Dynamics of Bioactive Compounds under the Influence of Yellow, Blue, and Violet Light Filters on Hippophae rhamnoides L. (Sea Buckthorn) Fruits

Abstract

The current study was carried out to monitor the dynamics of phenolic compounds and vitamin C variations in Hippophae rhamnoides L. (sea buckthorn) under the influence of different color filters, as follows: yellow (590 nm), blue (450 nm), and violet (400 nm). The fruits were harvested at maturity from different parts of the canopy (i.e., base, middle, and top), immediately stored at −18 °C, and afterward lyophilized to reduce the loss of compounds for preparing the chemical assays that were carried out. HPLC-DAD-ESI⁺ was used to determine the phenolic compounds and vitamin C content of the fruits. EPR (electron paramagnetic resonance) measurements were also carried out to confirm the antioxidant character of the berries. This is the first study to examine the effect of different color filters on the accumulation of phenolic compounds and vitamin C content in the fruits of sea buckthorn. Among the three color filters used, the violet filter proved to be the most beneficial for the accumulation of total phenolic compounds (3.326 mg/g) and vitamin C (1.550 mg/g) in the berries. To reach high contents of phenolic compounds and vitamin C, the best setup included using very-high-energy emission LEDs as close as possible to blue and violet (400–450 nm). Therefore, the different light color intensities and temperatures on each level of a canopy play key roles in enhancing the phenolic compound content, antioxidant activity, and vitamin C content of sea buckthorn fruits. This knowledge will help provide insights into the accumulation of secondary metabolites and improve future production strategies in sea buckthorn.

1. Introduction

Hippophae rhamnoides (sea buckthorn) is a thorny deciduous shrub, part of the Eleagnaceae family, and indigenous to Asia and Europe. It is an important plant due to its high medicinal and therapeutic potential; its use dates back to as early as the first half of 800 BC in the Tibetan medical classic “Somaratsa”, which mentions the therapeutic potential of sea buckthorn [1,2,3]. The ancient Greeks used sea buckthorn not only as a treatment against different skin disorders, but also in the diets of racehorses to alleviate their digestive system, hence its denomination ‘hippophae’ which means ‘shiny horse’ [4]. Traditionally, sea buckthorn has been used as food and in nutraceuticals, cosmetics, and drugs, which may be attributed to the fact that sea buckthorn contains significant amounts of vitamins, polyphenols, minerals, and fatty acids [5,6,7,8]. Numerous studies have demonstrated sea buckthorn’s pharmacological effects, including its capacity to serve as a cardiovascular protector and its anticancer, anti-inflammatory, antibacterial, and antiviral properties [3,9,10,11,12]. Furthermore, several therapeutic formulations containing various plant components, from both cultivated and wild sources, have been used as a treatment against multiple disorders, such as wounds, radiation-induced damage, mouth inflammation, and stomach ulcers [13,14]. Various research has been carried out already to reveal the chemical profile of the berries and their effects on human health [10]. Based on these research studies, sea buckthorn has proved to be one of the greatest natural sources of vitamin C, receiving the designation of “King of vitamin C” [15]. Furthermore, sea buckthorn is known for its significant antioxidant properties, but it is also applicable to the alleviation of a variety of clinical symptoms; for instance, it can repair mucosa damage or injuries, serve as a co-substrate for several crucial dioxygenases, and indirectly affect mRNA transcription [1,5,15].

In the specialty literature, other authors have emphasized the main physiological changes that plants experience under different lighting conditions [16,17,18,19]; therefore, the authors state that plants have a shorter developmental stage under dark conditions, and the main physiological change that plants undergo may be that of the transparency of plant tissues [17]. During various stages of growth and development, an increasingly accelerated stem growth occurs, which indicates that depending on the biological processes occurring, plants require certain light radiations with different wavelengths. A similar process can be seen when a plant is exposed to a light source with a very narrow spectra in wavelength.

