DETERMINATION OF WATER-SOLUBLE VITAMINS IN THE FRUIT PERICARP EXTRACT OF HORSE CHESTNUT (Aesculus hippocastanum) BY HPLC METHOD

ABSTRACT

This study focuses on the quantitative determination of water-soluble vitamins in the fruit pericarp extract of horse chestnut (Aesculus hippocastanum L.) using high-performance liquid chromatography (HPLC). The concentrations of vitamins B₁, B₂, B₆, B₉, B₁₂, and C were measured in the aqueous extract of the plant material. The results revealed the presence of all investigated vitamins, with particularly high levels of folic acid (B₉) and vitamin B₁₂ compared to other vitamins, indicating strong biological activity of the pericarp extract. Unlike previous studies that primarily focused on seeds and utilized alcoholic extraction, this research employed a water-based extraction of the fruit pericarp, offering a novel scientific approach. The findings suggest that the fruit pericarp of horse chestnut is a valuable natural source of essential vitamins and can be considered a promising raw material for the development of health-promoting preparations, dietary supplements, and functional food products.

АННОТАЦИЯ

Данное исследование посвящено количественному определению водорастворимых витаминов в экстракте околоплодника плодов конского каштана (Aesculus hippocastanum L.) с использованием метода высокоэффективной жидкостной хроматографии (ВЭЖХ). В водном экстракте растительного сырья были измерены концентрации витаминов B₁, B₂, B₆, B₉, B₁₂ и C. Результаты показали наличие всех исследуемых витаминов, причём особенно высокие уровни фолиевой кислоты (B₉) и витамина B₁₂ по сравнению с другими витаминами, что указывает на высокую биологическую активность экстракта околоплодника. В отличие от предыдущих исследований, в которых основное внимание уделялось семенам и применялась алкогольная экстракция, в настоящей работе использовался водный экстракт околоплодника плодов, что представляет собой новый научный подход. Полученные результаты свидетельствуют о том, что околоплодник конского каштана является ценным природным источником незаменимых витаминов и может рассматриваться как перспективное сырьё для разработки оздоровительных препаратов, биологически активных добавок и функциональных пищевых продуктов.

Keywords: vitamin B-complex, vitamin C, functional food, natural antioxidants, phytochemical composition

Ключевые слова: витаминный комплекс группы B, витамин C, функциональное питание, природные антиоксиданты, фитохимический состав.

Introduction.

The development of pharmaceutical products based on natural sources is considered one of the key priorities in modern pharmaceutical and biotechnological research. Among these, plant-derived extracts rich in vitamins are of particular interest, as they play a significant role in health restoration and disease prevention. Natural vitamins are well absorbed by the human body, cause fewer side effects, and often exhibit synergistic effects [1,2]. The common horse chestnut (Aesculus pocastanum L.) is widely used in traditional medicine as a venotonic, anti-inflammatory, and analgesic agent. Its seeds, flowers, and pericarp are known to contain saponins, flavonoids, tannins, coumarins, sterols, vitamins, and other biologically active compounds. While there have been numerous scientific studies on the seeds and flowers of horse chestnut, the vitamin composition of the fruit pericarp remains insufficiently explored [3,4,8]. In this study, the quantitative content of water-soluble vitamins (B₁, B₂, B₆, B₉, B₁₂, and C) in the fruit pericarp extract of horse chestnut was determined using high-performance liquid chromatography (HPLC), and their composition was analyzed [4,5]. Additionally, the results were compared with those of previous studies by Russian scientists, and significant differences were noted.

Materials and Methods.

The sample of horse chestnut fruit pericarp was selected for analysis. Using an NV222 analytical balance (OHAUS, USA) with 0.01 g accuracy, 2 g of the sample was weighed and placed in a 100 ml conical flask. Subsequently, 50 ml of 0.1 N hydrochloric acid (HCl) solution was added, and the mixture was subjected to extraction at 60°C for 15 minutes using a magnetic stirrer-heater (MS-H280 Pro, DLAB, China). After cooling, the extract was filtered and diluted with distilled water to a final volume of 100 ml [6,7]. From the prepared extract, 1.5 ml was filtered through a 0.45 µm syringe filter and transferred to a vial for chromatographic analysis.
Standard solutions of vitamins C, B₁, B₂, B₆, B₉, and B₁₂ were prepared. Each vitamin was dissolved in water at a concentration of 100 mg/l, and working solutions were prepared in the range of 5, 10, 15, and 20 mg/l. Chromatographic analyses were performed on a Shimadzu LC-40 Nexera Lite high-performance liquid chromatography system equipped with an LC-40D pump, SIL-40 autosampler, SPD-M40 photodiode array detector, and Shim-pack GIST C18 analytical column [9,10]. A gradient elution program was used with acetonitrile (A) and 0.5% aqueous acetic acid solution (B) as mobile phases. Detection was performed at three wavelengths: 280 nm, 265 nm, and 244 nm. Quantitative analysis was based on retention times and peak maxima of standard vitamins. All measurements were performed in triplicate, and the mean values were calculated using standard statistical methods.

Gradient Program of the Mobile Phase

a

b

c

Figure 1. Chromatograms of standard solutions of vitamins B₁, B₂, and B₉ at 265 nm(a), vitamins B₆ and B₁₂ at 280 nm(b) and  vitamin C at 244 nm(c)

Results.

The analysis of water-soluble vitamins in the fruit pericarp extract of horse chestnut by high-performance liquid chromatography (HPLC) showed a clear separation of the investigated components. Standard vitamin samples were used for identification, which allowed for accurate determination of the retention time of each compound.

The chromatograms (Fig. 2) display peaks corresponding to vitamins B₁, B₂, B₆, B₉, B₁₂, and vitamin C. The retention time of each vitamin remained stable, confirming the reliability of the analysis performed. The obtained chromatograms demonstrate good resolution and high sensitivity of the method.

a

b

c

Figure 2. Chromatograms for the determination of vitamins B₁, B₉, B₂(a), vitamins B₆, B₁₂(b), and vitamin C(c) in horse chestnut seed extract

Detailed and generalized values of the calculated amounts of water-soluble vitamins in horse chestnut extract are given in the Table 1.

Table 1.

 Vitamin content and retention times in horse chestnut seed extracts

Discussion.

According to the analytical results, all investigated vitamins were detected in the fruit pericarp of horse chestnut (Aesculus hippocastanum L.). Among them, the content of vitamin B₉ (folic acid) and vitamin B₁₂ was significantly higher compared to other vitamins [6,8]. This indicates that the pericarp possesses high biological activity. In previous studies conducted by Russian researchers, a 15% extract of horse chestnut seeds was prepared using 70% ethanol and ethyl acetate as extraction solvents. In contrast, in our study, a water extract of the fruit pericarp was specifically analyzed. This represents a novel scientific approach and provides new data on the composition of this plant material. The obtained results demonstrated that the fruit pericarp of horse chestnut can serve as a valuable natural source of essential vitamins.

Conclusion.

The present study confirmed the presence of water-soluble vitamins B₁, B₂, B₆, B₉, B₁₂, and C in the aqueous extract of horse chestnut fruit pericarp. Notably, the significantly high levels of folate (B₉) and vitamin B₁₂ highlight the strong biological activity and pharmacological potential of this plant material. Due to its richness in natural vitamins, horse chestnut pericarp is considered a promising raw material to produce health-promoting preparations, dietary supplements, and functional food products.

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