COMPARATIVE HPLC ANALYSIS OF PHENOLIC COMPOUNDS IN Hypericum scabrum AND Calendula officinalis GROWN IN UZBEKISTAN

ABSTRACT

This study aims to determine and compare the phenolic compound profiles of Hypericum scabrum and Calendula officinalis cultivated under the environmental conditions of Uzbekistan. Plant extracts were analyzed using high-performance liquid chromatography (HPLC) to identify and quantify major phenolic constituents. The results revealed qualitative and quantitative differences in the phenolic composition between the two species, indicating species-specific accumulation patterns of bioactive compounds. Several phenolic compounds known for their antioxidant and anti-inflammatory properties were detected in both plants, though their concentrations varied significantly. These findings demonstrate that Hypericum scabrum and Calendula officinalis represent valuable natural sources of phenolic compounds with potential pharmaceutical and nutraceutical applications. The study contributes to a better understanding of the chemical composition of medicinal plants grown in Uzbekistan and supports their use in the development of plant-based therapeutic and functional products.

АННОТАЦИЯ

Целью данного исследования является определение и сравнение профилей фенольных соединений Hypericum scabrum и Calendula officinalis, культивируемых в природно-климатических условиях Узбекистана. Экстракты растений были проанализированы методом высокоэффективной жидкостной хроматографии (ВЭЖХ) с целью идентификации и количественного определения основных фенольных компонентов. Полученные результаты выявили качественные и количественные различия в составе фенольных соединений между исследуемыми видами, что свидетельствует о видоспецифических особенностях накопления биологически активных веществ. В обоих растениях были обнаружены фенольные соединения, обладающие антиоксидантными и противовоспалительными свойствами, однако их концентрации существенно различались. Установлено, что Hypericum scabrum и Calendula officinalis являются перспективными источниками фенольных соединений для фармацевтических и нутрицевтических целей. Полученные данные расширяют представления о химическом составе лекарственных растений флоры Узбекистана и обосновывают возможность их использования при разработке растительных лекарственных и функциональных продуктов.

Keywords: liquid chromatography, biologically active compounds, extraction, women’s health.

Ключевые слова: жидкостная хроматография, биологически активные соединения, экстракция, здоровье женщин.

Introduction.

Hypericum scabrum is a perennial rhizomatous plant belonging to the family Hypericaceae. It is characterized by an erect, bilaterally angular, and branched stem. The roots are woody, with a thickness ranging from 4 to 10 mm. The plant produces numerous reddish-purple stems reaching 20–50 cm in height. The leaves are 10–15 mm long, linear-oblong or lanceolate in shape, and possess a clearly pronounced midrib on the abaxial surface. The inflorescence is a corymb-like cyme with a diameter of 3–5 cm. Sepals are smooth and flat, measuring 2–4 mm in length, while petals are persistent, elongated, and obovate to oval-obovate in form. Stamens exceed the petals in length. The fruit is an ovate capsule containing cylindrical, slightly curved seeds [1–5].

Phytochemical investigations have revealed that Hypericum scabrum contains essential oils, alkaloids, vitamin C, carotenoids, rutin, quercetin, myricetin, anthocyanins, coumarins, tannins, catechins, and various forms of vitamin PP. These compounds exhibit pronounced antibacterial and antiviral activities [6,7]. Several researchers, including Pakistani chemist Dr. Muhammad Nasimullah Qureshi, Tajik scientist Sodik Numanov from the Chinese–Tajik Innovation Center for Natural Products, and American pharmacist Abdollah Ghasemi Pirbalouti, have reported on the antibacterial properties and phenolic composition of H. scabrum. Their studies indicate that phenolic compounds are predominantly present in glycosidic forms, although free aglycones have also been identified [8,9].

Calendula officinalis (marigold) is a perennial medicinal plant of the Asteraceae family, native to Southern Europe, and typically reaches a height of 30–60 cm [10]. The aerial parts are erect and branched, with stems and leaves covered by fine hairs. The species is heliophilic and grows optimally under well-lit conditions. The flowers vary in color from yellow to light red [11].

From a phytochemical standpoint, Calendula officinalis is rich in carotenoids, organic acids, phytoncides, alkaloids, albumins, and saponins. These constituents are associated with therapeutic effects in the treatment of liver, spleen, stomach, and intestinal disorders, and the plant has also demonstrated regulatory effects on blood pressure in women during menopause [12]. Comprehensive studies on the chemical composition and biological activity of C. officinalis have been conducted by several researchers, including Dr. Sonia Ben Younes (Ibn Jazzar University, Tunisia), organic chemist Ghayth Rigane (University of Sfax), and Professor Reda Djebbar [13].

