Effect of drying methods on phenolic compounds and antioxidant activity of Capparis spinosa L. fruits

Abstract

Background

Drying is a critical post-harvest process for medicinal plants, which are typically high in moisture and microorganisms. To prevent spoilage and quality loss, it is essential to dry these plants promptly. The drying method significantly impacts the levels of secondary metabolites and the organoleptic characteristics of medicinal plants. This study aimed to investigate the effects of various drying methods on the total phenolics, flavonoids, anthocyanins, antioxidant activity, and phenolic acids in caper (Capparis spinosa L.) fruits. The experiment was performed using a completely randomized design with three replications and included 11 treatments: shade drying, sun drying, oven drying (at 50 °C, 60 °C, and 70 °C), microwave drying (at 300 W, 600 W, and 900 W), freeze-drying, salt-drying, and a fresh plant sample as a control.

Results

Among the drying methods tested, microwave drying consistently produced the highest levels of flavonoids, anthocyanins, and antioxidant activity, regardless of wattage. Specifically, the highest total phenol content was observed in samples dried at 900 W microwave, 600 W microwave, and 70 °C in the oven (5.3, 5.37, and 5.31 mg GAE/g DW, respectively). Drying at 600 W microwave yielded the highest levels of caffeic, cinnamic, ferulic, vanillic, and protocatechuic acids (13.03, 3.85, 4.28, 9.73, and 5.6 µg/g, respectively) while drying at 900 W microwave also resulted in elevated levels of caffeic, ferulic, protocatechuic, and p-coumaric acids. The 70 °C oven drying method also showed high levels of caffeic, cinnamic, ferulic, vanillic, and protocatechuic acids. Freeze-drying achieved the highest levels of rosmarinic, gallic, and m-coumaric acids (320.17, 175.3, and 12.99 µg/g, respectively), while shade drying produced high levels of p-hydroxybenzoic, cinnamic, ferulic, m-coumaric, protocatechuic, and p-coumaric acids.

Conclusions

Overall, microwave drying (especially at 600 W), oven drying at 70 °C, and freeze-drying emerged as effective alternatives to traditional drying methods. These methods not only preserved the color, texture, and taste of the fruits but also enhanced their bioactive compound levels.

Introduction

Medicinal plants have been used since ancient times for their therapeutic properties to enhance health and treat illnesses, due to their health-promoting effects and bioactive compounds [1]. Beyond their medicinal uses, these plants are also employed as food preservatives and flavorings. Plant extracts are known for their antioxidant, antibacterial, and anti-inflammatory properties, which can offer significant health benefits [2]. The oxidation of cellular components by free radicals is a key contributor to heart disease and cancer, and some plant-derived compounds can help counteract this process [3]. Consuming plants rich in antioxidants can bolster our defenses against free radical damage and lower the risk of heart disease and cancer [4]. Due to their potent antioxidant phytochemicals, medicinal plants have garnered interest from the biotechnology, pharmaceutical, cosmetic, and food industries, where they are used in drugs, flavorings, and fragrances [5].

The caper bush (Capparis spinosa L.) is a small shrub from the Capparidaceae family, typically growing close to the ground with its branches and leaves often covering soil or rocks. Its flowering is from early to late summer, completing its reproductive cycle within this period. Native to Mediterranean regions, caper bush also thrives in various other climates, including tropical and subtropical zones [6, 7]. Its buds and fruits are used in cuisines around the world, and the plant is also a staple in traditional medicine across many cultures for treating and managing various health conditions. The methods of using capers differ by country, influenced by the specific plant parts used and local traditions [8].

Dried caper fruit is commonly used to treat diabetes and lower blood pressure. The pickle made from caper buds and fruits is valued for its high content of phenolic acids, flavonoids, phytosterols, alkaloids, sweeteners, organic acids, and vitamins, which contribute to its rich array of bioactive compounds with therapeutic benefits. Notably, caper buds and fruits are particularly rich in phenolic and flavonoid compounds, which are significant for their biological activities and ability to scavenge free radicals [9]. A study by Aksay et al. [6] identified 19 phenolic acids in both fresh and fermented caper fruits and buds. Key phenolic compounds included quercetin-3-O-rutinoside, quercetin-O-galloly-O-hexoside, and kaempferol-3-O-rutinoside, with quercetin-3-O-rutinoside being found exclusively in buds. Additionally, research by Francesca et al. [10] identified five other phenolic compounds including gallic acid, catechin, caffeic acid, epi-catechin, and vanillic acid in caper fruits. Furthermore, both alcoholic and aqueous extracts of caper fruit exhibit antibacterial, hepatoprotective, and anti-diabetic properties, and help protect the body from oxidative stress [1].

