiang et al., 2020). Perennial rhubarb has eatable stalks, it has hard stems with a characteristic woody appearance, long leaves, and clusters of small, wind-pollinated flowers (Lee et al., 2017). Rheum officinale Baill., belonging to the Polygonaceae family, is widely cultivated in TCM (Xiong et al., 2019). Known in China as the “ruler or king of herbs,” rhubarb has been used for more than 2,000 years in traditional medicine due to its wide range of pharmacological properties (Wang et al., 2018). Its name in Arabic is “Raound, الرواند.” Rhubarb is a source of biologically active ingredients necessary for the treatment and prevention of lifestyle related diseases due to its laxative, diuretic, antidiabetic, antibacterial, hemostatic, anti-inflammatory, antiviral, immunosuppressive and antitumor properties (Huang et al., 2019; Jintao et al., 2018; Shang et al., 2019; Stompor–gorący, 2021). It contains several valuable bioactive phytochemicals such as anthraquinones, dianthrones, stilbenes, and flavonoids (Xie et al., 2020; Zhang et al., 2024), which contribute to improving the health status of humans and animals. It also has a high content of dietary fiber (Goel et al., 1999). It should be noted that the fresh shoots and stems of Rheum officinale are used for the treatment of many diseases, but rhubarb leaves can be poisonous, as they contain a high concentration of oxalates, unlike stems and petioles (Clementi and Misiti, 2010). R. officinale has been shown to possess antioxidant (Emen Tanrikut et al., 2013; Kalisz et al., 2020), antimicrobial (Alaadin et al., 2007), and antihyperglycemic properties (Kasabri et al., 2011).

In North America, Europe, and several Middle Eastern regions, some Rheum species are traditionally used in sweet, fruit-based preparations. Their value in the diet stems from their richness in bioactive constituents and dietary fiber. In particular, Rheum rhabarbarum is widely used in culinary applications for the preparation of desserts, cakes, mousses, juices, wines, and fruit teas (Dai et al., 2022). In Algeria, people use R. officinale stems as food prepared similarly to spinach dishes, valued for its beneficial effects against indigestion, stomach pain, haemorrhoids, and diarrhea. In our research, the phytochemical profile of the hydromethanolic extract derived from local R. officinale stems was analyzed using LC-ESI-MS/MS. Furthermore, its biological potential was assessed through evaluations of radical-scavenging ability, antimicrobial effectiveness, and inhibition of key metabolic enzymes.

 Materials and methods Reagents and chemicals

All reagents, solvents, and standards used throughout the experiments were supplied by Sigma-Aldrich (French).

 Extraction of plant material

Rhubarb stems were collected in January 2020 from the Babur Mountains (Easten Algeria) and authenticated at the Botanic Authentication Laboratory of Ahmed Ben Bella University (Oran1). The stems were first rinsed, air-dried and finely powdered. A portion of 10 g of the ground plant material was macerated in 100 mL of 80% methanol, filtered and evaporated with (Buchi, Germany) to obtain the hydromethanolic extract of rhubarb stems (HMERS).

 Phytochemical analysis of HMERS

The total phenolic content of HMERS was determined using the Folin–Ciocalteu method (Singleton and Rossi, 1965) with slight modifications (Müller et al., 2010). The mixture was freshly prepared by combining 100 μL of the diluted Folin–Ciocalteu reactive solution (1:10) and sodium carbonate solution at 75 g/L with 20 μL of the extract. The prepared reaction mixture was subjected to incubation for 2 h and then the reading was taken at 740 nm. The amount of total phenolic was determined and presented as gallic acid (mg GAE per g of extract).

For flavonoid quantification, the procedure performed using the method of the aluminum chloride colorimetric method described by (Djeridane et al., 2006). HMERS solution was prepared (0.125 mg/mL) and combined with 2% AlCl3 in methanol. Spectrophotometric readings were taken at 430 nm. The calibration curve was established using quercetin standards (5–50 μg/mL), and results were reported as mg QE per g (DW).

 <italic>In vitro</italic> evaluations of antioxidant activity The β-carotene bleaching test

The antioxidant activity of HMERS was evaluated using β-carotene linoleic acid model system (Benahmed et al., 2021). The β-carotene stock solution was prepared in chloroform (0.5 mg/mL), mixed with Tween 40 (200 mg) and linoleic acid (25 μL), and then evaporated under vacuum. 100 mL of H202 was introduced with vigorous shaking to obtain a stable emulsion. Aliquots (4 mL) were combined with different extract concentrations, and the values were read at 470 nm (0 h and 2 h of incubation at 50 °C). A control was used for correction (without β-carotene) and inhibition rate was determined according to the next equation: PI  % = 1 ‐ As   t 0 ‐ t 120   Ac   t 0 ‐ t 120 ×   100

Where, As is the absorbance values of the sample, whereas Ac refers to the control absorbance.

 DPPH test

DPPH free radical scavenging test of HMERS was determined by the assay described by Blois (1958). For the assay, a DPPH solution was freshly prepared (0.1 mM in methanol) and mixed with sample (160 μL: 40 μL) in different dilutions. After an incubation period of 30 min at room temperature in the dark, the absorbance at 517 nm was recorded using a 96-well microplate reader (EnSpire Multimode Plate Reader, PerkinElmer). The inhibitory potency was represented by IC50 values. Inhibition rate (%) was obtained using the formula below: I P   % = A b   c o n t r o l − A b   s a m p l e A b   c o n t r o l x 100

Where: Ab = absorbance.

 Reduction of copper cation test

The cupric reducing antioxidant capacity of HMERS was assed following the procedure adopted by Apak et al. (2004). To perform the assay, 40 µL of the extract was mixed with 60 µL of ammonium acetate (CH3COONH4), 50 µL of neocupronin, and 50 µL of copper (II) chloride dihydrate (CuCl2, 2H2O) in a suitable reaction vessel. After gentle homogenization, the blend was maintained 1 h under incubation, and the measured absorbance was recorded at 450 nm.

 ABTS cation decolorization test

The spectrophotometric test of ABTS+ scavenging ability was assessed as initiated by Re et al. (1999). For this assay, a stock solution of ABTS+ (160 μL) was combined with sample (40 μL) in methanol at varying dilutions. After incubation of the prepared mixture, the optical density was measured at 734 nm and the relative activity (%) was estimated according to the equation: Inhibition    e f f e c t   % = A   control   ‐   A   sample A   control ×   100 where A: is the absorbance.