Exposure to different parts of the light spectrum plays a key role not only in the plants’ growth and development, but also regarding the accumulation of secondary metabolites [20] and postharvest behavior and physiology [21]. According to the maturity stage, responses to various light spectra may vary amongst plant species or cultivars [22]. White and red LEDs have been shown to increase tomato yield [23], while blue LED light has been shown to improve tomato growth and development [24]. Similar results have been shown in pepper crops, as supplementary blue LED light irradiation improved the growth of the stems, leaves, and plant biomass [25]. Considering its impact on plant physiology, yellow light (with a wavelength of about 580 nm) is scarcely studied compared with other researched wavelengths. However, several findings have suggested that ascorbic acid and anthocyanin levels increase when plants are exposed to light with a wavelength between 500 and 600 nm [26], and that yellow light with a wavelength between 580 and 600 nm may affect gene expression in plants during growth [27]. Conversely, other investigations have revealed that ascorbic acid content significantly increased in tomato and strawberry plants under the application of blue light [28,29]. Furthermore, blue and blue/violet materials shorten the development periods in mango fruits, enhance peel appearance, increase their size and sphericity, and minimize the possible occurrence of diseases and peel imperfections [30].

In the current study, the effect of light at different wavelengths was assessed by the use of colored filters that covered sea buckthorn bushes. In order to simulate the improper light conditions, over the course of the whole experiment the plants were covered with three different color filters. Furthermore, the present study also aimed to examine whether the phenolic acid, vitamin C, and antioxidant contents vary depending on the exposure of the sea buckthorn to certain wavelengths of radiation and based on the fruits’ harvesting location on the plant canopy (i.e., base, middle, or top of the plant). The dynamics of the antioxidant activity of the berries were analyzed by EPR spectroscopy to confirm whether sea buckthorn is a suitable candidate to be grown in such conditions.

2. Materials and Methods

2.1. Raw Material and Reagents

The biological material was collected from Hippophae rhamnoides L. species, from a local cultivar created at the University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, named Golden Abundant. This variety has an average plant vigor, with an average leaf size of 6.08 cm long, 0.59 wide and an approximate leaf area of 3.04 cm². The leaves’ surface is shiny with a dark green color and silver spots on the lower part of the leaf blade. The fruits are characterized by a light-yellow color, and an oval shape.

The experiment was carried out in a sea buckthorn orchard at the Horticultural Research Station of the University of Agricultural Sciences and Veterinary Medicine of Cluj-Napoca. Ten shrubs were randomly selected and were covered with different color filters as follows: blue, yellow, and violet; the control was uncovered (without filter). Color filters were installed in early June, after fruit formation. In terms of cultivation technology, planting distances were as follows: 1.2 m between plants/row and 3 m between rows. The cuttings of the sea buckthorn plants were acquired at the end of winter (mid February). The role of pruning is to maintain the size, shape, and architecture of the plants to facilitate harvesting of the fruit, establishing an optimum balance between fruiting and vegetative branches, better light capture, and keeping the crop healthy.

The berries were harvested at full maturity, in early October and were then lyophilized (Christ, Alpha 1-2 LD Plus, Osterode am Harz, Germany). Parameters of freeze-drying process were as follows: freezing step for 18 h, main drying at −12 °C, 1.6 mbar, and the last step of final drying at −5 °C, 4.0 mbar. In order to carry out a rigorous study and to reduce loss of compounds, after lyophilization the fruits were stored at −18 °C until further analysis.

Acetonitrile, acetic acid for HPLC, was purchased from Merck Chemicals GmbH (Gesellschaft mit beschränkter Haftung, Darmstadt, Germany), while ultrapure water was obtained with Direct-Q UV from Millipore (Burlington, VT, USA). Rutin and gallic acid standards (purity > 99% HPLC) were purchased from Sigma-Aldrich (Burlington, VT, USA). All of the extraction solvents employed were of analytical grade. All the other chemicals were purchased from Merck Chemicals GmbH (Darmstadt, Germany).

