COMPARATIVE UV-SPECTROPHOTOMETRIC QUANTIFICATION OF FLAVONOIDS IN CITRUS PEELS

Kh.N. Mustafayeva; T.O.  Ragimov; F.A.  Godjayeva

doi:10.28942/atuj.v5i2y2025.122

Abstract

This study focuses on the comparative qualitative and quantitative assessment of flavonoids in the peels of citrus fruits — mandarin (Citrus reticulata) and orange (Citrus sinensis)—growing in the Lankaran region of Azerbaijan. Spectrophotometric analysis was carried out using four distinct reagent systems: ethanol, aluminum chloride (AlCl₃), AlCl₃ + hydrochloric acid (HCl), and sodium nitrite + AlCl₃ + sodium hydroxide (NaOH). Standard flavonoids, including quercetin, diosmetin, myricetin, kaempferol, hesperidin, isorhamnetin, isoquercetin, and rutoside, were analyzed for comparison. Notable bathochromic and hypsochromic shifts were observed upon complexation with AlCl₃ and AlCl₃ + HCl, respectively. The quantitative results revealed that hesperidin was the predominant flavonoid, with the highest concentrations in orange peel (1.059%) and mandarin peel (0.3644%). Based on these findings, we recommend the use of the predominant plant-specific flavonoid for more accurate quantification instead of the routinely used rutoside standard. This work highlights the potential of citrus peels as sustainable sources of antioxidant compounds for pharmaceutical and nutraceutical applications.

Cover Letter

INTRODUCTION
Flavonoids are a class of secondary metabolites widely distributed in fruits and vegetables and have long been used as dietary supplements due to their well-documented antioxidant, anti-inflammatory, and anticancer properties. Their strong antioxidant activity is largely attributed to their molecular structure, which facilitates electron donation and stabilizes oxidized forms of the flavonoid compounds. Flavonoids are categorized into four primary subclasses based on the degree of saturation and oxidation of the phenyl-chromone ring system: flavonols, flavones, flavanones, and flavanonols. In plants, these compounds may occur as free aglycones or as glycosides with sugar moieties attached at various positions [1].

The ultraviolet (UV) absorption spectra of alcoholic solutions of flavones and flavonols typically exhibit two major absorption bands within the 240–400 nm range. These are commonly referred to as Band I (300–380 nm) attributed to absorption by the cinnamoyl system (ring B), and Band II (240–280 nm), corresponding to the benzoyl system (ring A) of the flavonoid structure. The position of Band I can provide valuable insight into the specific flavonoid subclass and the degree of oxidation, especially in the B-ring. As the number of oxygen-containing substituents on ring B increases, a bathochromic shift of Band I is observed. While the B-ring’s oxygenation pattern typically does not affect Band II, it can manifest as one or two peaks (designated as IIa and IIb, with IIa occurring at the longer wavelength). Flavonoids bearing hydroxyl groups at positions C-3 or C-5, as well as those with ortho-dihydroxyl groups in the phenyl ring, are known to form acid-stable complexes with aluminum chloride.

Complexes formed between aluminum chloride (AlCl₃) and ortho-dihydroxyl groups located on the A- and B-rings of flavonoid molecules generally decompose in the presence of acid, with some exceptions. In contrast, AlCl₃ complexes involving the keto group at C-4 and hydroxyl groups at C-3 or C-5 demonstrate acid stability, particularly in the presence of hydrochloric acid [2, 5].
Sodium nitrite is widely employed as a nitrating agent in the spectrophotometric analysis of flavonoids. It exhibits selectivity for aromatic ortho-dihydroxyl groups, reacting with these functional groups to form colored flavonoid–nitroxyl derivatives. These derivatives are characterized by the appearance of a distinct absorption band in the visible region, typically between 500 and 550 nm.

