INVESTIGATION OF FLAVONOID COMPOSITION IN DIFFERENT MORPHOLOGICAL PARTS OF CULTIVATED CAPER (Capparis spinosa L.). USING HPLC

ABSTRACT

This article investigates the composition and content of flavonoid compounds in the flowers, buds, aerial parts, and roots of the caper plant Cultivated caper (Capparis spinosa L.).Various morphological parts of the plant were selected as research objects, and qualitative as well as quantitative analysis of flavonoids was carried out using high-performance liquid chromatography (HPLC). According to the research results, biologically active flavonoids such as apigenin, rutin, luteolin, hyperoside, and gallic acid were identified, and their amounts were found to vary significantly across different parts of the plant. The highest concentrations of flavonoids were observed in the flower and bud samples. The results of the study indicate that the caper plant can serve as a rich source of flavonoids with potential applications in pharmaceutical and medical practice.         

АННОТАЦИЯ

В данной статье исследуется состав и содержание флавоноидных соединений в цветках, бутонах, надземных частях и корнях каперса (Capparis spinosa L.). В качестве объектов исследования были выбраны различные морфологические части растения, а качественный и количественный анализ флавоноидов проводился с использованием высокоэффективной жидкостной хроматографии (ВЭЖХ). По результатам исследования были идентифицированы биологически активные флавоноиды, такие как апигенин, рутин, лютеолин, гиперозид и галловая кислота, и установлено, что их количество значительно варьируется в разных частях растения. Наибольшие концентрации флавоноидов наблюдались в образцах цветков и бутонов. Результаты исследования показывают, что каперс может служить богатым источником флавоноидов с потенциальным применением в фармацевтической и медицинской практике.

Keywords: Cultivated caper (Capparis spinosa L.)., flavonoids, HPLC, apigenin, rutin, luteolin.

Ключевые слова: Каперс (Capparis spinosa L.), флавоноиды, ВЭЖХ, апигенин, рутин, лютеолин.

Introduction. In recent years, interest in identifying, isolating, and evaluating the pharmacological activity of biologically active compounds in medicinal plants—particularly flavonoids—has been steadily increasing. Flavonoids possess strong antioxidant, anti-inflammatory, hepatoprotective, and capillary-strengthening properties, making them important in the treatment and prevention of various diseases.Cultivated caper (Capparis spinosa L.) is an aromatic plant widely distributed in the Mediterranean region, and its flower buds (capers) are commonly used as a culinary ingredient in Mediterranean cuisine. Previous studies have shown that capers are a source of flavonoids (rutin, kaempferol), phenolic acids, and glucosinolates (glucocapparin, glucoberin, sinigrin, glucobrassicin). These compounds exhibit antioxidant, anticancer, antibacterial, and antimutagenic properties and play a significant role in promoting health [1–5]. Studies have investigated the effects of cultivated C. spinosa extracts on various cancer cell lines. It has been found that the hydroalcoholic extract of cultivated C. spinosa (flower buds and leaves) reduces the growth and proliferation of cervical cancer (HeLa), breast cancer (MCF7), and osteosarcoma (Saos-2) cells compared to normal cells (fibroblasts) [6]. Additionally, the essential oil and aqueous infusion of cultivated C. spinosa (flower buds and leaves) have demonstrated inhibitory effects on the proliferation of colorectal adenocarcinoma (HT-29) cells [7].  The literature also reports a comparative phytochemical analysis of the flowers and fruits of Cultivated caper (Capparis spinosa L.). collected from the Basra region (southern Iraq) [8]. Extracts were prepared using Soxhlet extraction and maceration methods, and their yields were compared. The total phenolic and flavonoid contents were determined spectrophotometrically using standard samples. Furthermore, qualitative and quantitative analysis of certain antioxidant compounds, such as cinnamic acid, was performed using high-performance liquid chromatography (HPLC) [8]. The results showed that the content of phenolic compounds, flavonoids, and alkaloids in the flowers of cultivated Capparis spinosa was significantly higher than in the fruits. HPLC analysis also revealed the presence of important biologically active compounds such as kaempferol, quercetin, hexacosane, vitamin E, and stigmasterol in both flower and fruit extracts [8]. Cultivated caper (Capparis spinosa L.). has long been used in traditional medicine and is recognized in modern scientific studies for its high biological activity. Scientific literature indicates that this plant contains flavonoids, alkaloids, vitamins, and mineral elements [9].The antioxidant, anti-inflammatory, antidiabetic, and hepatoprotective properties of plant-based preparations are largely associated with flavonoid compounds [10].      However, the distribution and quantitative composition of flavonoids in different morphological parts of cultivated Capparis spinosa have not been sufficiently studied. Therefore, the aim of this research is to determine the flavonoid composition in the flowers (F1.2), buds (F1.4), aerial parts (F1.7), and roots (F1.9) of the caper plant using HPLC and to conduct a comparative quantitative analysis.