2.2. The HPLC-DAD-ESI+ Phenolic Compounds and Vitamin C

2.2.1. HPLC Analysis

HPLC Agilent 1200 was used in this experiment to analyze the prepared samples. The separation of the compounds was carried out according to the Kinetex XB C18 column with the following specifications: dimensions: 4.6 × 150 mm with 5 μm particles (Phenomenex, Torrance, CA, USA), with mobile phase (A) water with 0.1% acetic acid, (B) acetonitrile with 0.1% acetic acid, gradient (% B): 0 min, 5% B; 0–2 min, 5% B; 2–18 min, 5–40% B; 18–20 min, 40–90% B; 20–24 min, 90% B; 24–25 min, 5–90% B; 25–30 min, 5% B at a flow rate of 0.5 mL/min at 25 °C temperature. Spectral values were recorded in the range of 200–600 nm for all the peaks. The visual outputs of the chromatogram were recorded at the wavelength of λ = 280 si 340 nm. For MS, positive ion mode was chosen for electrospray ionization full scan under the following conditions: capillary pressure of 3000 V, temperature 350 °C, flow rate of N 7 L/min, and m/z 120–1200. The quantification of the data and interpretations were performed using Agilent ChemStation Rev B.04.02 SP1.

2.2.2. Phenolic Compounds

The method described by Diaconeasa et al., 2014 [31], with slight modifications was used to determine the phenolic compounds content. An amount of 0.15 g lyophilized and ground sea buckthorn berries was used. The extracts were prepared using 1.5 mL acidified methanol with 1% HCl concentration (v/v), vortexed for 1 min, sonicated for 30 min and then centrifuged at 10,000 rpm, for 10 min at T = 24 °C. The supernatant was filtered and 20 μL of sample was used for HPLC analyses. The total phenolic content includes the total quantity of di-gallic acid, gallic acid, protocatechuic acid, quercetin-acetyl-rhamnoside, isorhamnetin-glucosyl-rhamnoside, isorhamnetin-glucoside, isorhamnetin-rutinoside and isorhamnetin quantified in this experiment.

2.2.3. Vitamin C Assay

Vitamin C was quantified as the sum of ascorbic acid (AA) and dehydroascorbic acid (DHA). The method used was after Felfoldi et al., 2022 [32], with slight modifications imposed by the studied matrix. Briefly, 0.15 g of each sample was homogenized using an aqueous solution with 8% acetic acid and 3 mL of 3% H₃PO₄ followed by sonication using an Elmasonic E15H (Elma, Singen, Germany) bath for 30 min. Afterwards, the obtained solution was centrifuged for 10 min at 8000/min at 4 °C in an Eppendorf 5804 centrifuge (Eppendorf, Hamburg, Germany). The supernatant was filtered with Chromafil Xtra nylon 0.45 µm and 20 µL was injected into the HPLC-DAD-ESI-MS system equipped with a quaternary pump, autosampler, DAD detector, and coupled to an MS-detector single-quadrupole Agilent 6110 (Agilent-Technologies, Santa Clara, CA, USA). An XDB C18 Eclipse column (4.5 × 150 mm²) with a flow rate of 0.5 mL/min was used for separation and identification of the compounds, with 1% formic acid: acetonitrile (95:5) in distilled water (v/v) (A) and 1% formic acid in acetonitrile (B) as binary gradients. For MS fragmentation, a scanning range of 100–600 m/z in the ESI (+) mode was conducted with a capillary voltage of 3000 V, a temperature of 300 °C, and a nitrogen flow rate of 7 L/min. Chromatograms were recorded at a wavelength of 240 nm, and data acquisition was performed using Agilent ChemStation software (Rev B.04.02 SP1, Palo Alto, CA, USA). The analysis was conducted in triplicate. The total vitamin C content was calculated as the sum of (AA) and (DHA).

2.3. EPR Measurement of Antioxidant Activity

In addition to the determination of phenolic compounds and vitamin C content, another aim of this research was to investigate the fruits of sea buckthorn extracts by EPR spectroscopy. The free radical content of the samples was examined using electron paramagnetic resonance (EPR) spectroscopy by measuring free radical EPR signal intensities. The samples were assayed at room temperatures with a Bruker EMX EPR spectrometer in a standard chamber, continuous wave, in the X-band (9.4 GHz) with magnetic field modulation of 100 kHz and attenuation of 20 dB. The EPR spectra were recorded at room temperature. The EPR results were obtained by dividing the relative amplitude of the EPR signal (in arbitrary units) by the mass of each sample [33].