 Ferric cation reduction test

The reducing power of the extract was measured (Oyaizu, 1986). Different concentrations of the sample extract (10 μL each) were prepared and 0.2 M phosphate buffer (pH 6.6) containing 1% potassium ferricyanide were introduced into the sample. After reaction with trichloroacetic acid and ferric chloride, the spectrophotometric measurement was taken at 700 nm and the values were presented as the concentration (μg mL−1) required to achieve an absorbance of 0.5 (A0.5).

 O-phenanthroline test

The assay was carried out by combining 30 µL of 0.5% O-phenanthroline, 50 µL of FeCl3 (0.2%), 10 µL of sample extract, and 110 µL of methanol at various concentrations. The obtained mixture was maintained for 20 min at 30 °C and its reading absorbance was subsequently assessed at 510 nm. The percentage of inhibition was then calculated relative to an appropriate control (Khattabi et al., 2022).

 Enzymatic inhibition tests The <italic>in vitro</italic> anti-Alzheimer potential of HMERS

The anticholinesterase potential was performed by mixing the extract or galantamine (10 mL) with 20 μL portion of enzyme solution (6.85 × 10−3 U for BChE or 5.32 × 10−3 U for AChE) and 150 µL of phosphate buffer (100 mM, pH 8.0). After incubation, 10 μL of the substrate solution (acetyl or butyrylthiocholine) and an equal volume of DTNB (0.5 mM) were subsequently added to the first reaction, and measurement of absorbance was carried out at 412 nm. The percentage of enzyme inhibition was calculated using the formula: Inhibition  % = E   ‐   S / E × 100 where E is the enzyme activity in the absence of the test sample, and S is the enzyme activity in its presence (Ellman et al., 1961).

 <italic>In vitro</italic> anti-diabetic activity of <italic>R. officinale</italic> by alpha amylase inhibition assay

α-amylase inhibitory activity was performed using iodine/potassium iodide (IKI) method (Zengin et al., 2014), with some modifications. The assay involved incubating varying concentrations of the sample (extract or acarbose) with α-amylase (1 U) for (10 min; 37 °C), then added starch solution at 0.1% concentration, HCl and IKI. Sample absorbance was quantified at 630 nm and the inhibition percentage of the enzyme was resolute as: %  Inhibition = A   control   ‐   A   control   blanc   –   A   sample   –   A   sample   blanc A   control   –   A   control   blanc × 100

Where (A) are the absorbance values.

 Antimicrobial potential

Antimicrobial effect of HMERS was assessed against several strains, among them E. coli ATCC 8739, S. aureus ATCC 6538, E. faecalis ATCC 49452, P. s aeruginosa ATCC 27853, and the fungal strain C. albicans ATCC 90026, obtained from the Microbiology Laboratory of Tamanrasset University. Determination of the minimum inhibitory concentration (MIC) was performed according to the broth microdilution technique. Twofold serial dilutions of the extract (20–104 μg/mL) were prepared in DMSO (≤2%), which did not show any noticeable effect on microbial growth. An equal volume (100 µL) of the extract dilution and the microbial inoculum (106 CFU/mL) was added to each well. Negative (broth only) and positive (microorganism without extract) controls were included. The MIC was defined after being incubated for 24 h at 37 °C as the minimum concentration of the extract that prevented visible microbial growth. All experiments were carried out in three replicates.

 Mass spectrometer and chromatograph conditions

Authors used a Shimadzu-Nexera UHPLC system (SIL-30AC autosampler, CTO-10ASvp oven, LC-30AD pumps, DGU-20A3R degasser) and a Shimadzu LCMS-8040 triple quadrupole mass spectrometer to measure the amounts of 53 phytochemicals (Supplementary Table S1) (Yilmaz, 2020). Samples were separated on an Agilent Poroshell 120 EC-C18 column (150 × 2.1 mm, 2.7 μm) at 40 °C. Mobile phases were water (5 mM ammonium formate, 0.1% formic acid) as A and methanol with the same additives as B. The gradient progressed from 20% to 100% B over 0–25 min, held at 100% B until 35 min, then returned to 20% B by 45 min. Flow rate was 0.5 mL/min, injection volume 5 μL. Mass detection used electrospray ionization in positive and negative modes, with LabSolutions software for data processing. Quantification employed MRM with optimized precursor–product ion transitions. Ion source settings were: drying gas 15 L/min, nebulizing gas 3 L/min, interface 350 °C, desolvation line 250 °C, heat block 400 °C. Method validation parameters are listed in Supplementary Table S1.

 Data analysis

The experimental data were subjected to one-way analysis of variance (ANOVA). Mean comparisons were carried out using Duncan’s multiple range test, with statistical significance considered at p < 0.05.

 Results

As shown in Table 1, the TPC, TFC, and CTA assays revealed that the extract has considerable potential quantity of phenolic/flavonoid compounds and condensed tannins with IC50 value (373.10 ± 0.055 μg GAE/mg, 78.05 ± 0.004 μg QE/mg and 43.012 ± 0.05 μg CE/mg) of extract, respectively.

Total phenolics content in R. officinale extracts.

Extract Total phenolic (μg GAE mg−1 E) Total flavonoids (μg QE mg−1 E) Condensed tannins (μg CE mg−1 E)
HMERS 373.10 ± 0.055 78.05 ± 0.004 43.012 ± 0.05

TPC, total phenolic compounds; TFC, total flavonoids; CTA, condensed tannins. Data are presented as mean ± standard deviation from three independent replicates.

Tables 2 and 3 summarize the findings of the in vitro antioxidant and anti-enzymatic assays. Regarding the antioxidant performance of the extract, most of the assays revealed IC50 and A0.5 values comparable to the reference standards, with statistically significant effects (P < 0.05). Notably, in the ABTS assay, the extract exhibited a stronger response, reaching high significance (P < 0.01; IC50 = 0.42 ± 1.43 μg/mL). Likewise, for the enzyme inhibition tests, the IC50 values for cholinesterase inhibition (AChE and BCHE) were close to those of the reference compound, galantamine, which served as the positive control since it is clinically applied in the management of mild Alzheimer’s disease. In contrast, the anti-α-amylase test provided the most effective inhibition, as reflected by the lowest IC50 value (36.21 ± 0.56 μg/mL) lower than that of acarbose, indicating strong α-amylase inhibitory potential.

Antioxidant properties of HMERS presented as IC50 and A0.5 values (μg/mL).