Drying is a critical step in the post-harvest management and industrial processing of medicinal plants. It is one of the most common, cost-effective, and simplest methods for preserving these plants. Selecting the appropriate drying method for different plant parts is essential to maintain their quality and efficacy [11]. Using an unsuitable drying method can compromise the quality of plant materials and potentially destroy their bioactive compounds. Medicinal plants typically contain 75–80% water, and reducing this to below 15% is crucial for preserving secondary compounds and overall plant quality [12]. Proper drying methods inhibit microbial growth and prevent unwanted biochemical changes. However, the drying process can also lead to alterations in the quality of medicinal plants, such as the loss of color and aroma [13]. These changes may arise from phenolic compound degradation or new compound formation due to oxidation. Additionally, drying can reduce the levels of bioactive compounds with antioxidant properties, diminishing their antioxidant activity [14]. Despite these potential drawbacks, drying is essential for preventing microbial growth and aiding in the storage and transport of medicinal plants. Research indicates that both enzymatic and non-enzymatic reactions during drying can significantly affect the composition of bioactive ingredients in these plants [15].

Among various drying methods, sun drying is a traditional technique used for preserving crops and food; however, it often requires longer drying times compared with other methods. This extended drying period can lead to a significant loss of bioactive compounds, such as antioxidants and vitamins [16]. In contrast, hot air drying shortens the drying time and enhances processing efficiency, making it widely used for products with high water content [17]. Microwave drying, a more modern technique, offers higher efficiency by significantly reducing drying time. Microwave radiation ensures uniform heating of the plant material, resulting in a rapid reduction of its initial moisture content [18]. Freeze-drying, on the other hand, helps preserve the nutritional and sensory qualities of high-value, heat-sensitive raw materials [19]. By maintaining cellular structure and preventing the degradation of heat-sensitive compounds, freeze-drying can effectively retain antioxidants and flavonoids in the plant, which are often lost in traditional drying methods [20].

Research has shown that the drying method significantly influences both the content of active compounds and the appearance of medicinal plants. For example, a study comparing natural, microwave, convective, and combined drying methods on Cornus mas found that microwave drying at 300 W was the most effective for maintaining physical parameters and phytochemical properties [21]. In another study, various drying techniques—air drying, oven drying, microwave drying, and freeze-drying—were compared for their impact on ginger rhizomes (Zingiber officinale). Freeze-dried rhizomes had notably higher levels of total phenolics, antioxidant activity, and carotenoids. However, oven-dried rhizomes exhibited higher levels of phenolic acids. Overall, both oven drying and freeze-drying were found to produce higher levels of phenolic compounds and bioactivities [22]. A study on lavender flowers (Lavandula angustifolia) revealed that the heat-pump drying method resulted in higher levels of total phenolics, anthocyanins, and antioxidant activity compared with hot-air drying. The drying method also affected the phenolic acid levels in the extracts [23]. Additionally, research on rhubarb (Rheum palmatum L.) compared hot air drying, sun drying, and a pre-drying smoke method followed by hot air drying. The pre-drying with smoke method preserved the best levels of anthraquinone compounds and a glossy appearance while reducing the drying time by 10–15 days compared with sun drying [15]. A comparison of drying methods for Glycyrrhiza uralensis—including hot air drying, combined infrared and hot air, freeze vacuum, microwave vacuum, and pulsed vacuum—showed that freeze vacuum drying, despite its longer drying time, best preserved total phenolics, total flavonoids, and glycyrrhizin content. It also achieved the best color and least browning [24].

Since the drying method can greatly impact both the quantitative and qualitative properties of medicinal plants, selecting the appropriate drying technique based on the plant’s active ingredients is crucial [12]. Previous research on the drying of C. spinosa has predominantly focused on traditional drying methods. However, there is limited exploration of the effects of modern drying techniques, such as controlled temperature drying, freeze-drying, and microwave drying. Additionally, the impact of different drying methods on the preservation of phytochemical compounds and medicinal properties of this plant has not been thoroughly investigated. Considering the widespread use of C. spinosa in the food and pharmaceutical industries, along with its numerous health benefits, this study aims to examine how various drying methods influence the phytochemical properties and phenolic acid content of C. spinosa fruits. By comparing traditional and modern drying techniques, this research offers new insights into the preservation of the plant’s chemical and medicinal compounds.

Materials and methods

Plant materials

Caper (C. spinosa) fruits were manually harvested at the immature green stage from their natural habitats near Khorram Abad, Lorestan, Iran (33°28’N, 48°16’E, 1139 MASL) in late summer 2022. Plant identification was carried out by Dr. Farajollah Tarnian using the Flora of Iran. An herbarium specimen was also prepared and a voucher specimen number of LU-1004 was assigned by the Herbarium of the Faculty of Natural Resources, University of Lorestan. Upon arrival at the laboratory, the fruits were sorted to select healthy and uniform specimens for further processing. Before initiating the experiment, three 500 g samples of caper fruits were dried for 24 h in an oven at 105 °C to determine their dry matter content.

Drying treatments

The experiment was designed as a completely randomized design with ten different drying methods and three replications. The drying treatments included: room shade drying, sun drying, oven drying at 70, 60, and 50 °C, microwave drying at 900, 600, and 300 W, salt drying, freeze-drying, and a fresh plant sample (not dried). The drying temperatures for oven drying and microwave power levels were selected based on an extensive literature review and preliminary experiments [14, 21, 22]. For each replication, 1 kg of healthy, immature, and uniform caper fruits was used.