2.4. Statistical Analysis

The data obtained were analyzed using the analysis of variance (ANOVA). When the null hypothesis was rejected, post hoc tests were performed to determine significant differences between the means (n = 30). Tukey’s HSD test was performed to compare the means and identify statistically significant differences between the means at p < 0.05. Firstly, comparison tests were carried out to compare the means between compounds undergoing the same treatment. Secondly, comparisons were made to identify significant differences between the means of different filters and the same fruit position. The last comparisons were made to reveal significant differences between the filters against control. The statistical analyses were assessed using SPSS software (version 19). The data are represented as means ± standard error. Principal component analysis (PCA) was performed using the FactoMiner factoextra package [34]. Heatmap and dendrograms were generated using the Euclidean distance with complete linkage to emphasize the similarities and differences between the biologically active substances and different color filters and to assess the effects between the color filters and the accumulation of bioactive compounds depending on the fruit harvesting location on the plant canopy (i.e., base, middle, top) using the Cluster R (version 4.0.5) [35] and ggplot packages [36] from R (version 4.0.5).

3. Results

3.1. Phenolic Compounds and Vitamin C Content

The results obtained due to the application of different color filters and their impact on sea buckthorn chemical profile, shed light on the stimulating effect of violet and yellow filters on phenolic compounds and the vitamin C content of the fruits as compared to the control. To exclude external factors that can influence the accumulation of chemical compounds in fruits, three different parts of the canopy were bounded. Therefore, regarding the fruit’s position on the canopy, it was observed that the fruits harvested from the middle part of the canopy had the highest total phenolic content accumulation under all the filters applied (

Table 1. The influence of color filters and fruit position on phenolic compounds and vitamin C of sea buckthorn berries (μg/g). Different colors in the table header indicate the color filters used in this study.

Compound	Control Base	Control Middle	Control Top	F1 Base	F1 Middle	F1 Top	F2 Base	F2 Middle	F2 Top	F3 Base	F3 Middle	F3 Top
Di-Gallic acid	1.604 ± 0.12bA	3.159 ± 0.13eA	2.588 ± 0.32eA	2.834 ± 0.21fB *	3.578 ± 0.34eB *	3.368 ± 0.38eB *	3.576 ± 0.24dC*	4.419 ± 0.51eC *	3.900 ± 0.32eC *	3.330 ± 0.21dBC *	3.776 ± 0.09fB *	3.476± 0.11eB *
Gallic acid	0.538 ± 0.03aC	0.254 ± 0.01aB	0.193 ± 0.01aA	0.067± 0.02aA *	0.088± 0.021aA *	0.269± 0.012aB *	0.328± 0.03aB *	0.358 ± 0.04aC *	0.347 ± 0.05abC *	0.104 ± 0.01aA *	0.321 ± 0.03aBC	0.319 ± 0.04aC *
Protocatechuic acid	1.945 ± 0.03cC	2.157 ± 0.11dC	0.922 ± 0.02cdAB	0.965 ± 0.05bcdA *	1.01 ± 0.07bcA *	0.724 ± 0.08abA	1.411 ± 0.11cB *	1.529 ± 0.09cdB *	1.464 ± 0.06dC *	1.068 ± 0.09bA *	1.102 ± 0.11bdA *	1.051 ± 0.09bcB
Quercetin-acetyl-rhamnoside	0.243 ± 0.03aA	0.454 ± 0.04abB	0.623 ± 0.021bcC	0.603 ± 0.03bB *	0.823 ± 0.02bC *	0.529 ± 0.04aBC	0.306 ± 0.03aA	0.293 ± 0.08aA *	0.456 ± 0.06abB *	0.319 ± 0.04aA	0.556 ± 0.03abB *	0.293 ± 0.05aA *
Isorhamnetin-glucosyl-rhamnoside	2.064 ± 0.3cB	2.825 ± 0.5eC	2.759 ± 0.6eBC	2.118 ± 0.8eB	2.398 ± 0.7dB *	2.518 ± 0.43dB	1.33 ± 0.32cA *	1.923 ± 0.4dA *	1.421 ± 0.2dA *	2.094 ± 0.11cB	2.986 ± 0.43eC	3.083 ± 0.28eC
Isorhamnetin-glucoside	0.546 ± 0.08aA	0.803 ± 0.09bA	1.015 ± 0.17cdC	1.015 ± 0.11cdB *	1.056 ± 0.09bcB *	1.156 ± 0.07bcC *	0.754 ± 0.05abB *	0.733 ± 0.08abA	0.865 ± 0.05bcA *	0.867 ± 0.09bB *	1.376 ± 0.04dC*	1.200 ± 0.09cC *
Isorhamnetin-rutinoside	0.407 ± 0.05aA	0.477 ± 0.08abB	0.453 ± 0.05abB	0.721 ± 0.07bcB *	0.522 ± 0.07abB	0.685 ± 0.08abC *	0.278 ± 0.02aA	0.273 ± 0.02aA *	0.276 ± 0.03aA *	0.379 ± 0.05aA	0.566 ± 0.05abcB	0.533 ± 0.06abB
Isorhamnetin	1.443 ± 0.3bB	1.568 ± 0.6cB	1.224 ± 0.5dAB	1.33 ± 0.12dB	1.478 ± 0.17cB	1.365 ± 0.2cC	0.852 ± 0.12bA *	1.049 ± 0.13bcA *	1.002 ± 0.16cdA	0.921 ± 0.11bA *	1.611 ± 0.45dB	1.977 ± 0.28dC *
Total phenolic content	30.264			31.220			29.159			33.326
Ascorbic acid (AA)	0.185 ± 0.01aA	0.191 ± 0.01aA	0.186 ± 0.05aA	0.206 ± 0.07aB *	0.221 ± 0.05aB *	0.211 ± 0.04aB *	0.180 ± 0.06aA	0.192 ± 0.08aA	0.185 ± 0.02aA	0.225 ± 0.02aC *	0.237 ± 0.03aB *	0.228 ± 0.01aC *
Dehydroascorbic acid (DHA)	0.253 ± 0.03bA	0.278 ± 0.03bAB	0.263 ± 0.06bA	0.266 ± 0.04bB *	0.281 ± 0.01bB	0.278 ± 0.08bB *	0.256 ± 0.09bA	0.274 ± 0.04bA	0.261 ± 0.04bA	0.279 ± 0.02bC *	0.292 ± 0.03bC *	0.287 ± 0.05bC *
Total vitamin C	1.356			1.463			1.350			1.550