Materials and Methods

Plant material. For the purposes of this study, flowers (F1.2), buds (F1.4), aerial vegetative parts (F1.7), and roots (F1.9) of Cultivated caper (Capparis spinosa L.). were collected during the vegetation period. The collected plant material was cleaned of mechanical impurities and washed with distilled water. The samples were dried in a well-ventilated area at a temperature of 25–30°C, protected from direct sunlight. The dried plant parts were ground to a particle size of up to 1 mm using a laboratory grinder and stored under dry and dark conditions. This step is essential for preventing oxidation and degradation of flavonoid compounds.        

Extraction of flavonoids. To isolate flavonoids, 1.0 g of the ground plant sample was accurately weighed, and 20 mL of 70% ethanol solution was added. To ensure maximum extraction efficiency, the mixture was stirred on a magnetic stirrer for 30–40 minutes. The extraction process was carried out at a temperature of 40–50°C, as these conditions help preserve flavonoid stability and enhance extraction efficiency. After extraction, the solution was centrifuged at 5000 rpm for 10 minutes. The clear supernatant was collected and filtered through a 0.45 µm membrane filter. This step is important for protecting the HPLC column from mechanical particles and improving analytical accuracy. The filtered extract was directly subjected to HPLC analysis.

High-performance liquid chromatography (HPLC) conditions.

The qualitative and quantitative analysis of flavonoids in different morphological parts of Cultivated caper (Capparis spinosa L.) was performed using high-performance liquid chromatography (HPLC). Chromatographic separation was carried out on a reversed-phase C18 column (Poroshell 120 EC-C18, 2.7 µm, 4.6 × 100 mm). The mobile phase consisted of a binary system of 0.5% acetic acid solution (A) and acetonitrile (B), and separation was performed under isocratic conditions with a ratio of A:B (75:25). These isocratic conditions ensured efficient separation of flavonoids. The flow rate of the mobile phase was set at 0.6 mL/min. Flavonoids were detected using a UV detector at wavelengths of 254, 265, and 281 nm. Identification was performed by comparing retention times with those of standard solutions, while quantitative analysis was carried out based on peak area comparison with standard samples.

Results and Discussion

The flavonoid composition of extracts obtained from different morphological parts of Cultivated caper (Capparis spinosa L.). (flowers, buds, aerial parts, and roots) was analyzed using high-performance liquid chromatography (HPLC). The obtained HPLC chromatograms reflect the degree of separation of flavonoid compounds, the presence of individual flavonoids, and their distribution across various parts of the plant. The chromatographic analysis revealed that both the composition and quantity of flavonoids differ significantly among the morphological parts of the caper plant. Identification of the detected flavonoids was carried out by comparing their retention times and peak areas with those of standard samples.

Below are the HPLC chromatograms illustrating the flavonoid composition of extracts obtained from the flowers, buds, aerial parts, and roots of Cultivated caper (Capparis spinosa L.).

Figure 1. HPLC chromatogram of the flower extract (F1.2) of Cultivated caper (Capparis spinosa L.)

This chromatogram (Figure 1) indicates the presence of several flavonoid compounds in the flower extract of the caper plant. In particular, peaks corresponding to apigenin, luteolin, and gallic acid were identified based on comparisons of retention times with those of standard samples.The high-intensity peak of apigenin suggests that this compound is present in significant amounts in the flowers. This finding confirms that the flowers represent an important morphological part for flavonoid biosynthesis.