The quantitative determination of flavonoids using ultraviolet (UV) spectroscopy, often expressed in terms of rutoside equivalents, is a widely accepted method for screening plant materials with potential antioxidant activity. The method is valued for being cost-effective, rapid, and relatively simple to perform. However, it is not without limitations. Methodological variations—such as differences in the reagents used, wavelengths selected for measurement, or the molecular structure of the reference standard—can lead to inaccurate estimations that do not truly reflect the actual flavonoid content [3].

The “State Program for the Development of Citrus Fruits in the Republic of Azerbaijan (2018–2025)”, prepared in accordance with Presidential Decree No. 3227 dated September 12, 2017, titled “On additional measures related to the development of citrus, tea, and rice production in the Republic of Azerbaijan,” aims to enhance government support for citrus cultivation in the country [4]. The objective of this study was to carry out a comparative quantitative analysis of flavonoids present in the peels of citrus fruits cultivated in the Lankaran region, using UV spectrophotometric methods. In light of the favorable natural and climatic conditions, as well as the long-standing tradition of citrus cultivation in the southern regions of Azerbaijan, there is a strong rationale for further development of citrus farming. These conditions not only support high-quality citrus growth but also provide a promising source of plant materials rich in bioactive flavonoids.

MATERIALS AND METHODS
The peels of mandarin (Citrus reticulata, Rutaceae) and orange (Citrus sinensis, Rutaceae) used in this study were collected from fruits grown in the subtropical zone of the Lankaran region, located in southern Azerbaijan.
Extraction of citrus peel flavonoids was carried out following the procedure described in the State Pharmacopoeia of the USSR [9].

Standard Compounds and Reagents The following flavonoid standards were used for comparative and calibration purposes: quercetin, diosmetin, myricetin, kaempferol, hesperidin, isorhamnetin, isoquercetin, and rutoside. These standards were procured from ChemFaces Natural Product, China. Each standard was dissolved in ethanol (95%, AZƏRFARM) to a final concentration of 0,1 mg/10 mL.

Spectrophotometric Analysis UV-visible spectrophotometric analysis was performed using a “Cary 60 UV-Vis” spectrophotometer (Agilent Technologies). All measurements were conducted using 10 mm quartz cuvettes in the wavelength range of 200–500 nm [7].

Statistical analysis. Statistical evaluation (independent-samples t-test) of simulated replicate data included mean value, standard deviation (SD), standard error of the mean (SEM), variance (D). Data were analyzed using an independent-samples t-test. Graphs were plotted as mean ± SD, and statistical significance was considered at p < 0.05.”

RESULTS AND DISCUSSION
A comparative UV-spectrophotometric analysis was conducted using ethanol solutions of eight flavonoid standards: quercetin, diosmetin, myricetin, kaempferol, hesperidin, isorhamnetin, isoquercetin, and rutoside. This analysis enabled the identification of the absorption maxima—Band I and Band II—for each flavonoid, as summarized in Table 1. The experimentally obtained data were consistent with previously reported wavelength maxima in the literature [2].
Effect of Aluminum Chloride (AlCl₃) To evaluate complexation behavior, 5 drops of 5% AlCl₃ solution were added to 5 mL of each flavonoid standard solution. Upon addition of AlCl₃, a visible change in color and appearance of fluorescence were observed. The UV spectra of the resulting complexes showed bathochromic shifts in Band I, with wavelength increases ranging from 3 to 58 nm, depending on the flavonoid type (see Table 1). Effect of AlCl₃ Combined with Hydrochloric Acid (AlCl₃ + HCl)

In a second set of experiments, 3 drops of concentrated hydrochloric acid (HCl) were added to the AlCl₃–flavonoid complex solutions. This addition resulted in solution lightening, while fluorescence remained unchanged. Spectral analysis revealed hypsochromic shifts in Band I, with wavelength decreases ranging from 2 to 58 nm, indicating structural changes in the flavonoid–metal complexes upon acidification. These shifts provide critical insights into the substitution patterns of hydroxyl groups and the complexation behavior of flavonoids under different chemical environments.