Notes: Data are presented as means ± standard error (n = 30). Different lowercase letters in a column indicate significant differences between the compounds undergoing the same treatment; different capital letters in a row denote significant differences among the filters, but same position; Asterisk indicate significant differences between the filters against control according to Tukey’s HSD test (p < 0.05).

) as compared to the other parts. Regarding individual phenolic compounds, the highest content in gallic acid (0.538 ± 0.03 mg/g dw) was observed in the control fruits positioned at the base of the canopy, whereas the lowest accumulation was noticed in the fruits under the yellow filter (0.067–0.088 mg/g dw) positioned at both the base and middle parts of the canopy. Surprisingly, lower values in gallic acid were also observed under the violet filter in the fruits collected from the base of the canopy. A similar trend was observed regarding the accumulation of protocatechuic acid, with the highest values in the fruits collected from the base and middle parts of the canopy (1.945–2.157 mg/g dw) under control conditions. Conversely, the lowest level in protocatechuic acid was observed under the yellow filter in the top of the canopy. Quercetin-acetyl-rhamnoside accumulated in high amounts in the fruits situated in the middle part of the canopy (0.823 ± 0.02 mg/g dw) under the influence of yellow filters, whereas significantly lower levels were noticed under the effects of blue and violet filters.

Regarding the compounds from the isorhamnetin group, significantly high levels were detected in the fruits under the influence of violet filters, particularly those situated in the middle and top parts of the canopy. However, the blue filter had a negative effect regarding the accumulation of these compounds with significantly lower levels compared with the other filters and the control group.

Subsequent to phenolic acid content, vitamin C is another important parameter when evaluating fruit quality. Due to the determination of ascorbic acid content in sea buckthorn samples, it was observed that its content ranged between 0.237 mg/g blue filter/base and 0.237 mg/g violet filter/middle (Table 1). Thus, similar results with the phenolic compounds were observed under the influence of the violet filter, particularly in the fruits collected from the top and middle parts of the canopy. The highest levels in both ascorbic acid (0.237 ± 0.03 mg/g dw) and dehydroascorbic acid (0.292 ± 0.03 mg/g dw) accumulated in the fruits harvested from the middle part of the canopy. Interestingly, similar levels were noticed under the influence of the yellow filter, particularly in the fruits harvested from the middle part. Lower levels of vitamin C were noticed under the influence of the blue filter, mostly in the berries collected from both the top (0.185−0.262 mg/g dw) and base (0.180−0.256 mg/g dw) of the canopy.