Extract/Standard A0.5 IC50
CUPRAC FRAP Phenanthroline β-carotene DPPH ABTS
HMERS 18.47 ± 0.34a 16.21 ± 0.56a 10.84 ± 0.88a 18.61 ± 0.70a 13.36 ± 0.78a 0.42 ± 1.43a
BHT* 5.35 ± 0.71b nt 0.93 ± 0.07b 0.91 ± 0.01b 6.14 ± 0.41b 1.29 ± 0.30b
BHA* 8.97 ± 3.94c nt 2.24 ± 0.17c 1.05 ± 0.03c 12.99 ± 0.4c 1.81 ± 0.10c
Ascorbic acid* nt 6.77 ± 1.15b nt nt nt nt
αTocopherolb* nt 34.93 ± 2.3c nt nt 13.02 ± 5.1c nt

Standard compounds. nt, not tested. The IC50 and A0.5 values were derived from linear regression analysis and are presented as mean ± standard deviation from three independent replicates. Values with different superscript letters (a, b, c) in the same parameter were significantly different (P < 0.05).

Anti-enzymatic activity of HMERS represented as IC50 (μg/mL).

Stems extract Anti- AChE Anti-BChE Anti-alpha amelase
HMERS 28.14 ± 2.22a 73.71 ± 1.48a 36.21 ± 0.56a
Galantamine* 6.27 ± 1.15b 34.75 ± 1.99b -
Acarbose* - - 3650.93 ± 10.70b

Standard compounds. -, no activity. The IC50 values values were derived from linear regression analysis and reported as mean ± standard deviation from three replicates. Values with distinct superscripts letters (a, b) in a column denote significant variation (p < 0.05).

The antimicrobial activity result of our extract at different concentrations also the positive controls (ampicillin, gentamicin and amphotericin B) are showed in Table 4. The funding indicate that HMERS exhibited a significant antimicrobial effect (p < 0.05) against the different microorganisms studied, although the degree of activity varied comparing to the standard antibiotics (Figure 1).

HMERS’s antibacterial activity (mean ± SD. n = 5).

Microbial strains HMERS (mg L−1) Ampicillin (μL) Gentamicin (μL) Amphotericin B (µg)
0.1 0.01 0.001 10 10 20
E. faecalis 10 ± 0.01b 9 ± 0.00c 8 ± 0.00c 18 ± 0.01a - -
S. aureus 12 ± 0.00b 11 ± 0.01b 9 ± 0.00c 18 ± 0.01a 20 ± 0.01e -
P. aeruginosa 16 ± 0.02b 14 ± 0.0c 10 ± 0.02d 17 ± 0.01a 20 ± 0.01a -
E. coli. 13 ± 0.01b 11 ± 0.01c 9 ± 0.01c 17 ± 0.01a 17 ± 0.01a -
Candida albicans 12 ± 0.00a 10 ± 0.00b 8 ± 0.02c - - 10 ± 0.02b

Diameter of disc used is equal to 6 mm. Values are reported as mean ± standard deviation from three replicates, those with different letters (a, b, c, d) above the bars denote significant differences (p < 0.05) and values with the same letters are not significantly different (p > 0.05) among treatments within each microbial strain. Comparisons were not made between different microorganisms.

Antimicrobial activity of EMERS.

Bar chart comparing the antimicrobial activity of HMERS and various antibiotics against five microbial strains. Inhibition zones, measured in millimeters, are displayed for E. faecalis, S. aureus, P. aeruginosa, E. coli, and Candida albicans. Different concentrations of HMERS and commonly used antibiotics like Ampicillin, Gentamicin, and Amphotericin B are represented by color-coded bars, with labels indicating statistical significance.

Diameter of disc used is equal to 6 mm. Different letters above the bars denote significant differences (p < 0.05) among treatments within each microbial strain. Comparisons were not made between different microorganisms.

The identity of the medicinal plant was established using a rigorously validated LC-MS/MS methodology. Among the 53 phytochemicals included in the developed method, several phenolic (fifty) and non-phenolic (tree) were detected in our extract. The LC–MS/MS total ion current (TIC) chromatograms of the 53 standards phytochemicals and HMERS exposed in Figures 2, 3, respectively). In addition, the quantitative LC–MS/MS data are presented in (Table 5). The investigation revealed that phenolic acids represented the major group of polyphenols in this plant, with quinic, gallic, chlorogenic, protocatechuic, caffeic, p-coumaric, and tannic acids identified; quinic acid being the predominant coumpond in HMERS.

Total ion chromatogram of standard phenolic compounds analysed by the developed LC–MS/MS method (Karagecili et al., 2023).

Chromatogram displaying multiple peaks numbered from one to fifty-six, indicating compound elution times over a forty-five-minute period. The highest peaks are labeled twenty-one, twenty-two, fifty-three, and fifty-six. The y-axis shows intensity, while the x-axis shows time in minutes.

Chemical profile of EMERS using LC–MS/MS.

Chromatogram showing various peaks labeled with numbers along the x-axis, which represents time in minutes. Notable peaks include those at numbers 1, 4, 6, and peaks labeled as internal standards (IS) at 25, 32, 46, and 48. The y-axis represents intensity, with a maximum value of 8.0 x 10,000.

Quantitative screening of phytochemicals in HMERS.