In each drying method, the fruits were divided into two halves and uniformly on trays. For the salt-drying treatment, the samples were placed in a beaker with salt at room temperature (averaging 24 °C with a relative humidity of 60%), with the salt being refreshed daily throughout the drying process. The freeze-drying was performed using a SUBLIMATOR-VACO5 freeze-dryer, which can reach temperatures as low as -50 °C. For microwave drying, the samples were dried in a Samsung MG402MADXBB microwave at three different power levels: 300 W, 600 W, and 900 W. For the oven drying treatments, a German oven (UNpa110, Memmert) was used, with temperatures set at 50, 60, and 70 °C. For the shade and sun drying treatments, the samples were spread on trays, with shade drying conducted at room temperature and sun drying exposed to direct sunlight. The average room temperature for shade drying was 24 °C with a relative humidity of 60%, while sun drying occurred at an average temperature of 40 °C and an average relative humidity of 32%.

Throughout all drying methods, the plant materials were weighed continuously until the moisture content reached approximately 14% based on dry matter, at which point the drying process was stopped. The drying time for each treatment was recorded. After drying, the samples were stored in paper bags at 4 °C until phytochemical analyses could be conducted. Freshly harvested fruits, stored at -80 °C, were also used as a control treatment.

Total phenolics

The total phenolic content in the samples was determined following the method described by Singleton et al. [25]. To begin, 3 g of the powdered sample was mixed with 10 mL of 80% methanol and allowed to incubate at room temperature for 72 h. Afterward, the plant residues were filtered out using filter paper, leaving behind the extract [26].

Next, 400 µL of 7.5% sodium carbonate (W/V) and 400 µL of 10% folin-ciocalteu reagent were mixed with 200 µL of the diluted extract. The mixture was vortexed for 10 s and then allowed to react in the dark at room temperature for 30 min. The absorption spectrum of the solution was noted at 765 nm using a UV-1800 spectrophotometer (Mapada). A standard curve was created with gallic acid at 0, 10, 20, 40, 80, 160, and 320 mg/L concentrations, and the total phenol content in the samples was reported as mg of gallic acid (GAE)/g dry weight (DW). All measurements were performed in triplicate. The calibration curve equation for phenolics was: Y = 0.0016X + 1.9811, with a determination coefficient (R²) of 0.9867.

Total flavonoids

The total flavonoid content was determined using the aluminum chloride colorimetric method described by Hang et al. [27]. To 200 µL of plant extract, 100 µL of potassium acetate and 100 µL of aluminum chloride were added. The mixture was vortexed for 30 s and subsequently left to incubate for 30 min at room temperature in the dark. The absorbance was noted at 415 nm using a UV-1800 spectrophotometer (Mapada). A standard curve was generated with rutin at 0, 10, 20, 40, 80, 160, and 320 mg/L concentrations. The level of total flavonoid was reported as mg of rutin (RE)/ g DW. All analyses were performed in triplicate. The calibration curve equation for flavnoids was: Y = 0.0069X − 0.0109, with a determination coefficient (R²) of 0.9961.

Antioxidant activity

Antioxidant activity was assessed using the Ferric Reducing Antioxidant Power (FRAP) method, following the protocol of Bouvier et al. [28]. First, The FRAP solution was prepared by combining the acetate buffer (300 mM, pH = 3.6), iron chloride (20 mM) and TPTZ solution (10 mM in 40 mM hydrochloric acid) in a ratio of 10:1:1. For the assay, 180 µL of the FRAP solution was combined with 20 µL of the methanolic extract and incubated at 37 °C for 8 min. The absorbance was recorded at 593 nm using a UV-1800 spectrophotometer (Mapada). Iron sulfate heptahydrate was utilized as the standard, and antioxidant capacity was reported as mmol of Fe/g DW. All analyses were conducted in triplicate. The calibration curve equation for antioxidant activity was: Y = 0.0002X + 0.1029, with a determination coefficient (R²) of 0.9651.

Anthocyanin

The total anthocyanin content was assessed based on the differential pH method according to Giusti and Wrolstad [29]. First, two buffers were prepared: Buffer 1 with a pH of 1, made by dissolving 0.186 g of potassium chloride in distilled water to a final volume of 100 mL, and Buffer 2 with a pH of 4.5, prepared by dissolving 4.435 g of sodium acetate in distilled water to a final volume of 100 mL. For the assay, 200 µL of the extract was mixed with 1000 µL of each buffer. The absorbance of the extract was recorded at 510 nm and 700 nm using a UV-1800 spectrophotometer (Mapada) for both buffers. Distilled water was used as a blank. The anthocyanin content was calculated using a related formula and reported as µmol/g DW. All analyses were carried out with three replicates to ensure accuracy and reliability of the results.

Measurement of phenolic acids

Extraction

Two grams of powdered caper fruit were mixed with 20 mL of n-hexane and subjected to ultrasonic extraction for 30 min. Following filtering through filter paper, the residue was subsequently treated with 20 mL of 80% methanol and again subjected to ultrasonic extraction for 30 min. This methanolic solution was filtered to remove the residue. After the solvent evaporation, 10 mL of distilled water was added to the extract, and the pH was adjusted to 2 with dropwise additions of 6 N hydrochloric acid solution. The resulting colloidal solution was centrifuged at 6000 rpm for 5 min using a refrigerated centrifuge. The clear supernatant was collected, and n-hexane was added (hexane-to-aqueous phase ratio of 1:1 v/v). This mixture was vortexed for 2 min and repeated twice, after which the aqueous phase was separated from the organic phase. The aqueous phase was then mixed with diethyl ether and ethyl acetate (solvent-to-aqueous phase ratio of 1:1 v/v). This mixture was shaken for 2 min and repeated twice, and the organic phase was separated from the aqueous phase. The organic phase obtained was evaporated using a rotary evaporator and then dried in an oven at 50 °C [30].

