Phenolic Compound Content and Antioxidant Activity of Rheum ribes Shells

. Te antioxidative and therapeutic properties of Rheum ribes , a plant indigenous to Turkey, have been extensively researched. However, little attention has been paid to compounds extracted from R. ribes shells. Tis study focused on assessing the phenolic compound content and antioxidative capabilities of R. ribes shells. We identifed 44 out of 88 phytochemical compounds present in these shells using liquid chromatography-high-resolution mass spectrometry. Among these compounds, rutin hydrate M-OH 2 , kuromanine, and procyanidin B2 emerged as the most abundant, whereas sinapic acid had the lowest concentration. Furthermore, antioxidant activity was determined using the 2,2-diphenyl-1-picrylhydrazyl assay, with the R. ribes shell extract exhibiting an activity level of 1.06 ± 0.3mg Trolox equivalent/g of sample. In summary, this research explored the potential health advantages of R. ribes shells, thereby ofering valuable insights for discovering novel bioactive compounds in natural resources for future drug development.


Introduction
Phytochemicals are plant-derived chemicals primarily composed of polyphenols.In addition to essential nutrients, vitamins, and minerals, polyphenols are important for human health.Te spectrum of phytochemicals within the human diet is estimated to encompass a wide range, from 5,000 to 25,000 phytochemicals, found abundantly in various food sources, including fruits, vegetables, seeds, and other plant parts.Te bioactivity of these phytochemicals extends to a myriad of health benefts, contributing to the mitigation of health risks, such as Alzheimer's disease, heart diseases, cancers, and diabetes [1][2][3][4].Nonetheless, among approximately 250,000 fowering plants cultivated globally, only a scant fraction has been studied for active substances.Consequently, numerous unexplored phytochemical compounds exist within plant sources, awaiting discovery and characterization [5][6][7][8].
Rheum ribes L., also known as Us ¸gun, belongs to the Polygonaceae family.Us ¸gun grows in rocky and hilly areas at altitudes of 2100-2800 m.Exclusive to Turkey's eastern Anatolia region, this plant is the only species within the Rheum genus.
Te stems of this plant have thick rhizomes and can grow up to a height of 50 cm.Te leaves are characterized by their green and red coloration, wide kidney-shaped appearance, and the presence of stems.Te fowers are small and yellow.Petioles and fresh stems can be consumed raw or after being cooked [9].
Free radicals are composed of atoms and molecules with unshared electrons.Free radicals are either generated endogenously (via mitochondrial propagation, respiration, enzymes, and auto-oxidation reactions) or exogenously (in response to drugs, UV rays, alcohol, ionizing radiation, xenobiotics, environmental pollution, and cigarette smoke).In living systems, oxygen usage generates free radicals, which scavenge electrons from vital cellular components and disrupt membrane structures [10,11].Terefore, to prevent the damage caused by toxic free radicals, organisms have developed defense systems based on antioxidants that can neutralize free radicals, which can contribute to a variety of health issues, including premature aging, cardiovascular diseases, diabetes, and cancer.Antioxidants prolong food shelf life, preserve sensory qualities, and enhance overall quality by preventing rancidity in oily foods.Tey play a crucial role in improving nutritional value and meeting consumer preferences in the food sector [12][13][14].Phenolic compounds are prevalent in plant-and animal-derived foods, and this is valuable in the context of both human and animal health.Tey exert antioxidant, antiinfammatory, antibacterial, and antiviral efects, thereby contributing to disease prevention and bolstering overall health [15].Due to cost considerations, synthetic antioxidants are generally preferred over their natural counterparts.However, some studies have revealed that synthetic antioxidants can be toxic and carcinogenic.Previous studies have investigated the applicability of natural substances as antioxidants in food, reporting them to be more efective than synthetic antioxidants [16][17][18].According to the World Health Organization (WHO), approximately 20,000 plants are utilized for their medicinal properties.Research conducted by the Food and Agricultural Organization revealed 21,000 medicinal plant varieties, 5000 of which are commercially traded.In Turkey, only 500 of the 9000 available medicinal plants are used for therapeutic purposes.Between 2001 and 2005, the WHO initiated a program to support and improve traditional treatment methods in developing countries [19][20][21][22].Hence, organic sources, such as plants abundant in antioxidants, hold signifcant value for human health and scientifc research [23].
Traditional uses of Rheum ribes include the utilization of fresh shoots and stems in the amelioration of diverse medical conditions, such as hemorrhoids, measles, smallpox, gastrointestinal ailments, and diarrhea.Moreover, the plant exhibits therapeutic efcacy against disorders, such as diabetes, ulcers, hypertension, obesity, and digestive disorders.In countries other than Turkey, this plant is cultivated from young shoots.Notably, the roots and shoots of R. ribes exhibit high antioxidant activity [24][25][26][27].In Turkey, where R. ribes proliferates abundantly in its natural habitat, it is a staple in the local diet, leading to the routine disposal of considerable quantities of its shells on an annual basis.Te discarded shells of R. ribes carry components with promising applications in health, cosmetics, food, and pharmaceutical sectors.Surprisingly, despite their potential, there is little scientifc evidence regarding the properties of compounds derived from these shells.Tis unexplored territory presents an opportunity for further scientifc investigation that may lead to potential innovations in various industries.
In this study, our main goal was to analyze the phenolic composition and antioxidant capacity of the R. ribes shells.We evaluated the antioxidant activity using the 2,2diphenyl-1-picrylhydrazyl (DPPH) assay and determined the phenolic composition of the R. ribes shells using liquid chromatography-high-resolution mass spectrometry (LC-HRMS).Tis investigation aimed to identify new nutritional resources that could be instrumental in the prevention of various diseases.Tis study can serve as a guide for the production of herbal medicines and ofers a valuable resource, namely, the shell of R. ribes, for future research in this area.