No Analytes R.T. M.I. (m/z) F.I. (m/z) Amount (mg/g) No Analytes R.T. M.I. (m/z) F.I. (m/z) Amount (mg/g)
1 Quinic acid 3.0 190.8 93.0 5.059 29 Salicylic acid 21.8 137.2 65.0 N.D.
2 Fumaric aid 3.9 115.2 40.9 N.D. 30 Cyranoside 23.7 447.0 284.0 0.082
3 Aconitic acid 4.0 172.8 129.0 N.D. 31 Miquelianin 24.1 477.0 150.9 N.D.
4 Gallic acid 4.4 168.8 79.0 2.601 32 Rutin-D3-IS 25.5 612.2 304.1 N.A.
5 Epigallocatechin 6.7 304.8 219.0 N.D. 33 Rutin 25.6 608.9 301.0 N.D.
6 Protocatechuic acid 6.8 152.8 108.0 0.764 34 Isoquercitrin 25.6 463.0 271.0 N.D.
7 Catechin 7.4 288.8 203.1 0.355 35 Hesperidin 25.8 611.2 449.0 0.036
8 Gentisic acid 8.3 152.8 109.0 N.D. 36 o-Coumaric acid 26.1 162.8 93.0 N.D.
9 Chlorogenic acid 8.4 353.0 85.0 0.201 37 Genistin 26.3 431.0 239.0 N.D.
10 Protocatechuicaldehyde 8.5 137.2 92.0 N.D. 38 Rosmarinic acid 26.6 359.0 197.0 N.D.
11 Tannic acid 9.2 182.8 78.0 0.266 39 Ellagic acid 27.6 301.0 284.0 N.D.
12 Epigallocatechin gallate 9.4 457.0 305.1 N.D. 40 Cosmosiin 28.2 431.0 269.0 N.D.
13 Cynarin 9.8 515.0 191.0 N.D. 41 Quercitrin 29.8 447.0 301.0 N.D.
14 4-OH Benzoic acid 10.5 137.2 65.0 N.D. 42 Astragalin 30.4 447.0 255.0 N.D.
15 Epicatechin 11.6 289.0 203.0 3.734 43 Nicotiflorin 30.6 592.9 255.0/284.0 N.D.
16 Vanilic acid 11.8 166.8 108.0 N.D. 44 Fisetin 30.6 285.0 163.0 N.D.
17 Caffeic acid 12.1 179.0 134.0 0.055 45 Daidzein 34.0 253.0 223.0 N.D.
18 Syringic acid 12.6 196.8 166.9 N.D. 46 Quercetin-D3-IS 35.6 304.0 275.9 N.A.
19 Vanillin 13.9 153.1 125.0 N.D. 47 Quercetin 35.7 301.0 272.9 N.D.
20 Syringic aldehyde 14.6 181.0 151.1 N.D. 48 Naringenin 35.9 270.9 119.0 0.022
21 Daidzin 15.2 417.1 199.0 N.D. 49 Hesperetin 36.7 301.0 136.0/286.0 N.D.
22 Epicatechin gallate 15.5 441.0 289.0 1.499 50 Luteolin 36.7 284.8 151.0/175.0 N.D.
23 Piceid 17.2 391.0 135/106.9 N.D. 51 Genistein 36.9 269.0 135.0 N.D.
24 p-Coumaric acid 17.8 163.0 93.0 0.067 52 Kaempferol 37.9 285.0 239.0 N.D.
25 Ferulic acid-D3-ISh 18.8 196.2 152.1 N.A. 53 Apigenin 38.2 268.8 151.0/149.0 N.D.
26 Ferulic acid 18.8 192.8 149.0 N.D. 54 Amentoflavone 39.7 537.0 417.0 N.D.
27 Sinapic acid 18.9 222.8 193.0 N.D. 55 Chrysin 40.5 252.8 145.0/119.0 N.D.
28 Coumarin 20.9 146.9 103.1 N.D. 56 Acacetin 40.7 283.0 239.0 N.D.

RT, retention time; M.I., molecular ion; F.I., fragment ion; N.A., not applicable; N.D., not detected.

Bold values indicate the major compounds in the extract.

According to the Table 5, R. officinale stems exhibited a notably high quinic acid content (5.059 analyte/g extract), highlighting this species as a significant natural source of quinic acid, followed by epicatechin, gallic acid and epicatechin gallate (Figure 4).

Chemical structures of the major bioactive compounds detected in HMERS.

Chemical structures of four compounds. From left to right: Epiactechin, a bicyclic flavonoid with hydroxyl groups; Gallic acid, a phenolic acid with three hydroxyl groups on a benzene ring; Quinic acid, a cyclohexane carboxylic acid with multiple hydroxyl groups; Epiactechin gallate, a complex of epiactechin and gallic acid. Labels are beneath each structure. Discussion

Our survey aimed at profiling the phytochemical content of Rheum officinale stems cultivated in Algeria using a hydromethanolic extract and to assess their inhibitory effect on cholinesterase enzymes (AChE and BChE), and digestive carbohydrate enzymes, mainly α-amylase, along with their antioxidant and antimicrobial potentials. The LC–MS/MS profiling of HMERS identified a set of bioactive phytochemicals, even though, numesrous molecules were not detected (N.D.) or present in trace quantities. The profile highlights both phenolic acids and flavonoids, which are well-known as key contributors to antioxidant, antimicrobial, enzyme-inhibitory, and protective biological activities. Quinic acid, present at the highest concentration (5.059 mg/g), was the predominant compound in HMERS. As an important metabolite with antioxidant, anticancer, antidiabetic, hepatoprotective, and neuroprotective properties (Li et al., 2024; Samimi et al., 2021), its high presence in the rhubarb extract likely explains the observed antioxidant activity and suggests a role in attenuating oxidative stress-related conditions, including Alzheimer’s disease and metabolic dysfunctions. According to our LC-MS/MS analyses acquired data, study of Benali et al. (2024) identified that quinic acid as the most abundant phenolic compound in Rheum officinale leaf extracts, with a concentration of 129.686 mg/g. This finding suggests that quinic acid may contribute to the antioxidant and anti-inflammatory properties of the plant. The other most abundant constituent within the extract was gallic acid, with 2.601 mg/g. As a potent antioxidant, it also exhibits anti-inflammatory, anticancer, and antimicrobial properties (Kahkeshani et al., 2019; Keyvani-Ghamsari et al., 2023). Its high concentration likely underlies part of the extract’s strong radical-scavenging activity. Followed by epicatechin (3.734 mg/g) and epicatechin gallate (1.499 mg/g), these flavan-3-ols are well-documented antioxidants having cardiovascular, antidiabetic, and neuroprotective benefits (Márquez Campos et al., 2020). They are known also to modulate carbohydrate metabolism and neurotransmitter degradation, supporting the inhibitory activity against α-amylase and cholinesterases. Their presence in stems of rhubarb further reinforces the plant’s pharmacological value in both traditional and modern medicinal applications. Similar study reported that rhubarb extracts (Rheum officinale leaf) contain flavan-3-ols at concentrations ranging from 86.57 to 195.98 mg per 100 g of dry matter, depending on the variety and harvest period (Dai et al., 2024). Protocatechuic acid (0.764 mg/g) and catechin (0.355 mg/g), both contribute to antioxidant and antimicrobial effects. Protocatechuic acid, particularly, is associated with hepatoprotective and nephroprotective activities, consistent with rhubarb’s traditional use against renal disorders. Other minor compounds detected in trace amounts (<0.1 mg/g), such as, caffeic acid (0.055 mg/g), p-coumaric acid (0.067 mg/g), cyranoside (0.082 mg/g), hesperidin (0.036 mg/g), and naringenin (0.022 mg/g). Although present in low concentrations, these molecules are bioactive flavonoids and phenolic acids with anti-inflammatory, antimicrobial, and metabolic regulatory functions. Their synergistic interactions with major compounds may enhance the pharmacological profile of the extract (Kakkar and Bais, 2014; Kassab et al., 2022; Khattabi et al., 2022).