RP-HPLC analysis

The identification and quantification of phenolic compounds were performed using high-performance liquid chromatography (HPLC) (Knauer, Berlin, Germany). The analysis employed a reversed-phase Welch Ultisil XB-C18 column (150 mm × 4.6 mm × 5 μm). Methanol (solvent A) and acidic water (solvent B) were used as the mobile phases. The initial solvent composition was 20% solvent A and 80% solvent B, which was gradually adjusted to 100% solvent A over 40 min and maintained for an additional 5 min. The flow rate was 0.5 mL/min. A 20 µL sample of the extract, with a concentration of 500 ppm, was injected into the HPLC system. The total analysis time for each sample was 45 min with a detection wavelength of 280 nm. The identification of phenolic compounds was achieved by comparing the retention times of the sample peaks with those of standards. Quantification was performed using the calibration curves of the standard solutions and the area under the curve for each peak. The concentration of each phenolic compound in the fruit was reported as µg/g DW [31]. All analyses were conducted with three replicates.

Data analysis

Data was analyzed based on the experimental design using SAS software, and the comparison of treatment means was carried out using the LSD test at 0.05 level.

Results

Drying time

Analysis of variance indicated that the drying time of the samples was significantly influenced by the drying method used (Table 1). The longest drying time was observed with salt drying, which took 7200 min. This was followed by shade drying at 2880 min and freeze-drying at 1800 min. Microwave drying, regardless of power settings, was the fastest method, with drying times significantly shorter than those of shade and freeze-drying. Oven drying at 70 °C also provided relatively quick results (Fig. 1A).

Table 1 Analysis of variance of the effect of different drying methods on phytochemical properties of caper berries

Fig. 1

Comparison of (A) drying time, (B) total phenol, (C) total flavonoid, (D) anthocyanin, and (E) antioxidant activity of caper berries in the various drying methods; X1, X2: ambient-drying (shade-drying at room and sun-drying, respectively); X3, X4, X5: oven-drying (at 70, 60 and 50 °C, respectively); X6, X7, X8: microwave-drying (at 900, 600 and 300 watts, respectively), X9: salt drying; X10: freeze drying; and X11: fresh sample

Total phenolics

Analysis of variance revealed that the effect of different drying methods on the total phenolics content of caper fruits was significant (p ≤ 0.01) (Table 1). Mean comparisons indicated that the highest total phenol content was found in samples dried using microwave at 900 W (5.3 mg GAE/g DW), microwave at 600 W (5.37 mg GAE/g DW), and oven drying at 70 °C (5.31 mg GAE/g DW). These treatments did not differ significantly from sun-dried samples, microwave drying at 300 W, or oven drying at 50 °C and 60 °C. The lowest total phenolic content was observed in salt-dried samples, with freeze-dried and fresh samples also showing lower phenolic levels (Fig. 1B).

Total flavonoids

Analysis of variance revealed that the total flavonoid content of caper berries was significantly influenced by the different drying methods (p ≤ 0.01) (Table 1). Microwave-dried samples exhibited the highest levels, regardless of power settings. Oven-dried, shade-dried, sun-dried, and freeze-dried samples had intermediate levels of flavonoids, while the lowest levels were observed in fresh and salt-dried samples (Fig. 1C).

Anthocyanins

The analysis of variance indicated that antioxidant activity in caper fruits was significantly affected by the different drying methods (Table 1). The highest anthocyanin levels were found in microwave-dried samples at 300 W (0.625 µmol/g DW) and 600 W (0.612 µmol/g DW), followed by microwave drying at 900 W and oven drying at 70 °C. Anthocyanin content in samples dried at 50 °C and 60 °C in the oven, as well as those dried by sun and shade methods, was moderate. Fresh, salt-dried, and freeze-dried samples had the lowest anthocyanin levels (Fig. 1D).

Antioxidant activity

Analysis of variance demonstrated that different drying methods had a significant effect on antioxidant activity in caper fruits (Table 1). The highest antioxidant activity was recorded in microwave-dried samples at 900 W (2.22 mmol Fe/g DW), followed by samples dried at 600 W (1.98 mmol Fe/g DW) and 300 W (1.86 mmol Fe/g DW). The lowest antioxidant activity was observed in samples dried at 50 °C in the oven, which did not significantly differ from those dried at 60 °C, salt-dried, shade-dried, and fresh samples (Fig. 1E).

