HPLC PROFILING OF BIOACTIVE COMPOUNDS IN LEMON PEEL EXTRACT

ABSTRACT

In the present study, the content of flavonoids and water-soluble vitamins in lemon peel extract was determined using high-performance liquid chromatography (HPLC). The analysis revealed the presence of flavonoids such as apigenin (17.893 mg/100 g) and rutin (5.583 mg/100 g), as well as water-soluble vitamins including vitamin B2 (9.485 mg/100 g), vitamin B6 (6.068 mg/100 g), and vitamin B1 (3.588 mg/100 g) in appreciable amounts.

The obtained results were compared with data from previously reported independent HPLC analyses of lemon peel. The comparison demonstrated general consistency in the qualitative composition of certain flavonoids and vitamins, while quantitative differences were mainly attributed to variations in extraction conditions, lemon variety, solvent type, and analytical parameters.

Overall, the findings confirm that lemon peel is a rich source of biologically active compounds and can be considered a promising raw material for the development of pharmaceutical preparations and functional food products.

АННОТАЦИЯ

 В настоящем исследовании методом высокоэффективной жидкостной хроматографии (ВЭЖХ) определено содержание флавоноидов и водорастворимых витаминов в экстракте лимонной кожуры. В результате проведённого анализа установлено наличие флавоноидов апигенина (17,893 мг/100 г) и рутина (5,583 мг/100 г), а также витаминов группы B, включая витамин B2 (9,485 мг/100 г), витамин B6 (6,068 мг/100 г) и витамин B1 (3,588 мг/100 г), в значимых количествах.

Полученные данные были сопоставлены с результатами ранее опубликованных независимых ВЭЖХ-исследований лимонной кожуры. В ходе сравнительного анализа выявлено совпадение качественного состава ряда флавоноидов и витаминов, тогда как количественные различия обусловлены, в основном, вариациями условий экстракции, сортом лимона, типом используемого растворителя и параметрами хроматографического анализа.

Результаты исследования подтверждают, что лимонная кожура является перспективным источником биологически активных веществ и может быть использована при разработке фармацевтических препаратов и функциональных пищевых продуктов.

Keywords: lemon peel, flavonoids, water-soluble vitamins, HPLC analysis, biologically active compounds.

Ключевые слова: лимонная кожура, флавоноиды, водорастворимые витамины, ВЭЖХ-анализ, биологически активные вещества.

Introduction. Lemon (Citrus limon L.) is an evergreen perennial fruit tree belonging to the Rutaceae family and is widely cultivated in subtropical climatic regions. The tree typically reaches a height of 3–7 m, with spreading branches, some of which may bear thorns. The leaves are aromatic, green in color, 10–15 cm in length and 5–8 cm in width, with a glossy upper surface and a lighter green lower surface. The flowers are small, white, and consist of five petals. The fruit is oval or round in shape, measuring approximately 6–9 cm in length and 4–6 cm in diameter, with an average weight ranging from 120 to 400 g. The internal edible portion is referred to as the pulp, while ripe fruits are characterized by a yellow peel that may be smooth or rough, and a juicy, acidic flesh divided into 8–12 segments [1].

The peel of lemon (Citrus limon L.), which constitutes the outer part of the fruit, is composed of two main layers. The outer yellow layer, known as the flavedo, represents the smooth and glossy surface of the peel and is rich in essential oils, pigments such as flavonoids and carotenoids, and various aromatic compounds. The inner white layer, referred to as the albedo, is a fibrous and slightly bitter tissue containing high amounts of pectins, cellulose, polyphenols, and other dietary fibers.

Lemon peel is distinguished by its notable physiological, chemical, and phytotherapeutic properties. It is formed as a protective barrier by the plant and plays an essential role in determining the shelf life, aroma, and sensory characteristics of the fruit. Owing to its rich composition of biologically active compounds, lemon peel has attracted increasing scientific interest as a valuable natural raw material for pharmaceutical and functional food applications [2].

Figure 1. Citrus limon L. fruit peel

The nutritional composition and content of biologically active compounds in the pulp and peel of Citrus limon L. differ significantly. Lemon pulp is characterized by a highwater content (approximately 85–90%), enrichment with sugars, and a relatively high concentration of ascorbic acid (vitamin C), reaching up to about 5.96%, which distinguishes it from the peel. In contrast, the nutritional value of lemon peel is considerably higher than that of the pulp. Although water constitutes nearly 80% of the peel, the remaining fraction is rich in vitamins, minerals, fatty acids, and dietary fibers [3].

From this perspective, the present study was designed to investigate the content of water-soluble vitamins and flavonoids in lemon peel. The primary objective was to explore the potential of lemon peel not as a waste product, but as a valuable raw material with prospects for beneficial utilization.