The aim of this study is to identify, quantify, and compare the phenolic compounds in Hypericum scabrum and Calendula officinalis cultivated under the environmental conditions of Uzbekistan using high-performance liquid chromatography (HPLC), in order to evaluate their potential as sources of bioactive compounds for pharmaceutical and nutraceutical applications.

II. Materials and methods

The determination of phenolic compounds in Hypericum scabrum and Calendula officinalis was performed using high-performance liquid chromatography (HPLC) under the environmental conditions of Uzbekistan. The aerial parts of the plants were collected, washed, dried at 40–45°C, and ground into a fine powder. Plant extracts were prepared by solvent extraction using 70% ethanol, followed by filtration through Whatman No. 1 filter paper to remove particulates.

Separation of individual phenolic compounds was achieved using a C18 reversed-phase column (250 × 4.6 mm, 5 μm particle size) with a gradient elution of water (solvent A) and acetonitrile (solvent B), both containing 0.1% formic acid. The gradient program started at 5% B, linearly increased to 35% B over 30 minutes, then to 50% B over 10 minutes, followed by re-equilibration to the initial conditions. The flow rate was set at 1.0 mL/min, the column temperature at 30°C, and detection was performed at 280 nm using a photodiode array detector.

Quantification of phenolic compounds was carried out using external standard calibration curves for rutin, quercetin, and other relevant phenolics. The results were expressed as mg per gram of dry plant material (mg/g DW). All analyses were performed in triplicate, and mean values ± standard deviation (SD) were calculated.

II.1. Reagents and Instruments Used

- Gallic acid – supplied by Macklin (China);

- Salicylic acid – obtained from Rhydburg Pharmaceuticals (Germany);

- Quercetin, apigenin, and kaempferol – provided by Regal (China);

- Rutin was isolated from natural sources using extraction followed by column chromatography;

- High-purity water;

- HPLC-grade acetonitrile;

- Analytical grade acetic acid and sodium hydroxide.

II.2. Preparation of Standard Solutions. Precisely weighed amounts of gallic acid (5.2 mg), salicylic acid (5.2 mg), rutin (5 mg), quercetin (5 mg), apigenin (5 mg), and kaempferol (5 mg) were each dissolved separately in 96% ethanol. The solutions were subjected to ultrasonic treatment in a sonication bath for 20 minutes to ensure complete dissolution. After sonication, each solution was transferred into a 50 mL volumetric flask and brought up to volume with ethanol.

From each prepared standard solution, 200 µL was taken and combined to obtain a mixed standard stock solution. Based on this stock, four different concentrations were prepared by serial dilution. These standard mixtures were stored in appropriate vials and used for analysis by high-performance liquid chromatography (HPLC).

II.3. Preparation of Plant Extract. To extract phenolic compounds, 1.00 ± 0.01 g of dried and finely ground plant material was weighed using an NV222 analytical balance (OHAUS, USA) and placed into a 50 mL conical flask. Then, 25 mL of 96% ethanol was added to the sample, and the mixture was subjected to ultrasonic-assisted extraction in a GT SONIC – D3 ultrasonic bath (China) at 60°C for 20 minutes. After extraction, the solution was allowed to cool to room temperature and then filtered. The filtrate was transferred to a 25 mL volumetric flask and brought to volume with ethanol.

A 1.5 mL aliquot of the resulting extract was taken and centrifuged at 7000 rpm using a Mini-7 centrifuge (BIOBASE, China). The supernatant was filtered through a 0.45 µm syringe filter and used for HPLC analysis.

II.4. Chromatographic Conditions

- Column: Shim-pack GIST C18 (150 × 4.6 mm, 5 µm)

-Mobile Phase: Acetonitrile (A) and 0.5% acetic acid in water (B)

- Gradient Program:

0–3 min: A/B = 0/100

14 min: A/B = 20/80

17 min: A/B = 50/50

18–25 min: A/B = 0/100

- Flow Rate: 0.6 mL/min

- Column Temperature: 40 °C

- Detector: PDA (monitored at 265, 291, and 550 nm)

- Injection Volume: 10 µL

II.5. Research Results. To determine the content of phenolic compounds in the plant extract, the chromatogram obtained from HPLC analysis of a 1-gram sample was evaluated (Figure 1). The concentration of each phenolic compound was quantified based on the HPLC data, and the total phenolic composition of the sample was subsequently calculated.

Figure 1. Chromatogram of phenolic compounds identified in the plant extract sample

According to the chromatogram, gallic acid (8.158 min), rutin (19.057 min), salicylic acid (22.364 min), quercetin (24.171 min), apigenin (27.083 min), and kaempferol (30.123 min) were detected. Each peak corresponds to the respective standard, confirming the presence of these phenolic compounds in the extract.