Figure 2. HPLC chromatogram of the bud extract (F1.4) of Cultivated caper (Capparis spinosa L.)

The HPLC chromatogram of the bud extract (Figure 2) shows a relatively diverse spectrum of flavonoid compounds. Specifically, peaks corresponding to rutin, luteolin, gallic acid, and hyperoside were identified.The high-intensity peak of rutin indicates that this compound is accumulated in large amounts in the buds. This finding confirms that the buds are rich in biologically active compounds and represent a pharmacologically promising raw material.

Figure 3. HPLC chromatogram of the aerial part extract (F1.7) of Cultivated caper (Capparis spinosa L.)

The chromatogram of the aerial part extract (Figure 3) shows prominent peaks corresponding mainly to luteolin and gallic acid, while the content of other flavonoids appears relatively low.The distinct and stable peak of luteolin indicates that this compound is widely distributed in the vegetative organs. This finding confirms the important biological role of luteolin in plant metabolism.

Figure 4. HPLC chromatogram of the root extract (F1.9) of Cultivated caper (Capparis spinosa L.)

The HPLC chromatogram of the root extract (Figure 4) shows that the total flavonoid content is lower compared to other parts of the plant. The peaks corresponding mainly to luteolin and rutin exhibit relatively low intensity. This result indicates that flavonoids are predominantly accumulated in the generative and aerial parts, while their presence in the roots is comparatively low.

Analysis of the HPLC chromatograms demonstrated an uneven distribution of flavonoids across the morphological parts of Cultivated caper (Capparis spinosa L.).The quantitative content of flavonoid compounds in the different morphological parts was determined by comparing the peak areas of each sample with those of standard flavonoid solutions. For each sample, the area of peaks corresponding to specific flavonoids was compared with the peak areas of the respective standards, and the flavonoid content was calculated in mg per 100 g of plant material. This approach allowed the assessment of the quantitative distribution of flavonoids in the flowers, buds, aerial parts, and roots, and the results were analyzed in relation to the plant’s morphological characteristics.

Figure 5. Flavonoid composition of Cultivated caper (Capparis spinosa L.).

As shown in Figure 5, the flavonoid content varies significantly across the different morphological parts of Cultivated caper (Capparis spinosa L.). The study results indicate that the flowers (F1.2), buds (F1.4), aerial parts (F1.7), and roots (F1.9) differ in their accumulation of flavonoid compounds.

Apigenin was predominantly found in the generative parts of the plant, with the highest content observed in the flowers (105 mg/100 g). In the buds, apigenin content was 74.5 mg/100 g, while it was not detected in the aerial or root parts. This suggests that apigenin is primarily synthesized as part of the flowering-related metabolic processes.

Gallic acid was detected in all studied samples and appeared as a relatively widely distributed compound among the flavonoids. Its highest concentration was found in the buds (38.5 mg/100 g), while lower amounts were observed in flowers (6.5 mg/100 g), aerial parts (5.5 mg/100 g), and roots (7.5 mg/100 g), indicating its involvement in various metabolic processes across plant organs.

Rutin was detected only in the buds and roots, with the highest content in the buds (115 mg/100 g) and a much lower concentration in the roots (0.045 mg/100 g). The absence of rutin in flowers and aerial parts suggests that its accumulation is specific to certain morphological parts.

Hyperoside was identified exclusively in the buds, with a content of 1.25 mg/100 g, highlighting the buds as a specialized biosynthesis center for biologically active compounds.

Luteolin was found in all examined parts, with the highest concentrations in flowers (75 mg/100 g) and aerial parts (75 mg/100 g). Buds contained 72.5 mg/100 g, while roots had a relatively lower content of 65 mg/100 g. These results indicate that luteolin is a widely distributed and biologically significant flavonoid in Cultivated caper (Capparis spinosa L.).

According to literature data, various flavonoids, including rutin, apigenin, and other flavonols, have been detected in different parts of Capparis spinosa, with rutin generally found in higher concentrations in leaves and buds [11]. Our results corroborate these findings, showing that flavonoids such as rutin, luteolin, and apigenin accumulate differently across the plant’s morphological parts, reflecting organ-specific variations in plant metabolism.