Species of the genus Rheum, including R. officinale, are widely recognized for their therapeutic potential. Rich in phenolic acids, flavonoids and anthraquinones. This species possesses a broad spectrum of biological activities, including antioxidant, anti-inflammatory, antimicrobial, anticancer, antidiabetic, hepatoprotective, and laxative effects. Their bioactive compounds contribute to protection against oxidative stress, modulation of metabolic disorders, and support of digestive and liver health, which underlies their long-standing use in traditional medicine (Xiang et al., 2020; Yang et al., 2024). In our extract, several common phytochemicals, such as anthraquinones, rutin, quercetin, syringic acid, ferulic acid, and kaempferol, were undetected. Their absence may reflect species-specific phytochemistry, methanol extraction selectivity, or a lower accumulation in stems comparing to roots or leaves.

According to our results, rhubarb stems exhibit significant antimicrobial action, particularly against S. aureus, which is highly sensitive to its compounds such us quinic acid. Studies suggest that rhubarb exerts these effects by altering membrane permeability, inhibiting protein synthesis, and disrupting respiratory metabolism (Lingqing et al., 2021; Xiang et al., 2017). The study by Li et al. (2014) focused on the antibacterial activity of quinic acid against Staphylococcus aureus by demonstrating that quinic acid reduces membrane fluidity and interferes with the normal function of the bacterial cell membrane. The researchers found that quinic acid, along with chlorogenic acid, possessed wide-ranging antibacterial effects. Other molecules also extracted from different species of Rheum include anthraquinones and its derivatives, such as emodin, rhein, and aloe-emodin, show remarkable antibacterial effects in vitro against various strains, such as S. aureus, Lactobacillus, and E. coli (Ji et al., 2017; Jiang et al., 2019; Stompor–Gorący, 2021).

Starch is the main source of digestible carbohydrates in the human food and the major contributor to postprandial glucose levels. Its enzymatic hydrolysis is carried out by α-amylase and α-glucosidase, the main catalysts involved in carbohydrate digestion. Inhibiting these enzymes is a well-established therapeutic strategy to control hyperglycemia by limiting glucose absorption (Melakhessou et al., 2021). In parallel, Cholinesterases, namely (AChE) acetylcholinesterase and (BChE) butyrylcholinesterase, act as key enzymes involved in neurotransmission. Dysregulation of their activity is strongly associated with Alzheimer’s disease. In this study, rhubarb stem extract showed significant in vitro inhibition of AChE, BChE, and α-amylase, consistent with the reported neuroprotective and antidiabetic potential of its constituents. Our results suggest that the combined presence of phenolic acids (e.g., gallic, protocatechuic, caffeic, and p-coumaric acids) and flavan-3-ols (epicatechin, catechin) contributes to the observed enzyme inhibition, thereby supporting the therapeutic relevance of the (HMERS). Previous studies have similarly documented the effective inhibition of these enzymes by rhubarb-derived preparations. Moretheless (Xie et al., 2022), highlighted that anthraquinones, flavanols and their polymers, as well as phenolic acids such as gallic acid, represent core bioactive constituents of rhubarb responsible for its multifunctional pharmacological properties. Previous studies demonstrated that anthraquinones such as emodin, chrysophanol, rhein, and danthron have shown therapeutic potential in Alzheimer’s disease models (Cao et al., 2017; Li et al., 2019). Notably, rhein-derived hybrids inhibited key enzymes (AChE, BChE, BACE-1), reduced Aβ aggregation in vitro, and mitigated oxidative stress and tau pathology (Pérez-Areales et al., 2017). Similarly, tacrine–rhein hybrids further combined anti-amyloid and metal-chelating activities with fewer side effects (Li et al., 2014). Furthermore, rhubarb has long been used in traditional medicine for the management of diabetic nephropathy (DN) and is frequently combined with conventional drugs for enhanced efficacy (Cao et al., 2017; Huang et al., 2023). Clinical investigation have demonstrated that treatment with rhubarb-based compounds significantly improves biochemical markers including serum creatinine, blood urea nitrogen, albumin, and fasting plasma glucose. The nephroprotective effects of rhubarb are ascribed to its ability to reduce urinary protein excretion, regulate lipid metabolism, improve renal function, and modulate key molecular markers. These actions help suppress renal inflammation and fibrosis, thereby slowing the progression of DN (Gao and Nan, 2022; Zhang et al., 2023). In summary, our findings provide compelling evidence that rhubarb (R. officinale) contains bioactive compounds, including phenolic acids and flavan-3-ols, which collectively contribute to its antioxidant, antibacterial, antidiabetic, and neuroprotective activities. These results not only support traditional uses of rhubarb but also highlight its potential as a valuable reservoir of multifunctional molecules with therapeutic relevance.

 Conclusion

Rhubarb is recognized as one of the most valuable medicinal species, extensively applied in ancestral healing systems due to its therapeutic efficacy. The findings of this study demonstrate that Rheum officinale stems possess a remarkable profile of bioactive compounds, primarily phenolics and flavonoids, which contribute to their potent antioxidant, enzyme-inhibitory, and antimicrobial potential. These biological properties suggest that R. officinale could serve as a promising natural source for developing nutraceuticals and therapeutic agents targeting oxidative stress, neurodegenerative disorders, and metabolic diseases.

To fully harness its pharmacological potential, subsequent research should emphasize the purification and structural characterization of its bioactive molecules, combined with in vivo evaluations of their therapeutic effectiveness and safety. These additional investigations would contribute to a clearer understanding of the mechanisms underlying its biological effects and support its possible translation into clinical applications.

 Data availability statement

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

 Ethics statement

Written informed consent was obtained from the individual(s), and minor(s)’ legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.

