DETERMINATION AND ANALYSIS OF FLAVONOIDS IN MULBERRY LEAVES USING THE HPLC-DAD METHOD

ABSTRACT

In the study, the flavonoid composition of Morus alba leaves was determined using high-performance liquid chromatography with a diode-array detector (HPLC-DAD). The obtained samples were extracted with 70% ethanol, treated with ultrasound, and subjected to multiple filtration steps. The chromatographic analysis was performed under isocratic elution conditions. The spectral characteristics of flavonoids were recorded in the ultraviolet range of 200–400 nm, and identification was carried out based on their absorption maxima. The results demonstrated that the HPLC-DAD method is effective for the qualitative and quantitative evaluation of biologically active compounds with antioxidant properties in mulberry leaves.

АННОТАЦИЯ

В исследовании для определения флавоноидного состава листьев Morus alba применяли метод высокоэффективной жидкостной хроматографии с диодно-матричным детектором (HPLC-DAD). Полученные образцы экстрагировали 70% этиловым спиртом, обрабатывали ультразвуком и подвергали многократной фильтрации. Хроматографический анализ проводили в условиях изократической элюции. Спектральные характеристики флавоноидов регистрировали в ультрафиолетовом диапазоне 200–400 нм, а идентификацию осуществляли на основании максимумов их поглощения. Результаты показали, что метод HPLC-DAD является эффективным для качественной и количественной оценки биологически активных соединений с антиоксидантными свойствами в листьях шелковицы.

Keyword: HPLC-DAD, flavonoids, ethanol extraction, ultrasonic baths, membrane filter.

Ключевые слова: HPLC-DAD, флавоноиды, экстракция этанолом, ультразвуковые ванны, мембранный фильтр.

Introduction

Secondary metabolites in plants include flavonoids, which are widespread. Due to their antioxidant, antimicrobial, and anti-inflammatory properties, these compounds are considered important indicators when evaluating the quality of medicinal plants [1].

The qualitative and quantitative determination of flavonoids is an important analytical criterion in evaluating the quality indicators of medicinal plant raw materials. Their concentration depends on the plant species, vegetation stage, and processing methods. In scientific research, identifying and studying the flavonoid composition of raw materials is of great importance for ensuring the efficacy and safety of herbal medicinal preparations [2]. The flavonoid content in mulberry leaves is generally higher in young leaves. The total flavonoid content, calculated on a dry weight basis, can range from several mg/g to several tens of mg/g. The biological significance of mulberry leaf flavonoids lies in their strong antioxidant activity, as well as their antimicrobial and anti-inflammatory properties. In addition, they help strengthen blood vessel walls and may have a positive effect on glucose metabolism [3]. Therefore, reliable and sensitive analytical methods are required to accurately identify and analyze flavonoids. The high-performance liquid chromatography with a diode-array detector (HPLC-DAD) used in this study proved effective for separating flavonoids from mulberry leaves and assessing their spectral characteristics. This work describes the methodology for isolating flavonoids from mulberry leaf raw material and analyzing them using the HPLC-DAD method [4].

Materials and Methods. For analysis, dried mulberry leaves were taken and ground to a particle size of 0.1–1.5 mm. A 1.0000 g sample was placed in a flat-bottomed flask with a reflux condenser, and 99 ml of 70% ethanol was added. The mixture was extracted at 50–60°C for 2 hours with vigorous stirring. To enhance the extraction process, the flask containing the solution was placed in an ultrasonic bath [5]. The process was repeated twice at 35°C for 15 minutes with 10-minute intervals. The obtained extract was allowed to cool to room temperature. The cooled filtrate was filtered through a coarse filter. Then, 2 ml of the filtrate was taken and filtered through a 0.2 µm porous membrane filter. A portion of the resulting solution, either 100 µl of the total extract or a precisely measured small sample, was diluted to 1 ml with the eluent. Acetonitrile and buffer solution were used as the mobile phase. Spectral data were recorded in the 200–400 nm range.

Table 1.

Flavonoids in mulberry leaves

Figure 1. Flavonoid distribution chart of mulberry leaves

Figure 2. Chromatography of flavonoids in mulberry leaves

Figure 3. Chromatography of flavonoids in mulberry leaves

Results and discussion.

As a result of the analysis (HPLC-DAD), chromatographic peaks characteristic of flavonoids were observed in the mulberry leaf extract, and the compounds exhibited their specific maximum absorption wavelengths. During the chromatography process, the composition of the mobile phase (eluent) remained constant throughout the analysis, providing sufficient accuracy.

The proposed sample preparation method allowed for efficient isolation of flavonoids from the raw material. The chosen ethanol extraction and ultrasonic treatment were observed to enhance the transfer of flavonoids from the mulberry leaf extract into the solvent. Multi-stage filtration protected the chromatography system from mechanical impurities.

Quantitative analysis showed that the flavonoids in mulberry leaves are present in the following decreasing order:

Gall kislota>Izopamnetin>Kempferol >Giperazid> Rabinin>Apigenin

Gipolaetin 7-О-D Gly, Gipolaetin  flavonoids were not detected. The highest content was found for gallic acid, an organic compound (C₇H₆O₅, 3,4,5-trihydroxybenzoic acid) naturally occurring in tea, oak bark, berries, and many other plants. Gallic acid is a well-known natural antioxidant with anti-inflammatory and antimicrobial properties, widely used in medicine, food industry, and cosmetics. Chemically active, it dissolves well in hot water and ethanol, easily oxidizes and darkens in light, and decarboxylates to pyrogallol upon heating [6].

The second most abundant flavonoid was rutin, with a content of 19.074 mg. Known as vitamin P, rutin (C₂₇H₃₀O₁₆) is a natural flavonoid that strengthens capillary walls, reducing their fragility and permeability. It also has strong antioxidant and anti-inflammatory properties, protects cells from damage, and maintains healthy blood vessels. Rutin is found in citrus fruits (especially in the peel), buckwheat, berries, tea, and apples, and its absorption is enhanced when combined with vitamin C.

Conclusion

The HPLC-DAD–based analysis used for the isolation of flavonoids from mulberry leaves proved to be simple, reliable, and reproducible. The sample preparation, which included extraction with 70% ethanol, ultrasonic treatment, and multi-stage filtration, allowed for maximal recovery of biologically active compounds from mulberry leaves and provided a clean extract suitable for analysis.

Using the HPLC-DAD method, absorption spectra and chromatographic peaks characteristic of flavonoids were recorded in the 200–400 nm range. The analysis showed that mulberry leaves contain the highest amount of gallic acid, followed by rutin and other flavonoids [7]. Gipolacetin and its 7-O-D-glucoside forms were not detected.

The results indicate that mulberry leaves are rich in biologically active compounds, particularly phenolic substances.

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