3.2. EPR Investigations of the Antioxidant Activity

The first derivative of EPR absorption versus magnetic field is graphically presented for each type of filter. Analyzing the EPR spectra in the lyophilized fruits, it was observed that the concentration of the paramagnetic species present in the sample increased depending on the filter used in the following ascending order: violet, blue, and yellow, as seen in Figure 1.

The values presented in

Table 2. The influence of the filter on the amplitude of the EPR. Different colors in the table columns indicate the color filters used in this study.

Filters	C1 (Control) [a.u]	F1 (Yellow Filter) [a.u]	F2 (Blue Filter) [a.u]	F3 (Violet Filter) [a.u]
	The Amplitude of the EPR Signal, Measured from Figure 1
Base	0.25	0.55	0.27	0.25
Middle	1.38	0.93	1.37	1.21
Top	0.15	0.63	1.17	0.37

reflect the intensity of the first derivative of the EPR signal for the sea buckthorn fruits grouped in relation to the three filters and the canopy part from where the fruits were harvested. Since the EPR absorption width mostly remains constant, the signal intensity values are proportional to the area of the EPR signal in respect of the number of paramagnetic centers (the amount of free radicals) that generate the EPR absorption.

Several statistical methods were used to evaluate different correlations between the color filters, sea buckthorn samples, and their composition (Figure 2 and Figure 3). Based on the principal component analysis (PCA), the first two accounted for 68.20% of the total variation (Figure 2). Following the quadrants, the first quadrant highlights the samples from the control group situated at the base of the canopy with significant levels in gallic acid and protocatechuic acid. The second quadrant revealed the similarities between the fruits harvested from the top and middle parts of the canopy and under the influence of the violet filter. These samples were proved to accumulate high levels of isorhamnetin-glucosyl-rhamnoside and vitamin C. The subsequent quadrant emphasizes the samples under the influence of the yellow filter with significant accumulation in isorhamnetin-rutinoside in the fruits collected from the top and base parts of the canopy. Furthermore, the samples from the middle part accumulated high levels in quercetin-acetyl-rhamnoside. The last quadrant revealed the fruits under the influence of the blue filter which had the lowest phenolic acid accumulation irrespective of their position on the canopy.

Hierarchical clustering (HCA) and heat mapping were used to better visualize the similarities and differences between the biologically active substances and different color filters (Figure 3). The HCA revealed that the samples were mainly grouped into two clusters including sub-clusters (Figure 2). Unambiguous discrimination between the biologically active compounds and color filters is seen via the different cluster positions of violet and yellow color filters, followed by the control group and blue color filter. The violet- and yellow-colored filters at the base, middle, and top parts of H. rhamnoides plants were discriminated from the other filters used. Thus, following the importance score, the first sub-cluster (A1.a) highlights the accumulation of isorhamnetin-glucoside, ascorbic and dehydroascorbic acids in the fruits located in the middle part of the canopy (F3 middle), followed by a significant accumulation of isorhamnetin in the upper part of the canopy (F3 top).

The second cluster (A1.b) comprises the fruits from the base of the canopy under the influence of purple color filters, closely followed by the fruits subjected to yellow color filters. The fruits subjected to yellow color filters accumulated similar and high levels in quercetin-acetyl-rhamnoside and isorhamnetin-rutinoside compared with the other filters used. Conversely, lower values in gallic acid and protocatechuic acid were observed using the yellow filter. The following sub-cluster (B1.a) underlines the control fruits from the base of the canopy (C base), where the highest levels in gallic and protocatechuic acids were detected. However, significant lower levels in di-gallic acid, isorhamnetin-glucoside and ascorbic acid were identified. Subsequently, these were closely followed by the fruits located in the middle part of the canopy (C middle) with similar lower values in gallic acid and protocatechuic acid. The last sub-cluster (B1.b) comprises the sea buckthorn fruits subjected to blue filters which revealed dissimilarities in the accumulation of compounds from the isorhamnetin group (i.e., isorhamnetin-rutinoside, isorhamnetin, glucosyl-rhamnoside, and isorhamnetin). Conversely, slightly similar and lower levels in di-gallic acid were noticed particularly in the fruits located in the middle and lower parts of the canopy.