In recent years, several studies have focused on the chemical composition of lemon peel; however, reported data on the concentrations of flavonoids and water-soluble vitamins vary considerably across different sources. These discrepancies may be attributed to factors such as the geographical origin of the plant, methods of raw material preparation, and differences in extraction procedures and chromatographic conditions applied during analysis. Therefore, comparative evaluation of the results obtained in this study with data from independent analyses remains both relevant and scientifically justified.

Experience part.

Determination of phenolic compounds by HPLC

For the experiment, lemon fruits of the “Toshkent tong‘ichi” cultivar cultivated in the territory of Uzbekistan were used. The content of phenolic compounds in the extract was determined using high-performance liquid chromatography (HPLC). Gallic acid (Macklin, China), salicylic acid (Rhydburg Pharmaceuticals, Germany), quercetin, apigenin, and kaempferol (Regal, China), as well as rutin isolated from natural sources, were used as reference standards. HPLC-grade water, acetonitrile, acetic acid, and sodium hydroxide were employed for chromatographic analysis. Quantitative determination of polyphenols was carried out using a Shimadzu LC-40 Nexera Lite chromatographic system.

Standard stock solutions were prepared by dissolving accurately weighed amounts (5.0–5.2 mg) of each reference compound in 96% ethanol and adjusting the final volume to 50 mL. The obtained stock solutions were mixed and subsequently diluted stepwise to prepare four working standard solutions for calibration.

For sample preparation, 1 g of lemon peel was extracted with 25 mL of 96% ethanol using ultrasonic-assisted extraction at 60 °C for 20 min. The resulting extract was filtered and centrifuged at 7000 rpm, followed by filtration through a 0.45 µm membrane filter prior to HPLC analysis.

Phenolic compounds in both standard solutions and sample extracts were separated using a reversed-phase Shim-pack GIST C18 column (150 × 4.6 mm, 5 µm; Shimadzu, Japan). The mobile phase consisted of acetonitrile (A) and 0.5% aqueous acetic acid solution (B), applied in a gradient elution mode. The chromatographic conditions were as follows: injection volume of 10 µL, flow rate of 0.5 mL/min, and column temperature maintained at 40 °C. Detection of phenolic compounds was performed at a wavelength of 300 nm.

Table 1.

Gradient elution program of the mobile phase

Figure 2. Chromatogram of standards recorded at 300 nm

Determination of water-soluble vitamins by HPLC

The content of water-soluble vitamins in the extract was determined using high-performance liquid chromatography (HPLC). Reference standards of vitamins C, B1, B2, B3, B6, B9, B12, and PP were obtained from Rhydburg Pharmaceuticals, Carl Roth, DSM, and BLDPharm. HPLC-grade water, acetonitrile, acetic acid, and sodium hydroxide were used for chromatographic analysis. Quantitative determination of water-soluble vitamins was performed using a Shimadzu LC-40 Nexera Lite chromatographic system.

Standard solutions of vitamins C, B1, B3, B6, B12, and PP were prepared by dissolving the compounds in 0.1 N HCl, while vitamins B2 and B9 were dissolved in 0.025% NaOH. From the stock solutions, a series of working solutions at different concentrations was prepared by successive dilution, including a zero calibration point (0 mg/L) using purified water.

For sample preparation, 1 g of the sample was extracted with 25 mL of 0.1 N HCl using ultrasonic-assisted extraction at 60 °C for 20 min. The obtained extract was filtered and subsequently passed through a 0.22 µm membrane filter prior to HPLC analysis.

Chromatographic separation was carried out on a Shim-pack GIST C18 column using the LC-40 Nexera Lite system with a gradient elution of acetonitrile (A) and 0.25% aqueous acetic acid (B). The injection volume was 10 µL, the flow rate was set at 0.6 mL/min, and the column temperature was maintained at 40 °C. Detection of vitamins was performed at wavelengths of 265, 291, and 550 nm, while vitamin C was specifically monitored at 265 nm [4].

Table 2.

Gradient elution program of the mobile phase for the determination of vitamins

Table 3.

Mobile phase gradient program for vitamin C determination

Figure 3. HPLC chromatogram of vitamin standard solutions

Figure 4. HPLC chromatogram of vitamin C standard solution

Additional analytical data employed for comparison were evaluated as supplementary information in the present study.

Results and Discussion

Calculation of phenolic compound content

The chromatogram of the sample extract (1 g) was obtained (Figure 5), and based on the resulting data, the content of phenolic compounds in 100 g of the sample was calculated using the following equation. The calculated values are presented in Table 4.