Conclusion

The overall HPLC analysis results indicate that the flowers and buds of Cultivated caper (Capparis spinosa L.). are the richest morphological parts in flavonoid compounds. The high content of biologically active flavonoids such as apigenin, rutin, and luteolin in these parts scientifically supports their strong antioxidant, anti-inflammatory, and hepatoprotective effects. The data obtained in this study further highlight the potential of Capparis spinosa as a natural raw material rich in flavonoids for applications in the pharmaceutical industry and medical practice.

References:

1. Annaz, H.; Sane, Y.; Bitchagno, G.T.M.; Ben Bakrim,W.; Drissi, B.; Mahdi, I.; El Bouhssini, M.; Sobeh, M. Caper (Capparis spinose L.): An Updated Review on Its Phytochemistry, Nutritional Value, Traditional Uses, and Therapeutic Potential. Front. Pharmacol. 2022, 13, 878749.
2. Kdimy, A.; El Yadini, M.; Guaadaoui, A.; Bourais, I.; El Hajjaji, S.; Le, H.V. Phytochemistry, Biological Activities, Therapeutic Potential, and Socio-Economic Value of the Caper Bush  (Capparis spinosa L.). Chem. Biodivers. 2022; online ahead of print.
3. Lo Bosco, F.; Guarrasi, V.; Moschetti, M.; Germana, M.A.; Butera, D.; Corana, F.; Papetti, A. Nutraceutical Value of Pantelleria Capers (Capparis spinosa L.). J. Food Sci. 2019, 84, 2337–2346.
4. Wojdylo, A.; Nowicka, P.; Grimalt, M.; Legua, P.; Almansa, M.S.; Amoros, A.; Carbonell-Barrachina, A.A.; Hernandez, F. Polyphenol Compounds and Biological Activity of Caper  (Capparis spinosa L.) Flowers Buds. Plants 2019, 8, 539.
5. Zhang, H.; Ma, Z.F. Phytochemical and Pharmacological Properties of Capparis spinosa as a Medicinal Plant. Nutrients 2018, 10, 116.
6. Moghadamnia, Y.; Mousavi Kani, S.N.; Ghasemi-Kasman, M.; Kazemi Kani, M.T.; Kazemi, S. The Anti-cancer Effects of Capparis spinosa Hydroalcoholic Extract. Avicenna J. Med. Biotechnol. 2019, 11, 43–47.
7. Kulisic-Bilusic, T.; Schmoller, I.; Schnabele, K.; Siracusa, L.; Ruberto, G. The anticarcinogenic potential of essential oil and aqueous infusion from caper (Capparis spinosa L.). Food Chem. 2012, 132, 261–267.
8. Mohammed Abdul Mahdi Neama, Yessar Abdul Hussian Dawood, Hiba Najeh Alsaad. Exploring the photochemical composition of indigenous Capparis Genus species in Iraq. Multidiscip. Sci. J. RESEARCH ARTICLE. Published Online: April 2, 2024. https://doi.org/10.31893/multiscience.2024179
9. Tao Yang, Yu-Qing Liu, Chang-Hong Wang, Zheng-Tao Wang. Advances on investigation of chemical constituents, pharmacological activities and clinical applications of Capparis spinose. Zhongguo Zhong Yao Za Zhi. 2008 Nov;33(21):2453-8. Chinese. PMID: 19149246.
10. Mohamad Hesam Shahrajabian, Wenli Sun and Qi Cheng. Plant of the Millennium, Caper (Capparis spinosa L.), chemical composition and medicinal uses. Bull Natl Res Cent (2021) 45:131. P.1-9. https://doi.org/10.1186/s42269-021-00592-0.
11. Hassan Annaz, Yaya Sane, Gabin Thierry M Bitchagno, Widad Ben Bakrim, Badreddine Drissi, Ismail Mahdi, Mustapha El Bouhssini, Mansour Sobeh. Caper  (Capparis spinosa L.): An Updated Review on Its Phytochemistry, Nutritional Value, Traditional Uses, and Therapeutic Potential. Front Pharmacol. 2022 Jul 22;13:878749. doi: 10.3389/fphar.2022.878749