4. Discussion

The present study revealed that the violet filter stimulated most the accumulation of phenolic compounds except the fruits harvested from the base of the canopy, where the highest amount of phenolic compounds was detected under the effects of the yellow filter. Ullah et al., (2019) [37], investigated the effects of multispectral lightning (including control, red, green, blue, dark, white, and yellow light) on the accumulation of polyphenols in Lepidium sativum L., and observed the highest polyphenol content under the effect of white light (39.19 mg/g DW), followed by dark light (35.14 mg/g DW), blue (29.96 mg/g DW), green (29.45 mg/g DW), red (27.63 mg/g DW), control (26.11 mg/g DW), and yellow (23.64 mg/g DW) [37]. From the top of the canopy, the highest amounts of phenolic compounds of the fruits were also induced by the violet filter. It was noticed that fruits harvested from the bottom part of the canopy had lower phenolic contents compared with those harvested from the middle and top parts of the tree. In a different study, important quantities regarding phenolic content in 10 different varieties of sea buckthorn were also determined from different parts of the plant indicating similar quantities: skin (1293.30 mg/100 g dm), flesh (507.16 mg/100 g dm), endocarp (669.85 mg/100 g dm), seeds (1488.15 mg/100 g dm), branches (9318.65 mg/100 g dm), leaves (2026.37 mg/100 g dm) [38]. Various combined blue–red light treatments in Salvia miltiorrhiza Bunge. (red sage or dānshēn) revealed diverse growth development and accumulation of phenolic acids. Thus, the highest content in total phenolics and rosmarinic and salvianolic acids were noticed under blue and red (3B:7R) combined light treatments [39]. The same ascending trend in phenolic accumulation was observed in arugula (Eruca sativa Mill.) under both blue and blue-violet light treatments [40]. Consistent with the present study’s results, the content in protocatechuic acid of Verbena officinalis L. significantly decreased under the influence of different light filters, particularly under red and red–blue light treatments [41]. The lowest total phenolic content in sea buckthorn fruits was recorded under the effects of blue filters. However, the changes in phenolic content in response to UV-light may vary between species.

Previous studies showed an increase in anthocyanin levels as response to UV radiation in various fruits’ skin such as apple and sweet cherry [42,43]. Regarding different wavelengths of LED light, RBG light (red, blue, green) had a great influence on the antioxidant potential of the micro-culture of Nasturtium officinale W.T. Aiton [44]. Another study carried out by Ye et al. 2022 [45] suggested that the blue filter applied to Anoectochilus roxburghi (Wall.) Lindl. had a positive influence on plant development, while yellow and red colors had reverse effects. Muneer et al. (2014) [46] investigated the influence of green, red and blue light-emitting diodes and observed that blue LEDs of high intensity improved plant development, thus controlling the integrity of chloroplast proteins which leads to the optimization of photosynthetic performance under natural conditions. Liu and Van Iersel (2021) [47] reported that no synergic or antagonistic interactions between blue, green and red light intensities were shown considering the photosynthetic physiology of lettuce. Based on these results, the use of blue filters in plant growth represents a useful technique to improve plant growth and development. In addition, blue light technologies can be accessible to a wider range of interests since their use requires low investment. Furthermore, under the light radiation with the highest energies, for 400 nm (violet filter), the evolution is as expected, i.e., the highest amount of antioxidants prevails where the light is abundant, like the control sample.