 Author contributions

FK: Formal Analysis, Validation, Project administration, Methodology, Data curation, Supervision, Software, Conceptualization, Resources, Writing – review and editing, Visualization, Writing – original draft, Funding acquisition, Investigation. AiA: Visualization, Data curation, Validation, Investigation, Methodology, Conceptualization, Funding acquisition, Supervision, Project administration, Resources, Software, Writing – review and editing, Formal Analysis, Writing – original draft. LK: Funding acquisition, Writing – review and editing, Formal Analysis, Supervision, Investigation, Writing – original draft, Software, Project administration, Validation, Data curation, Resources, Methodology, Conceptualization, Visualization. MY: Formal Analysis, Visualization, Resources, Funding acquisition, Writing – original draft, Project administration, Investigation, Supervision, Data curation, Methodology, Writing – review and editing, Validation, Conceptualization, Software. OC: Funding acquisition, Supervision, Resources, Software, Formal Analysis, Writing – review and editing, Writing – original draft, Visualization, Data curation, Validation, Project administration, Conceptualization, Methodology, Investigation. AyA: Supervision, Writing – review and editing, Writing – original draft, Formal Analysis, Data curation, Software, Funding acquisition, Conceptualization, Resources, Investigation, Validation, Visualization, Project administration, Methodology. MM: Data curation, Methodology, Validation, Investigation, Writing – review and editing, Formal Analysis, Writing – original draft, Conceptualization, Resources, Supervision, Visualization, Funding acquisition, Project administration, Software.

 Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer IT declared a past co-authorship with the author MY at the time of review.

 Generative AI statement

The authors declare that no Generative AI was used in the creation of this manuscript.

Any alternative text (alt text) provided alongside figures in this article has been generated by Frontiers with the support of artificial intelligence and reasonable efforts have been made to ensure accuracy, including review by the authors wherever possible. If you identify any issues, please contact us.

 Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

 Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchem.2025.1661223/full#supplementary-material

Edited by: John D. Wade, University of Melbourne, Australia

Reviewed by: Ramesh Maruthi Chingle, National Institutes of Health (NIH), United States