RP-HPLC analysis of phenolic acids

RP-HPLC analysis of the caper fruit extract identified ten phenolic acids at varying concentrations. The chromatogram of this analysis is shown in Fig. 2. The predominant phenolic acids were rosmarinic acid, with an average concentration of 157.84 µg/g of extract, gallic acid at 58.64 µg/g, and p-coumaric acid at 21.06 µg/g. Other notable phenolic acids included caffeic acid (9.03 µg/g), m-coumaric acid (8.96 µg/g), and vanillic acid (1.6 µg/g). Additionally, protocatechuic, ferulic, cinnamic, and p-hydroxybenzoic acids were detected at lower concentrations.

Fig. 2

RP-HPLC base peak chromatogram of extract of caper berries (visualized at 280 nm). Peaks: (1) Gallic acid; (2) Protocatechuic acid; (3) Rosmarinic acid; (4) p-hydroxybenzoic acid; (5) Vanillic acid; (6) Caffeic acid; (7) p-Coumaric acid; (8) Ferulic acid; (9) m-Coumaric acid; and (10) Cinnamic acid

The effect of drying methods on the levels of all phenolic acids identified in caper fruits was significant (Table 1). Mean comparisons revealed that the highest level of caffeic acid was in caper fruits dried using an oven at 70 °C (14.49 µg/g of extract), which did not differ significantly from the microwave drying at 600 W (13.03 µg/g of extract). The level of caffeic acid was also notably high in samples dried in a microwave at 900 W. In contrast, the lowest levels of caffeic acid were observed in fresh samples (5.16 µg/g of extract) and in samples dried at 60 °C in the oven (6.09 µg/g of extract) (Fig. 2A). The analysis revealed significant variations in cinnamic acid and ferulic acid levels across different drying methods. The highest level of cinnamic acid was found in caper fruits dried using a microwave at 600 W (3.85 µg/g of extract) and an oven at 70 °C (3.17 µg/g of extract). Shade drying also resulted in a relatively high cinnamic acid level compared with other methods. In contrast, salt-drying yielded the lowest cinnamic acid concentration (0.03 µg/g of extract), which was not significantly different from the level in fresh samples (Fig. 2B). For ferulic acid, the highest levels were detected in caper fruits dried with a microwave at 600 W (4.28 µg/g of extract) and an oven at 70 °C (3.44 µg/g of extract), followed by drying with a microwave at 900 W and shade drying. The lowest ferulic acid concentration was observed in samples dried at 50 °C in the oven (0.7 µg/g) and with a microwave at 300 W (0.6 µg/g). These levels did not differ significantly from those found in fresh, salt-dried, or sun-dried samples (Fig. 2C).

The highest concentration of p-hydroxybenzoic acid was noted in shade-dried samples (2.2 µg/g of extract), followed by oven-dried samples at 50 °C and 60 °C, as well as salt-dried samples. The lowest levels were obtained in samples dried at 70 °C in the oven and in fresh samples (Fig. 2D). For protocatechuic acid, the highest concentrations were noted in samples dried with a microwave at 600 W (5.6 µg/g of extract) and 900 W (5.8 µg/g of extract), as well as in shade-dried samples. The lowest levels were found in salt-dried samples (1.39 µg/g of extract), which did not differ significantly from fresh samples or those dried at 50 °C and 60 °C in the oven (Fig. 2E).

The highest p-coumaric acid content was achieved in samples dried in a microwave at 600 W, with 49.1 µg/g of extract. This was followed by samples dried at 900 W in the microwave and those dried in the shade. Salt-drying resulted in the lowest level of p-coumaric acid (8.09 µg/g of extract), although this was not significantly different from the levels found in sun-dried and 60 °C oven-dried samples (Fig. 3F). For vanillic acid, the highest levels were observed in samples dried at 600 W in the microwave (9.73 µg/g of extract), as well as in salt-dried and 70 °C oven-dried samples (8.85 and 8.77 µg/g of extract, respectively). The lowest concentration of vanillic acid was recorded in samples dried at 50 °C in the oven (3.23 µg/g of extract), but this level did not differ significantly from those in fresh samples, shade-dried samples, and freeze-dried samples (Fig. 3G).

Fig. 3

Comparison of (A) caffeic acid, (B) cinnamic acid, (C) ferulic acid, (D) p-hydroxybenzoic acid, (E) protocatechuic acid, and (F) p-coumaric acid of caper berries in the various drying methods; X1, X2: ambient-drying (shade-drying at room and sun-drying, respectively); X3, X4, X5: oven-drying (at 70, 60 and 50 °C, respectively); X6, X7, X8: microwave-drying (at 900, 600 and 300 watts, respectively); X9: salt drying; X10: freeze drying; and X11: fresh sample. Comparison of (G) vanillic acid, (H) rosmarinic acid, (I) gallic acid, and (J) m-coumaric acid of caper berries in the various drying methods; X1, X2: ambient-drying (shade-drying at room and sun-drying, respectively); X3, X4, X5: oven-drying (at 70, 60 and 50 °C, respectively); X6, X7, X8: microwave-drying (at 900, 600 and 300 watts, respectively); X9: salt drying; X10: freeze drying; and X11: fresh sample

The levels of rosmarinic acid and gallic acid were the highest in freeze-dried samples, measuring 320.17 µg/g and 175.13 µg/g of extract, respectively. The lowest rosmarinic acid concentration was found in samples dried in an oven at 50 °C (101.6 µg/g of extract), although this was not significantly different from those obtained from sun drying, shade drying, and salt drying. The lowest level of gallic acid was noted in samples dried at 60 °C in the oven and at 300 W in the microwave (26.6 and 22.4 µg/g of extract, respectively) (Fig. 3.H, Fig. 3I). Freeze-dried, shade-dried, sun-dried, and fresh samples all exhibited high levels of m-coumaric acid, with 12.99, 12.26, 11.36, and 11.35 µg/g concentrations of extract, respectively. In contrast, salt-dried samples had the lowest level of m-coumaric acid, at 2.5 µg/g of extract (Fig. 3J).