The highest amounts of vitamin C were registered in fruits harvested from the middle part of the canopy, following the same pattern as phenolic compounds under the influence of the violet filter. Similarly, the highest vitamin C content in sea buckthorn was determined under the violet filter (1.550 mg/g) followed by the yellow filter (1.463 mg/g). Ilhan et al. (2021) determined the vitamin C and phenolic content in 10 different genotypes of sea buckthorn harvested from Eastern Anatolia in natural light conditions. The vitamin C content ranged between 40.10 and 50.62 mg/100 g while the phenolic content ranged between 450 and 622 mg/100 g [48]. Other studies have highlighted that the vitamin C content in sea buckthorn is influenced not only by the variety, but also the geographical area of the species. For example, the sea buckthorn grown in Europe in coastal dunes showed a vitamin C content of 120–315 mg/100 g, while those grown in the Alps had a higher vitamin C content (405–1100 mg/100 g). The fruits of Chinese buckthorn (Rhamnus utilis) are considered to have the highest vitamin C content, reaching levels up to 2500 mg/100 g [49]. According to Pop et al. (2013) [50], ascorbic acid can be found in many other fruits such as Fragaria x ananassa varying between 13.79 mg/100 g and 44.05 mg/100 g. In addition, vitamin C is an important component of other plant organs as well, such as roots in carrots ranging from 8.51 to 11.91 mg/100 g or tomatoes 19.76 mg/100 mL [51,52]. Regarding color filters, the vitamin C content of red-leaf pak choi significantly accumulated under the influence of blue LED light at 460 nm, compared with supplemental UV-A light at 400 nm and at 380 nm which significantly decreased the vitamin C content. Conversely, lower vitamin C content has been detected in green-leaf pak choi under the influence of blue LED light particularly at 460 nm [53]. As storage conditions might affect the vitamin C content, different LED light pulses have been used during postharvest storage conditions in raspberry and blackberry. The authors revealed that following seven days of cold preservation the contents of both malic and quinic acids were significantly enhanced under the influence of green, blue, and red light treatments, whereas green and blue LED sources further increased the ascorbic acid content. Additionally, after a storage period of 14 days, the citric acid levels were significantly higher in the blue, green, and blue + red LED light treatments [54].

The present findings indicate that all the four filters had a positive influence on vitamin C accumulation in the fruits regardless of their position in the canopy. However, several studies carried out on different fruits such as grapes, strawberry, kiwi, peach, and blueberry reveal that the change in ascorbate levels during fruit ripening is highly dependent on the species [55,56,57].

The experimental values for vitamin C were determined by the HPLC-DAD-ESI⁺ method [58,59,60]. Figure 1 reflects the accumulation of the chemical compounds analyzed in this study. The results show that the fruits harvested from the middle part of the plants exhibited the highest concentrations of paramagnetic centers, and antioxidants (phenolic compounds, vitamin C). Similar results were obtained in tomato, Chinese cabbage crops [61], and other crops [62,63]. These results can be explained by the different color filters and temperatures on each level of the canopy that might influence the accumulation of bioactive compounds. However, the values registered in the plants covered with yellow and blue filters are contrary to expectations regarding the amount of antioxidants which is more abundant at the base of the plant compared to the results obtained from the top part. These results might be due to several possible causes. The first explanation could be related to the cumulative effect between temperature and the wavelength of absorbed radiation, i.e., for these wavelengths of 450 nm and 590 nm, the plant synthesizes more antioxidants due to the higher temperature near the soil and telluric radiation than the influence of the two filters. The second possible explanation would be the shape and the smaller absorption surface of the light radiation at the top of the plant, combined with a lower absorption at these two wavelengths; this second hypothesis is in contradiction with the yield obtained in chili crops [64] and those of basil [65]. However, the authors of these studies refer to the size of the plants under similar light radiation, while the current study refers more to the number of antioxidants present in the fruits.

Considering the number of phenolic compounds, ascorbic acid and the investigation of the presence of paramagnetic species by the EPR method, all these are in agreement with similar studies and highlight the middle part of the canopy as where the highest values were recorded [14,66,67].

5. Conclusions

The highest possible antioxidant activity can be induced by the use of LEDs with emissions in ultraviolet or blue (with maximum efficiency of conversion from electrical energy to light) with a very good management of the spatial distribution of light. It can also be concluded that the size of the light absorption surface plays an important role. The most favorable results can be obtained with the application of blue and violet filters for fruits harvested from the middle and from the top parts of the canopy. To sum up, the best setup of LED light in order to reach a high content of chemical compounds is the use of very high energy emission LEDs as close as possible to blue and violet (400–450 nm). Consequently, the phenolic compounds, antioxidant activity, and vitamin C content of sea buckthorn fruits are enhanced by used color filters and differences in temperature. This is the first study to examine the effects of different color filters on the accumulation levels of phenolic acids and vitamin C content in sea buckthorn. Further investigation is required to conduct an in-depth assessment of these treatments using diverse LED lights.