Yang Xiaorong, Chinese Academy of Agricultural Sciences, China

Ibrahim Tegin, Siirt University, Türkiye

 References Alaadin A. M. Al-Khateeb E. H. Jäger A. K. (2007). Antibacterial activity of the Iraqi Rheum ribes. Root. Pharm. Biol. 45, 688–690. 10.1080/13880200701575049 Apak R. Güçlü K. Özyürek M. Karademir S. E. (2004). Novel total antioxidant capacity index for dietary polyphenols and vitamins C and E, using their cupric ion reducing capability in the presence of neocuproine: CUPRAC method. J. Agric. Food Chem. 52, 7970–7981. 10.1021/jf048741x 15612784 Benahmed F. Kerroum F. Benzakour S. djiré A. Kharoubi O. (2021). Antioxydant and hepatoprotective effect of aqueous extract of Pistacia atlantica Desf on Rat’s exposed to Mercury. Biosci. Res. 18 (2), 1238–1247. Benali T. Bakrim S. Ghchime R. Benkhaira N. El Omari N. (2024). Pharmacological insights into the multifaceted biological properties of quinic acid. PubMed. Biotechnol. Genet. Eng. Rev. 40 (2024), 4. 10.1080/02648725.2022.2122303 Blois M. S. (1958). Antioxidant determinations by the use of a stable free radical. Nature 181, 1199–1200. 10.1038/1811199a0 Cao Y. J. Pu Z. J. Tang Y. P. Shen J. Chen Y. Y. Kang A. (2017). Advances in bioactive constituents, pharmacology and clinical applications of rhubarb. Chin. Med. 12, 36. 10.1186/s13020-017-0158-5 29299052 Clementi E. M. Misiti F. (2010). Potential health benefits of rhubarb. Bioact. Foods Promot. Heal., 407–423. 10.1016/B978-0-12-374628-3.00027-X Dai L. X. Miao X. Yang X. R. Zuo L. P. Lan Z. H. Li B. (2022). High value-added application of two renewable sources as healthy food: the nutritional properties, chemical compositions, antioxidant, and antiinflammatory activities of the stalks of Rheum officinale baill. and Rheum tanguticum maxim. ex regel. Front. Nutr. 8, 1–14. 10.3389/fnut.2021.770264 35141263 Dai L. Cao X. Miao X. Yang X. Zhang J. Shang X. (2024). The chemical composition, protective effect of Rheum officinale leaf juice and its mechanism against dextran sulfate sodium-induced ulcerative colitis. Phytomedicine 129 (2024), 155653. 10.1016/j.phymed.2024.155653 38688143 Demir S. Koyu H. Yilmaz M. A. Tarhan A. Ozturk S. B. (2024). Antityrosinase activity and LC-MS/MS analysis of optimized ultrasound-assisted condition extracts and fractions from strawberry tree (Arbutus unedo L.). J. Food Drug Anal. 32, 200–218. 10.38212/2224-6614.3496 38934692 Djeridane A. Yousfi M. Nadjemi B. Boutassouna D. Stocker P. Vidal N. (2006). Antioxidant activity of some algerian medicinal plants extracts containing phenolic compounds. Food Chem. 97, 654–660. 10.1016/J.FOODCHEM.2005.04.028 Ellman G. L. Courtney K. D. Andres V. Featherstone R. M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88–95. 10.1016/0006-2952(61)90145-9 13726518 Emen Tanrikut S. Çeken B. Altaş S. Pirinççioǧlu M. Kizil G. Kizil M. (2013). DNA cleavage protecting activity and in vitro antioxidant potential of aqueous extract from fresh stems of Rheum ribes . Acta Aliment. 42, 461–472. 10.1556/AALIM.42.2013.4.1 Findik B. T. Yildiz H. Akdeniz M. Yener I. Yilmaz M. A. Cakir O. (2024). Phytochemical profile, enzyme inhibition, antioxidant, and antibacterial activity of Rosa pimpinellifolia L.: a comprehensive study to investigate the bioactivity of different parts (whole fruit, pulp, and seed part) of the fruit. Food Chem. 455, 139921. 10.1016/J.FOODCHEM.2024.139921 38843718 Gao Y. Nan Z. (2022). Mechanistic insights into the use of rhubarb in diabetic kidney disease treatment using network pharmacology. Med. Baltim. 101, e28465. 10.1097/MD.0000000000028465 35029893 Goel V. Cheema S. K. Agellon L. B. Ooraikul B. Basu T. K. (1999). Dietary rhubarb (Rheum rhaponticum) stalk fibre stimulates cholesterol 7α-hydroxylase gene expression and bile acid excretion in cholesterol-fed C57BL/6J mice. Br. J. Nutr. 81, 65–71. 10.1017/S0007114599000161 10341678 Huang Z. Xu Y. Wang Q. Gao X. (2019). Metabolism and mutual biotransformations of anthraquinones and anthrones in rhubarb by human intestinal flora using UPLC-Q-TOF/MS. J. Chromatogr. B 1104, 59–66. 10.1016/J.JCHROMB.2018.10.008 30445288 Huang W. Rao Y. Li L. Li C. An Y. (2023). Clinical effect of rhubarb on the treatment of chronic renal failure: a meta-analysis. Front. Pharmacol. 14, 1108861. 10.3389/fphar.2023.1108861 37153797 Ji X. Liu X. Peng Y. Zhan R. Xu H. Ge X. (2017). Comparative analysis of methicillin-sensitive and resistant Staphylococcus aureus exposed to emodin based on proteomic profiling. Biochem. Biophys. Res. Commun. 494, 318–324. 10.1016/J.BBRC.2017.10.033 29017918 Jiang L. Yi T. Shen Z. Teng Z. Wang J. (2019). Aloe-emodin attenuates staphylococcus aureus pathogenicity by interfering with the oligomerization of α-Toxin. Front. Cell. Infect. Microbiol. 9, 157. 10.3389/fcimb.2019.00157 31157174 Jintao X. Yongli S. Liming Y. Quanwei Y. Chunyan L. Xingyi C. (2018). Near-infrared spectroscopy for rapid and simultaneous determination of five main active components in rhubarb of different geographical origins and processing. Spectrochim. Acta Part A Mol. Biomol. Spectrosc. 205, 419–427. 10.1016/J.SAA.2018.07.055 30048943 Kahkeshani N. Farzaei F. Fotouhi M. Alavi S. S. Bahramsoltani R. Naseri R. (2019). Pharmacological effects of gallic acid in health and diseases: a mechanistic review. Iran. J. Basic Med. Sci. 22 (3), 225–237. 10.22038/ijbms.2019.32806.7897 31156781 Kakkar S. Bais S. (2014). A review on protocatechuic acid and its pharmacological potential. ISRN Pharmacol. 2014, 1–9. 10.1155/2014/952943 25006494 Kalisz S. Oszmiański J. Kolniak-Ostek J. Grobelna A. Kieliszek M. Cendrowski A. (2020). Effect of a variety of polyphenols compounds and antioxidant properties of rhubarb (Rheum rhabarbarum). LWT 118, 108775. 10.1016/J.LWT.2019.108775 Karagecili H. Yılmaz M. A. Ertürk A. Kiziltas H. Güven L. Alwasel S. H. (2023). Comprehensive metabolite profiling of berdav propolis using LC-MS/MS: determination of antioxidant, anticholinergic, antiglaucoma, and antidiabetic effects. Molecules 28, 1739. 10.3390/molecules28041739 36838726 Kasabri V. Afifi F. U. Hamdan I. (2011). In vitro and in vivo acute antihyperglycemic effects of five selected indigenous plants from Jordan used in traditional medicine. J. Ethnopharmacol. 133, 888–896. 10.1016/J.JEP.2010.11.025 21093568 Kassab R. B. Theyab A. Al-Ghamdy A. O. Algahtani M. Mufti A. H. Alsharif K. F. (2022). Protocatechuic acid abrogates oxidative insults, inflammation, and apoptosis in liver and kidney associated with monosodium glutamate intoxication in rats. Environ. Sci. Pollut. Res. 29, 12208–12221. 10.1007/s11356-021-16578-4 34562213 Keyvani-Ghamsari S. Rahimi M. Khorsandi K. (2023). An update on the potential mechanism of gallic acid as an antibacterial and anticancer agent. Food Sci. Nutr. 11 (10), 5856–5872. 10.1002/fsn3.3615 37823155 Khattabi L. Boudiar T. Bouhenna M. M. Chettoum A. Chebrouk F. Chader H. (2022). RP‐HPLC‐ESI‐QTOF‐MS qualitative profiling, antioxidant, anti‐enzymatic, anti‐inflammatory and non‐cytotoxic properties of Ephedra alata monjauzeana. Foods 11, 145. 