Principal component analysis (PCA)

The PCA was carried out based on phytochemical alterations of C. spinosa samples in different drying regimes. The results demonstrated that PC1 and PC2 included 64.32% and 23.95% of the variance, respectively. Compared with the control treatment, microwave drying at 600 and 900 W. and oven drying at 70 °C were linked to a higher accumulation of cinnamic acid, ferulic acid, vanillic acid, protocatechuic acid, caffeic acid, p-coumaric acid, anthocyanin, total phenol, total flavonoid, and antioxidant activity. Nevertheless, a freeze-drying method was linked to a higher accumulation of gallic acid, rosmarinic acid, and m-coumaric acid. The highest accumulation of p-hydroxybenzoic acid was observed in shade-dried, 60 °C oven-dried, 50 °C oven-dried, and salt-dried samples. In this diagram, compounds that are grouped and have an angle of less than 90 degrees with each other have a direct and positive correlation. This is because an angle of less than 90 degrees in a PCA biplot is an approximation of the correlation coefficient, which is consistent with the Pearson correlation coefficient results between the attributes (Fig. 4).

Fig. 4

Principal component analysis (PC1 and PC2) of phytochemical alterations of different drying treatments in caper berries. RO: drying at room; SU: sun-drying; AV: Oven-drying (at 70, 60 and 50 °C); MW: Microwave-drying (at 900, 600 and 300 watts); SA: salt drying; FD: freeze drying; FR: fresh sample; CAF: caffeic acid; CIN: cinnamic acid; FER: ferulic acid; PHID: p-hydroxybenzoic acid; PRT: protocatechuic acid; PCO: p-coumaric acid; Van: vanillic acid; ROS: rosmarinic acid; GAL: gallic acid; MCO: m-coumaric acid; PHE: total phenol; FLA: total flavonoid; ANTO: anthocyanin; and ANTI: antioxidant activity

Discussion

Phenolic compounds are found in various plant organs and serve multiple functions, including protection against pests, diseases, environmental stresses, ultraviolet radiation, and free radicals, as well as attracting pollinating insects. Previous studies have demonstrated that phenolic compounds in food, particularly fruits, are beneficial for consumer health [32, 33]. There is increasing interest in polyphenolic compounds due to their therapeutic benefits and ability to prevent diseases like cardiovascular disorders, diabetes, hyperlipidemia, and cancer. Additionally, these compounds are known for their anti-obesity, anti-inflammatory, antibacterial, anti-tumor, and hepatoprotective properties [33, 34]. Plants, especially fruits and vegetables, are excellent sources of antioxidants, which are crucial for human health. Antioxidant compounds are widely used as preservatives in the food industry and pharmaceuticals for the prevention and treatment of cancer, diabetes, and cardiovascular diseases [35, 36].

In the present study, remarkable levels of phenolic compounds such as rosmarinic, gallic, p-coumaric, caffeic, m-coumaric, and vanillic acids were identified in caper fruit extract. Additionally, protocatechuic, ferulic, cinnamic, and p-hydroxybenzoic acids were detected in lower amounts. Francesca et al. [10] identified nine phenolic compounds in caper fruits, including catechin, epi-catechin, caffeic acid, gallic acid, ferulic acid, rutin, vanillic acid, coumaric acid, and myricetin, with rutin being the predominant compound. Another study reported the presence of five phenolic compounds—catechin, epi-catechin, quercetin, rutin, and kaempferol—in caper fruits, with quercetin, kaempferol, and rutin being the most predominant [7]. Aksay et al. [6] identified eighteen phenolic compounds in caper fruits, including protocatechuic acid, p-coumaric acid, catechin, ferulic acid, kaempferol, epi-catechin, and quercetin, with quercetin and kaempferol being the principal ones. Variations in the phenolic compounds identified across different studies may be attributed to genetic differences among plant populations, climatic conditions at the growth site, and the developmental stage of the samples collected [6, 36].

The drying method can have a significant impact on the content of secondary metabolites, such as phenolic compounds, and the biological activity of plants both after harvest and during storage [37]. This is largely due to the duration of the drying process and the environmental conditions involved such as temperature and relative humidity [6, 38, 39]. In the present study, microwave drying, across all three power levels tested, resulted in the highest concentrations of total phenolics, anthocyanins, total flavonoids, and antioxidant activity. Specifically, microwave drying at 600 W significantly increased the levels of caffeic, cinnamic, ferulic, vanillic, and protocatechuic acids compared with other methods. Similarly, microwave drying at 900 W produced high levels of caffeic, ferulic, protocatechuic, and p-coumaric acids. Consistent with our findings, several studies have reported increased antioxidant activity and phenolic content in plants subjected to microwave drying, including research on Cardiospermum halicacabum [40], Daucus carota [41], and Thai Red Curry Powder [42]. An evaluation of different drying methods, including microwave drying for 5, 8, and 10 min at 900 W and hot air heating for 60, 120, and 180 min at 100 °C, on Leptadenia pyrotechnica revealed that microwave heating for 8 min resulted in higher total phenolic content and antioxidant properties, along with a significant increase in phenolic compounds [43].