Collection of Plant Materials. R. ribes was collected in the
Pervari district of Siirt province, Turkey, where it grows naturally, at the beginning of May 2023.R. ribes plant is a plant known to everyone in the region, like other vegetables and fruits widely consumed in the region.After collection, the shells were left to dry in a dark room at 24 °C for 20 d.Once dried, the shells were ground into a powder and utilized for subsequent experiments.

DPPH Radical-Scavenging Capacity.
Te extract was prepared by combining 0.2 g powdered shell samples with 5 mL methanol solution (75%, with 0.1% phosphoric acid).Phosphoric acid is used in the DPPH method to provide acidic conditions, increase solubility, control reaction rate, and ensure stability.Te mixture was thoroughly homogenized using an Ultra-Turrax (MS3-MaxiHomo35, Staufen, Germany) device at 600 rpm for 30 s. Next, the mixture was centrifuged at 3600 × g, 5000 rpm (Archer LC-05A; Archer, San Jose, CA, USA) for 10 min at 24 °C, followed by incubation of the resulting supernatant in an ultrasonic water bath at 25 °C for 15 min.After incubation, the supernatant was extracted twice, and the combined extracts were adjusted to a fnal volume of 10 mL using methanol.Subsequently, the prepared extract was transferred to a 100 μL tube.

Antioxidant Activity Analysis.
Te assessment of the DPPH radical-scavenging activity of the shell samples was conducted by adapting a method described earlier [28].Briefy, 0.1 mL extract was placed into a 10 mL tube, and then, 3.9 mL of a 60 μM DPPH solution, prepared with methanol as the solvent, was added.Te mixture was incubated at 21 °C for 30 min.After incubation, the absorbance was measured at 517 nm using a Libra S70 double-beam spectrophotometer (Biochrom, Cambridge, England).In this study, Trolox was used as the blank reference, and the DPPH solution was regarded as the control.Te results were then compared with the standard antioxidant compound, Trolox.Te content of the plant extract was measured in milligrams of Trolox equivalents per gram (mg Trolox equivalent/g).

Calibration Curve.
Te DPPH calibration analysis included concentrations of 20, 40, 60, 80, and 100 μg/mL.Using these concentrations, a calibration curve for DPPH was constructed, and the IC 50 value was calculated from the curve.A Trolox calibration curve was constructed using Trolox standard solutions of diferent concentrations.Te IC 50 value for DPPH was calculated as the Trolox equivalent based on the Trolox and DPPH curves.Te calculation of percentage inhibition was performed using the following formula: where Ak signifes the absorbance value of the control sample (lacking antioxidants) and A0 denotes the absorbance value of the sample containing antioxidants.Te IC 50 values of the samples (50% scavenging concentration of DPPH radicals) were obtained by calculating the percentage inhibition values from the plotted graph.