10.3390/foods11020145 35053877 Krajewska A. Dziki D. Yilmaz M. A. Özdemir F. A. (2024). Physicochemical properties of dried and powdered pear pomace. Molecules 29, 742. 10.3390/molecules29030742 38338485 Lee M. Hutcheon J. Dukan E. Milne I. (2017). Rhubarb (Rheum species): the role of Edinburgh in its cultivation and development. J. R. Coll. Physicians Edinb 47, 102–109. 10.4997/jrcpe.2017.121 28569293 Li S. Y. Jiang N. Xie S. S. Wang K. D. G. Wang X. B. Kong L. Y. (2014). Design, synthesis and evaluation of novel tacrine–rhein hybrids as multifunctional agents for the treatment of Alzheimer’s disease. Org. Biomol. Chem. 12, 801–814. 10.1039/C3OB42010H 24310227 Li X. Chu S. Liu Y. Chen N. (2019). Neuroprotective effects of anthraquinones from rhubarb in central nervous system diseases. Evidence-Based Complement. Altern. Med. 2019, 1–12. 10.1155/2019/3790728 31223328 Li S. Cai Y. Guan T. Zhang Y. Huang K. Zhang Z. (2024). Quinic acid alleviates high-fat diet-induced neuroinflammation by inhibiting DR3/IKK/NF-κB signaling via gut microbial tryptophan metabolites. Gut Microbes 16 (1), 2374608. 10.1080/19490976.2024.2374608 38972055 Lingqing X. Yanmei M. Yonghuan J. Zinan L. Qianjun H. Weihong W. (2021). Study on the antibacterial effect and mechanism of rhubarb and monomer on Staphylococcus aureus . Guoji Jianyan Yixue Zazhi 42, 1929–1934. Márquez Campos E. Jakobs L. Simon M. C. (2020). Antidiabetic effects of Flavan-3-ols and their microbial metabolites. Nutrients 12, 1592. 10.3390/nu12061592 32485837 Melakhessou M. A. Marref S. E. Benkiki N. Marref C. Becheker I. Khattabi L. (2021). In vitro,, acute and subchronic evaluation of the antidiabetic activity of Atractylis flava Desf n-butanol extract in alloxan-diabetic rats. Futur. J. Pharm. Sci. 7, 206. 10.1186/s43094-021-00358-5 Müller L. Gnoyke S. Popken A. M. Böhm V. (2010). Antioxidant capacity and related parameters of different fruit formulations. LWT - Food Sci. Technol. 43, 992–999. 10.1016/j.lwt.2010.02.004 Oncu S. Becit-Kizilkaya M. Bilir A. Saritas A. Arikan-Soylemez E. S. Koca H. B. (2024). Anti-cataract effect of the traditional aqueous extract of yerba mate (Ilex paraguariensis A. St.-Hil.): an in ovo perspective. Life 14, 994. 10.3390/life14080994 39202736 Oyaizu M. (1986). Studies on products of browning reaction. Antioxidative activities of products of browning reaction prepared from glucosamine. Jpn. J. Nutr. Diet. 44, 307–315. 10.5264/eiyogakuzashi.44.307 Pérez-Areales F. J. Betari N. Viayna A. Pont C. Espargaró A. Bartolini M. (2017). Design, synthesis and multitarget biological profiling of second-generation anti-alzheimer rhein–huprine hybrids. Future Med. Chem. 9, 965–981. 10.4155/FMC-2017-0049 28632395 Re R. Pellegrini N. Proteggente A. Pannala A. Yang M. Rice-Evans C. (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol. Med. 26, 1231–1237. 10.1016/s0891-5849(98)00315-3 10381194 Samimi S. Ardestani M. S. Dorkoosh F. A. (2021). Preparation of carbon quantum dots-quinic acid for drug delivery of gemcitabine to breast cancer cells. J. Drug Deliv. Sci. Technol. 61, 102287. 10.1016/J.JDDST.2020.102287 Shang X. F. Zhao Z. M. Li J. C. Yang G. Z. Liu Y. Q. Dai L. X. (2019). Insecticidal and antifungal activities of Rheum palmatum L. anthraquinones and structurally related compounds. Ind. Crops Prod. 137, 508–520. 10.1016/J.INDCROP.2019.05.055 Singleton V. L. Rossi J. A. J. (1965). Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am. J. Enology Vitic. 16, 144–158. 10.5344/ajev.1965.16.3.144 Stompor–gorący M. (2021). The health benefits of Emodin, a natural anthraquinone derived from Rhubarb-A summary update. Int. J. Mol. Sci. 22, 9522. 10.3390/IJMS22179522 34502424 Wang X. Feng L. Zhou T. Ruhsam M. Huang L. Hou X. (2018). Genetic and chemical differentiation characterizes top-geoherb and non-top-geoherb areas in the TCM herb rhubarb. Sci. Rep. 81 (8), 9424. 10.1038/s41598-018-27510-1 29930263 Xiang H. Cao F. Ming D. Zheng Y. Dong X. Zhong X. (2017). Aloe-emodin inhibits Staphylococcus aureus biofilms and extracellular protein production at the initial adhesion stage of biofilm development. Appl. Microbiol. Biotechnol. 101, 6671–6681. 10.1007/s00253-017-8403-5 28710559 Xiang H. Zuo J. Guo F. Dong D. (2020). What we already know about rhubarb: a comprehensive review. Chin. Med. 15, 88. 10.1186/s13020-020-00370-6 32863857 Xie L. Wang Y. Luo G. Zhou W. Miao J. Tang S. (2020). Identification of the multiple bioactive derivatives and their endogenous molecular targets that may mediate the laxative effect of rhubarb in rats. J. Tradit. Chin. Med. Sci. 7, 210–220. 10.1016/J.JTCMS.2020.04.004 Xie J. Xiong J. Ding L. S. Chen L. Zhou H. Liu L. (2022). A efficient method to identify cardioprotective components of Astragali Radix using a combination of molecularly imprinted polymers-based knockout extract and activity evaluation. J. Chromatogr. A 1576, 10–18. 10.1016/j.chroma.2018.09.027 30245072 Xiong F. Nie X. Zhao X. Yang L. Zhou G. (2019). Effects of different nitrogen fertilizer levels on growth and active compounds of rhubarb from Qinghai plateau. J. Sci. Food Agric. 99, 2874–2882. 10.1002/JSFA.9500 30460686 Yang X. Dai L. Yan F. Ma Y. Guo X. Jenis J. (2024). The phytochemistry and pharmacology of three Rheum species: a comprehensive review with future perspectives. Phytomedicine 131, 155772. 10.1016/j.phymed.2024.155772 38852474 Yener I. Yilmaz M. A. Olmez O. T. Akdeniz M. Tekin F. Hasimi N. (2020). A detailed biological and chemical investigation of Sixteen Achillea species’ essential oils via chemometric approach. Chem. Biodivers. 17, e1900484. 10.1002/CBDV.201900484 31999042 Yilmaz M. A. (2020). Simultaneous quantitative screening of 53 phytochemicals in 33 species of medicinal and aromatic plants: a detailed, robust and comprehensive LC–MS/MS method validation. Ind. Crops Prod. 149, 112347. 10.1016/J.INDCROP.2020.112347 Yilmaz M. A. Cakir O. Zengin G. Izol E. Behcet L. (2024). The uprisal of a lost endemic edible species, Micromeria cymuligera: comprehensive elucidation of its biological activities and phytochemical composition. Food Biosci. 61, 104690. 10.1016/J.FBIO.2024.104690 Zengin G. Sarikurkcu C. Aktumsek A. Ceylan R. Ceylan O. (2014). A comprehensive study on phytochemical characterization of Haplophyllum myrtifolium Boiss. endemic to Turkey and its inhibitory potential against key enzymes involved in Alzheimer, skin diseases and type II diabetes. Ind. Crops Prod. 53, 244–251. 10.1016/j.indcrop.2013.12.043 Zhang F. Wu R. Liu Y. Dai S. Xue X. Li Y. (2023). Nephroprotective and nephrotoxic effects of Rhubarb and their molecular mechanisms. Biomed. Pharmacother. 160, 114297. 10.1016/J.BIOPHA.2023.114297 36716659 Zhang H. He Q. Xing L. Wang R. Wang Y. Liu Y. (2024). The haplotype-resolved genome assembly of autotetraploid rhubarb Rheum officinale provides insights into its genome evolution and massive accumulation of anthraquinones. Plant Commun. 5, 100677. 10.1016/j.xplc.2023.100677 37634079