Microwave drying is a modern and more efficient method compared with traditional techniques due to its reduced drying time. Microwave waves cause polar molecules, such as water molecules, to oscillate. This oscillation allows the water molecules within the plant material to absorb microwave energy uniformly, resulting in a rapid increase in temperature. Molecular vibrations generated by microwaves also disrupt the bonds between water and cell components. Consequently, the initial moisture content of the material is reduced quickly [44, 45]. The impact of microwave power on active ingredients varies among medicinal plants. While some plants exhibit the highest levels of active compounds at high microwave power, others may experience a reduction in these compounds due to excessive heat. In this study, microwave drying at 600 W (medium power) leads to the highest levels of total phenolics, anthocyanins, flavonoids, antioxidant activity, and phenolic acids. Typically, phenolic compounds are bound within plant cells. During heating, if these compounds are in a simple (unbound) form, they may degrade. However, if they are bound and esterified, the heating process can break these bonds, converting them into simpler, more extractable forms, thereby increasing their content. Thus, microwave drying facilitates the breakdown of bonds between complex phenolics, leading to an increase in free phenolic acids at the cellular level [46]. The observed increase in phenolic acids in caper fruits dried at 600 W is likely due to the conversion of phenolic compounds from a non-extractable to an extractable form during the drying process. For instance, hydrolyzable tannins degrade during microwave drying, producing simpler phenolic compounds like gallic acid [47].

In line with this research, microwave drying at 600 W yielded the highest levels of total phenolics, flavonoids, color parameters, chlorophyll, and nutrients in Thymus vulgaris L. leaves. Researchers attributed the increased levels of flavonoids and phenolics with this drying method to preserve compounds in their natural state and minimal disruption of plant tissues [48]. Similarly, using microwave drying for pistachio fruits also enhanced the levels of phenolics and flavonoids. Microwave-dried Cornus mas fruits exhibited higher anthocyanin levels compared with those dried using other methods, such as natural drying, microwave drying, convective drying, and combined microwave-convective drying. Extended drying periods with other methods often lead to the oxidation of phenolic compounds, reducing antioxidant capacity and anthocyanin content [21]. Additionally, microwave drying is more energy-efficient compared with other methods. It rapidly removes moisture from plant materials by causing the cells to swell and creating additional pores in the tissues, which leads to shorter drying times and minimal alterations to the plant samples [49]. Our findings indicate that microwave drying is the fastest method for drying caper fruits (Fig. 2A). This method enhances moisture transfer and removal by leveraging the difference in water vapor pressure between the internal and surface areas of the material [50]. However, research on essential oil-bearing plants suggests that while microwave drying offers rapid processing and preserves plant color, it may not be ideal for retaining volatile oils [5].

In the present study, samples dried in an oven at 70 °C also demonstrated favorable levels of total phenols, flavonoids, anthocyanins, antioxidant activity, and phenolic acids including caffeic, cinnamic, ferulic, protocatechuic, and vanillic acids compared with other drying methods. This aligns with findings from previous studies, which indicated that combining microwave drying with subsequent oven drying at 70 °C increased the content of total phenolics, flavonoids, antioxidant activity, and extractable phenolic compounds in Myrtus communis L [44]. Li et al. [51] also reported that drying chicory leaves at 70 °C enhanced the levels of phenolic acids and antioxidant activity. High temperatures during drying inactivate enzymes, preventing oxidative reactions and thus better preserving the plant’s active ingredients. For instance, drying thyme and rosemary leaves at 70 °C resulted in the highest levels of phenolic acids and antioxidant activity. Researchers attributed this increase to the collapse of the intercellular spaces in plant leaves due to heat, which facilitates the release of phenolic compounds [52]. Consistent with our results, the levels of phenolic acids in oven-dried rhizomes of Z. officinale were higher than those in samples dried using other methods [22]. Drying Coleus amboinicus in an oven at 70 °C produced the highest levels of thymol compared with other temperatures (50 °C, 60 °C, and 80 °C). This positive outcome is attributed to the rapid and efficient drying process at 70 °C, which avoids the damage associated with higher temperatures [53]. Additionally, a study examining the effects of various drying methods— oven drying at 50 °C, sun drying, and a combined oven-microwave drying at 700 W—on the phytochemical properties of peppermint found that the oven-microwave method resulted in the shortest drying time, the highest levels of total phenolics, and the best color quality [54].