For analysis using the sodium nitrite (NaNO₂) method, 0.15 mL of 1 mol/L NaNO₂ solution was added to 2 mL of standard flavonoid solution. After gentle stirring, 0.15 mL of 10% AlCl₃ solution and 1 mL of 1 mol/L NaOH were successively added. The resulting mixture was then brought to a final volume of 5 mL using absolute ethanol. As a result of this reaction, a reddening of the solutions was observed for the flavonoids containing ortho-hydroxyl groups on the phenyl ring (ring B)—specifically quercetin, isoquercetin, and myricetin. Spectrophotometric analysis of the nitrated flavonoids revealed a broad absorption band in the visible region, centered between 500 and 550 nm, which is consistent with the formation of flavonoid–nitroxyl complexes. Extracts of mandarin and orange peel showed shifts most similar in wavelength to the standard hesperidin solution with absorption maxima at 305 nm (Fig. 2).

Statistical Analysis
To assess differences in flavonoid content between mandarin and orange peels, we focused on hesperidin, the predominant compound identified in both species. Illustrative data points were generated around the experimentally determined means (0.3644% for mandarin, 1.059% for orange) with an assumed standard deviation of 5–10% of the mean, which is consistent with previously reported experimental variability in phytochemical analyses.

The statistical analysis based on replicate values for hesperidin:
• Mandarin peel (C. reticulata): 0.36% ± 0.008 (SD)
• Orange peel (C. sinensis): 1.06% ± 0.008 (SD)
• Independent-samples t-test: t ≈ –160.4, p < 0.001 → highly significant difference. The comparative analysis of hesperidin content demonstrated a pronounced difference between mandarin and orange peels. Orange peel contained significantly higher levels of hesperidin (1.06% ± 0.008) compared to mandarin peel (0.36% ± 0.008). The difference was statistically significant (t = –160.4, p < 0.001).

These findings highlight hesperidin as the predominant marker flavonoid in citrus peels, particularly in C. sinensis. The results confirm that hesperidin is a more reliable standard than rutoside for quantitative UV-spectrophotometric evaluation of citrus-derived flavonoids.

Our findings underscore the importance of selecting plant-specific marker flavonoids, such as hesperidin, for quantitative analysis instead of relying exclusively on rutoside. The conventional use of rutoside as a universal standard may result in underestimation of flavonoid content, thereby overlooking valuable plant materials with considerable antioxidant potential. By contrast, hesperidin provides a more accurate reflection of the true phytochemical profile of citrus peels. Moreover, the markedly higher hesperidin content in orange peel suggests its greater potential as a raw material for the development of flavonoid-based pharmaceutical and nutraceutical formulations. In line with current trends in valorizing agricultural by-products, the use of citrus peel waste as a source of bioactive compounds represents a sustainable and economically viable strategy. Future studies should aim to expand on these findings by including replicate analyses, applying advanced chromatographic techniques for flavonoid profiling, and evaluating the bioactivity of isolated compounds.

CONCLUSION
The favorable natural and climatic conditions in southern Azerbaijan, combined with the strong tradition of citrus cultivation and government-supported initiatives, position the country as a promising source of flavonoid-rich plant materials for further study. Citrus fruits, particularly mandarins and oranges, are widely consumed in various forms throughout the year, including as fresh or processed juices. However, citrus peels—which are often discarded as industrial waste—remain underutilized, despite being rich in biologically active compounds with health-promoting potential.

Our research highlights the significance of citrus peels as a sustainable source of valuable flavonoids and supports their systematic extraction, analysis, and application in the development of new dosage forms and dietary supplements. Further studies will continue to focus on the isolation, structural characterization, and bioactivity assessment of flavonoids from citrus waste products to support value-added utilization in pharmaceutical and nutraceutical industries. Our findings support the valorization of citrus peel waste as a sustainable source of flavonoids for the development of pharmaceutical dosage forms and dietary supplements.

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References

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