Here, X – the amount of phenolic compounds in 100 grams of the sample, mg; C_phen – the concentration of phenolic compounds in the extract determined by the HPLC method, mg/l; V_extract – the volume of the sample extract, l; m_sample – the mass of the sample taken for extract preparation.

Figure 5. Chromatogram for the determination of polyphenols in the sample extract

Table 4.

Content and retention times of polyphenols in the extract

The chromatographic analysis demonstrated that the examined sample is rich in polyphenolic compounds. The highest concentrations were observed for apigenin (17.893 mg/100 g) and rutin (5.583 mg/100 g), indicating pronounced antioxidant and biologically active properties of the plant material.

In addition, salicylic acid (3.040 mg/100 g) and quercetin (2.063 mg/100 g) were detected in considerable amounts, which may further enhance the anti-inflammatory and protective effects of the extract [5]. Although gallic acid and kaempferol were present at lower concentrations, they play an important role in contributing to the overall antioxidant activity of the extract [6, 7].

The retention times obtained under the selected chromatographic conditions confirmed the suitability of the method and the reliable separation of the target compounds.

For comparative purposes, the concentrations of flavonoids previously reported in lemon peel using other independent HPLC analyses are presented below.

Table 5.

 Flavonoid contents in lemon peel determined by independent HPLC analyses (mg/mL) [9]

Note. It should be noted that independent HPLC analyses of lemon peel reported the presence of rutin and apigenin, which is in qualitative agreement with the results obtained in the present study. However, direct quantitative comparison was not performed due to differences in extraction conditions and calculation units.

Determination of vitamins in the sample extract

The chromatograms of the sample extract were obtained (Figures 6 and 7), and based on the acquired data, the vitamin contents in 100 g of the sample were calculated using the following equation. The calculated values are presented in Table 6.

Where X represents the vitamin content in 100 g of the sample (mg); C_vit is the concentration of the vitamin in the extract determined by HPLC (mg/L); V_extract denotes the volume of the sample extract (L); m_sample is the mass of the sample used for extraction (g).

Figure 6. HPLC chromatogram of vitamins in the sample extract

Figure 7. Chromatogram for the determination of vitamin C in the sample extract

Table 6.

Vitamin contents and retention times in the extract

According to the chromatographic analysis, the investigated sample was found to be rich in water-soluble vitamins. The highest concentration was observed for vitamin B2, reaching 9.485 mg/100 g, which indicates the biological significance of the sample in supporting metabolic processes.

In addition, appreciable amounts of vitamin B6 (6.068 mg/100 g) and vitamin B1 (3.588 mg/100 g) were detected. Vitamin C and vitamin PP were present at moderate levels, whereas vitamins B3 and B9 were found in relatively low amounts. Vitamin B12 was not detected, suggesting that its concentration in the sample was below the analytical detection limit of the method.

The retention times (2.784–18.677 s) confirmed the suitability of the selected chromatographic conditions and the reliable separation of the analyzed vitamins.

It should be noted that the results obtained for water-soluble vitamins in lemon peel were qualitatively compared with data from other independent HPLC analyses. During the comparison, the presence of vitamins B2, B6, and C was consistently reported in both analyses. The absence of certain vitamins, particularly B1 and B12, in other studies may be attributed to differences in extraction conditions, solvent systems, and analytical sensitivity. Direct quantitative comparison was not performed due to differences in calculation units.

Table 7.

Contents of water-soluble vitamins in lemon peel determined by independent HPLC analyses (mg/mL) [9]

Note. The data presented in this table are provided for qualitative comparison purposes only, and quantitative comparison was not performed due to differences in extraction procedures and calculation units.

Overall, the conducted analyses demonstrated that lemon peel is rich in flavonoids and water-soluble vitamins. The obtained results showed qualitative agreement with data from other independent HPLC analyses, confirming the presence of major biologically active components. Variations in quantitative values can be attributed to differences in extraction conditions, solvents used, and calculation units. These factors highlight the importance of methodological aspects in the evaluation of bioactive compounds in lemon peel.

Conclusion. Based on the results of the present study, lemon peel was identified as an important source of flavonoids and water-soluble vitamins. HPLC analysis revealed apigenin and rutin as the predominant flavonoids in the extract, while vitamins B2, B6, and B1 were detected in relatively high amounts, confirming the biological activity of lemon peel.

Qualitative comparison of the obtained results with data from other independent HPLC analyses demonstrated the consistent presence of these biologically active compounds. Observed quantitative differences for certain components can be explained by variations in extraction and analytical conditions, as well as differences in calculation units.

In general, lemon peel should not be regarded as a waste product but rather as a promising natural raw material with potential applications in the pharmaceutical industry and the development of functional food products.

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