In the present study, freeze-drying resulted in relatively high levels of antioxidant activity. Additionally, the highest concentrations of rosmarinic, gallic, and m-coumaric acids were observed with the freeze-drying method. Consistent with our findings, an evaluation of the effects of different drying methods (sun drying, hot air drying, microwave drying, vacuum drying, and freeze drying) on the bioactive compounds, antioxidant activity, and antidiabetic effects of wild guava leaves revealed that freeze-dried leaves had the highest levels of total phenolic content, gallic acid, total flavonoids, and antioxidant capacity compared with the other methods. In contrast, sun-dried leaves exhibited the lowest values for these parameters [55]. Freeze-drying, known as lyophilization, involves freezing water into ice under low pressure and then removing it through sublimation. This technique is widely used to stabilize high-quality foods, biological materials, and pharmaceuticals while preserving the product’s biological, nutritional, and organoleptic qualities [56]. The low temperatures used in freeze-drying minimize the likelihood of degradation reactions in phenolic compounds, thereby more effectively preserving phytochemical content [57, 58]. A study comparing seven drying methods (oven, vacuum oven, shade, sun, microwave, and freeze-drying) on Citrus sinensis L. Osbeck peel found that freeze-drying enhanced both physical properties (such as density and color) and phytochemical properties (including yield, chemical composition of essential oil, antioxidant activity, and total phenolics) more than the other methods [59]. Park et al. [39] investigated the effects of freeze-drying and oven drying at 25 °C, 50 °C, and 80 °C on Agastache rugosa and reported that freeze-drying yielded the highest content of phenolic acids, including rosmarinic acid, aligning with our study’s findings. Freeze-drying enhances the levels of some phenolic compounds because it effectively releases low molecular weight phenolics [60]. For example, freeze-drying increased the levels of phenolic acids, total phenolics, flavonoids, and antioxidant activity in Litchi chinensis L. fruit. In freeze-dried fruits, 20 polyphenols, including hydroxybenzoic acid and coumaric acid, were identified, and rutin levels were significantly elevated [61]. Moreover, freeze-drying enhances the extraction of phenolic compounds from plants by breaking the ice crystals in the sample matrix, which disrupts the cell structure and aids in the release of cellular materials and access to solvents [56, 60].

In the present research, sun-dried caper fruits exhibited relatively high levels of total phenolics and antioxidant activity. Conversely, shade-drying resulted in higher levels of cinnamic, ferulic, m-coumaric, protocatechuic, and p-coumaric acids, with the highest concentration of p-hydroxybenzoic acid also observed in shade-dried fruits. In line with our findings, Bettaieb Rebey et al. [38] reported that shade-drying enhanced the phenolic acids in anise (Pimpinella anisum) seeds. Additionally, Liang et al. [62] found that shade-drying and oven-drying at 40–70 °C were optimal for increasing phenolic acid levels in Angelica dahurica leaves. Another study demonstrated that conventional sun-drying of dates (Phoenix dactylifera) for 6 to 8 days elevated total phenolics, flavonoids, antioxidant activity, and total soluble solid content [14]. This increase may be attributed to the production or release of certain phenolic acids during drying, as the tissue structure is damaged and degradative enzymes, such as those involved in oxidation, hydrolysis, and glycolysis, become activated [63].

However, this study found that the total phenols content, antioxidant activity, anthocyanins, and flavonoids were lower in fresh and salt-dried caper fruits compared with other drying methods. Fresh fruits had lower levels of phenolic acids, including vanillic, p-coumaric, protocatechuic, ferulic, cinnamic, and caffeic acids. Salt-dried samples also showed very low levels of p-coumaric, p-hydroxybenzoic, protocatechuic, ferulic, cinnamic, caffeic, m-coumaric, rosmarinic, and gallic acids. It seems that the slow drying process used in salt-drying makes the fruits more vulnerable to destructive oxidation reactions from external oxygen and internal enzymatic degradation, leading to a reduction in bioactive compounds [64]. M’hiri et al. [65] also found that fresh orange peel samples contained lower levels of phenolics, flavonoids, and phenolic acids compared with oven-dried samples, suggesting that heat treatment facilitates the release of low molecular weight phenolic compounds, a process that does not occur in fresh samples.

Conclusions

Caper fruits are rich in bioactive compounds, offering significant nutritional and medicinal value. However, the drying method significantly affects the levels of secondary metabolites in these fruits. Fresh and salt-dried fruits performed poorly compared with other drying methods, suggesting that the traditional salt-drying method used in Iran is likely unsuitable for caper fruits. In this study, microwave drying at 600 W resulted in substantial increases in many of the phytochemical properties of C. spinosa fruits. Oven drying at 70 °C, freeze-drying, and shade drying also achieved high levels of various phytochemical compounds. Therefore, microwave drying (particularly at 600 W), oven drying at 70 °C, and freeze-drying could likely be considered superior alternatives to traditional drying methods. However, both a microwave system and a laboratory freeze dryer were used. While these methods yielded promising results at the laboratory scale, challenges such as cost and technical limitations may arise when scaling up to industrial or commercial levels. To evaluate scalability, further research is required, employing industrial-grade equipment and optimizing the process for large-scale applications. For future research, conducting sensory evaluations of the fruit could help determine the impact of different drying methods on its final quality. Additionally, evaluating the scalability of microwave drying and freeze-drying under industrial conditions would provide valuable insights into the potential of these methods for commercial use.