Chemical Composition Analysis.
A comprehensive analysis of the R. ribes shell using LC-HRMS led to the identifcation of 44 phytochemical compounds (Table 1).Te compounds present in the highest concentrations were rutin hydrate M-OH 2 (8917.999mg/kg), kuromanine (5243.637mg/kg), and procyanidin B2 (2930.503mg/kg), whereas sinapic acid (0.409 mg/kg) was the compound with the lowest detection limit.Catechin, one of the compounds present in the shells, ofers various benefts in preventing lung and cardiovascular diseases including cancer [29].Quercetin also inhibits cancer cell growth [30].Moreover, a diet enriched with cyanidin may be efective in preventing diabetic arterial damage and diabetic nephropathy in obese [31].Additionally, the intake of cyanidin-3-O-glycoside can reduce body fat accumulation in mice fed a high-fat diet, regulate blood sugar, and increase insulin sensitivity [32].
Phenolic compounds are potent chain-breaking antioxidants [45], and their concentrations show strong linear correlations with their efectiveness with respect to neutralizing free radicals, particularly the DPPH radical [26].Terefore, the abundance of phenolic compounds in the shells of R. ribes ofers clear health benefts.[21,26].In this study, the antioxidant capacity of the plant samples was assessed using the DPPH assay, revealing an antioxidant activity of 1.06 ± 0.3 mg Trolox equivalent/g per sample.

Journal of Chemistry
Te antioxidant capacities of R. ribes root, stem, and aqueous extracts were examined in a previous study.Te ethanolic root extract demonstrated the strongest antioxidant activity, with an IC 50 value of 4.73 ± 0.21 μg/mL.However, the aqueous root extract displayed the lowest antioxidant activity, with an IC 50 value of 25.62 ± 0.85 μg/mL [42].Te DPPH radical-scavenging abilities of R. ribes stem extracts, prepared using water, ethanol, and methanol solvents, were 93.49%, 94.21%, and 95.86%, respectively [46].Previous investigations have reported DPPH activities in R. ribes stem extracts prepared using aqueous and methanol solvents, yielding values of 83.90% and 87.07%, respectively [47].Among the diverse extracts obtained from diferent parts of R. ribes in a previous study, including preparations with ether, ethanol, and aqueous extracts, the ethanol extracts of the shell have exhibited the most notable antioxidant activity [48].
Phenolic compounds are typically found in foods such as fruits, vegetables, and grains, and the antioxidant capabilities of these foods are directly linked to their phenolic content [26,48].Te fndings of this study also clearly demonstrate that phenolic compounds in R. ribes shells provide antioxidant efects.

. Conclusion
Tis study identifed 44 compounds phytochemical compounds in the R. ribes shell using LC-HRMS.Te primary compounds identifed in the analysis included rutin hydrate M-OH 2 , kuromanine, procyanidin B2, and gallic acid.Notably, this is a pioneering study investigating the composition of R. ribes shells.Te existence of these phytochemicals in R. ribes shells suggests their potential application in the cosmetic and food industries, as well as in the synthesis of essential fatty acids.Furthermore, our fndings indicate that R. ribes shells possess substantial antioxidant properties, potentially serving as a natural antioxidant source for the food and pharmaceutical industries.Te shells of R. ribes contain compounds with notable antioxidant properties, warranting further studies on this topic.
Te limitations of this study are the exclusive use of the DPPH antioxidant method and the sole reliance on LC-HRMS for determining chemical compositions.Future research should incorporate a diverse range of analytical methods for a more comprehensive assessment.Additionally, comparing the chemical compositions of R. ribes shells from diferent geographical regions could provide insights into potential variations in the antioxidant and overall chemical profles of the plant.Taking these steps is crucial for a more profound understanding of the chemical content and antioxidant potential of R. ribes shells.
2.3.2.Chromatography and High-Resolution Mass Spectrometry Conditions.Te samples were analyzed using a Phenomenex Gemini 3 μm NX-C18 110 Å (100 m × 2 mm) column (Phenomenex, Torrance, CA, USA) maintained at 30 °C.Te elution process involved mobile phase A, composed of 2% (v/v) glacial acetic acid in ultrapure water obtained from an ultrapure water system (GFL 2004/human power 1, company name, city, state, country), and mobile phase B, consisting of 99.9% pure LC-MS-grade methanol (Sigma-Aldrich, St. Louis, MO, USA).Te elution parameters included a fow rate of 0.3 mL/min, a sample injection volume of 20.0 µL, and a total analysis time of 20 min.Analysis was carried out using a heated electrospray ionization Orbitrap HRMS instrument (Exactive Plus ™ ; Termo Fisher Scientifc, Waltham, MA, USA) operating in both positive (full MS/ all-ion fragmentation [AIF]) and negative (full MS/AIF) modes.LTQ Velos ESI Positive Ion Calibration Solution.Both LC and MS processes were conducted simultaneously using Trace-Finder 3.2 (Termo Fisher Scientifc) during the LC-HRMS analyses.Data acquisition and recording were executed using Xcalibur version 2.1.0.I140 (Termo Fisher Scientifc).
3.2.DPPH Scavenging Capacity.Various components of R. ribes, including its roots and stems, demonstrate significant antioxidant properties