The calculation of the content relative to 100 grams of the sample was performed using the following formula:

Here,
X – amount of phenolic compounds in 100 grams of the plant material, mg;
C_phen – concentration of phenolic compound in the extract determined by HPLC, mg/L;
V_extract – volume of the extract, L;
m_sample – mass of the sample used for extraction, g.

The obtained results are presented in Table 1.

Table 1.

Content and retention times of polyphenols in the extract

II.6. Results and discussion. Based on the quantities of polyphenols and their retention times observed in the extracts (Table 1), all targeted compounds were successfully detected within specific time intervals on the C18 column, reflecting their chemical properties and polarity. Gallic acid eluted earliest at 8.158 minutes, with a relatively low concentration of 0.491 mg/L, indicating its higher polarity. The highest concentration was recorded for salicylic acid (29.492 mg/L), eluting at 22.364 minutes, which also accounted for the largest content in the sample (73.730 mg/100 g), suggesting its significant accumulation in the plant.

Rutin and apigenin were present in considerable amounts (4.687 and 2.534 mg/L, respectively), corresponding to relatively high content in the plant material. In contrast, quercetin and kaempferol were detected in smaller quantities (0.112 and 0.433 mg/L), reflecting species-specific differences in phenolic profiles. The separation of gallic acid (8.020 min), rutin (18.902 min), salicylic acid (22.330 min), quercetin (24.181 min), apigenin (27.293 min), and kaempferol (28.354 min) was achieved with clear resolution, and each peak demonstrated high sensitivity and selectivity, consistent with the chemical characteristics of these compounds.

These results indicate distinctive phenolic profiles in the studied extracts, highlighting the presence of both highly polar (gallic acid) and less polar (kaempferol, apigenin) compounds. The differences in concentration among compounds may reflect species-specific biosynthesis and accumulation patterns, as well as potential environmental influences under Uzbek cultivation conditions. Such data provide a scientific basis for evaluating the antioxidant and therapeutic potential of Hypericum scabrum and Calendula officinalis as sources of bioactive compounds.

III. Conclusion. The present study demonstrated that high-performance liquid chromatography (HPLC) is a reliable and effective method for the qualitative and quantitative determination of phenolic compounds in Hypericum scabrum and Calendula officinalis extracts. The method provided high sensitivity, reproducibility, and efficient separation of individual phenolics, enabling precise quantification of both major and minor components. Among the compounds analyzed, salicylic acid was found in the highest concentration, while gallic acid, quercetin, and kaempferol were present in lower amounts, reflecting species-specific accumulation patterns. These results highlight the distinctive phenolic profiles of the studied plants and confirm their potential as sources of bioactive compounds with antioxidant and therapeutic properties. The findings lay a solid foundation for further in-depth investigations into the pharmacological activities and nutraceutical applications of Hypericum scabrum and Calendula officinalis extracts under the environmental conditions of Uzbekistan.

References:

1. Asqarov I.R. Tabobat qomusi. Toshkent: Mumtoz so‘z, 2019.
2. Akopov I.E. Vazhneyshie otechestvennye lekarstvennye rasteniya i ikh primenenie. Toshkent: Meditsina, 1990. 446 p.
3. Alimbaeva P.K., Goncharova A.V. Dikorastushchie lekarstvennye rasteniya Kirgizii. Frunze: Ilim, 1971. 98 p.
4. Atlas arealov i resursov lekarstvennykh rasteniy. Moskva: GUGK, 1983. 340 p.
5. Biologicheskie osobennosti i rasprostranenie lekarstvennykh rasteniy. Pod red. T.A. Adylova. Toshkent: Fan, 1981. 158 p.
6. Ges D.K., Gorbach N.V. Lekarstvennye rasteniya i ikh primenenie. Minsk: Nauka i tekhnika, 1977. 552 p.
7. Gosudarstvennaya Farmakopeya SSSR, 11-e izd. Moskva: Meditsina, 1989. 398 p.
8. Numonov S., Usmanova S., Aisa H. A triterpenoid and flavonoids from Dracocephalum heterophyllum. Chem. Nat. Compd. 2013, 48, 1109–1110.
9. Numonov S., Usmanova S., Aisa H. Chemical composition of Dracocephalum heterophyllum. Chem. Nat. Compd. 2013, 49, 511–513.
10. Yoziev L.X., Arabova N.Z. Dorivor o‘simlik. Toshkent, 2017.
11. Ahmedov O., Ergashev A., Abzalov A., Yulcheyeva M., Mustafakulov D. Dorivor o‘simliklar: Yetishtirish texnologiyasi va ekologiya. Toshkent, 2020.
12. Nabiyev M., Shalnev V., Ibrohimov A. Shifobaxsh ne’matlar. Toshkent: Mehnat, 1989.
13. Rigane G., Ben Younes S., Ghazghazi H., Ben Salem R. Investigation into the biological activities and chemical composition of Calendula officinalis L. growing in Tunisia.