Antioxidative Response and Phenolic Content of Young Industrial Hemp Leaves at Different Light and Mycorrhiza

2.2. Pigment Content in Young Leaves

In general, for chlorophyll and carotenoid content, significant differences (p < 0.05) were determined for the light and interaction of light and biopreparations. Biopreparations significantly increased only chl/car content by 8.8% at treatment four on blue light. Also, at the blue light, all chlorophyll content and carotenoid parameters were higher than their content at the white light, except for the ratio of chl a/b (Figure 1a–f).

2.3. Antioxidant Activity

The 1-diphenyl-2-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) methods determined the antioxidant capacity of the young industrial hemp leaves. Light treatments and biopreparations did not significantly influence the DPPH and FRAP of young C. sativa leaves. Only interactions between light and biopreparations significantly influenced DPPH and FRAP. On blue light, values of DPPH were higher on all treatments 2–5 (13.9, 37.3, 56 and 22.5%, respectively) compared to control. On white light, lower values were recorded on treatments two (31.9%), three (8%), and five (21%), and higher values were recorded on treatment four (39.2%). Among all biopreparation treatments, the highest DPPH was determined in treatment four, where the determined volume of DPPH was 856.10 (Figure 2a). FRAP antioxidant potential was reduced by 43.8% on treatment four on blue light (p < 0.05) (Figure 2b).

3.1. Morphological Parameters of Young Hemp Plants

It is well known that plants are grown under blue light, inhibiting elongation to produce short, thick, dense plants with increasing root development, whereas isolated red light creates tall, stretched plants with thin leaves and long stems [54,55,56,57]. This is also confirmed in this study, as hemp seedlings were under significant (p < 0.05) influence of light treatment, whereas greater values of all morphological parameters were recorded on white light (FLUO). Likewise, Magagnini et al. [52] found significant differences in plant morphology of hemp plants (drug chemotype “G-170”) after 46 days, recording higher values of morphological parameters when plants grown under the HPS (high peruse sodium lamps) compared to plants grown under LED light treatments. In Glowacka’s research [58], shorter stems were found in tomatoes grown under blue light. Likewise, Javanmardi and Emami [59] claim that blue light, in contrast to white light, reduces the height of tomato and pepper seedlings. On the contrary, Cheng et al. [37] reported that the industrial hemp variety Bamahuoma had a higher number of leaves per plant, stem diameter and root length and stem height (by 13.7, 10.2, 6.8 and 2.3%, respectively) under blue light, as compared to white light treatment. Kakabouki et al. [60] investigated the influence of Trichoderma harzianum seed inoculation on the agronomical and quality properties of hemp. They stated that the presence of Trichoderma harzianum led to a statistically significant increment of the root density of the plants compared to controls, as well as to an increment of Arbuscular Mycorrhizal Fungi (AMF) percentage. Furthermore, the presence of T. harzianum affected the height and dry weight of plants, as well as the number, fresh weight, and moisture of buds. Except for beneficial fungi species, there is also the interesting finding of Balthazar et al. [61], who reported that beneficial bacteria of Pseudomonas spp. (P. fluorescens, Pseudomonas protegens, and P. putida), seem to be naturally present in hemp tissues and surrounding soil. In a study with arbuscular mycorrhizal fungi (Rhizophagus prolifer and R. aggregatus) Seemakram et al. [62] found a significant influence on plant length, leaf surface area (cm²) and root dry weight, but also increment of plant total cannabinoids content (CBD, CBDA, CBG, THC) with mycorrhizal fungi 60 days after sowing C. sativa (cultivar KKU05).

The negative effect of blue light on biopreparation treatment is determined in all treatments, resulting in lower values for all morphological parameters compared to control. Also, the main differences were recorded in treatments four and five, where biopreparations were applied both in the substrate and seed inoculation. Since LED blue light inhibits the formation of arbuscular mycorrhizal fungi and lowers plant growth [63], it is possible that this effect is more pronounced when a larger amount of biopreparation is applied.

3.2. Pigment Content in Young Leaves

The effect of blue and red wavelengths on the photosynthesis rate is widely known, so LED lighting producers choose these wavelengths. However, the indirect influence of green light on photosynthesis is often overlooked because it is considered that the green appearance of plants is due to the reflection of the green light spectra. Today, it is known that less than 50% of green light (500–600 nm range) is reflected by plant chloroplasts, while the rest is absorbed by plant pigments or transmitted to shaded parts of the plant [64,65]. Therefore, monochromatic lighting can be used only as supplementary lighting. However, it is necessary to determine the most photosynthetically effective spectral composition for each plant species and stage of development within the same plant species. Hogewoning et al. [66] investigated the influence of different light spectra on chlorophyll content in cucumber leaves (Cucumis sativus cv. Hoffmanns Giganta) grown in a hydroponic system. Plants were illuminated for 16 h with different proportions of blue (450 nm) and red (638 nm) LED lights. They found that the total chlorophyll content in the leaves of cucumber plants increased with an increase in the proportion of blue light. Snowden et al. [67] also found significantly increased chlorophyll concentration with increasing blue light in tomato, cucumber, radish and pepper at the higher light levels. Li and Kubota [68] showed an increase in chloroplast pigments in leaves of lettuce grown under blue light, and the authors stated that the concentration of carotenoids increased by 12% in leaves of plants grown under blue light compared to control plants grown under white light. An increase in value under the influence of blue light for chlorophylls and carotenoids, except for the chl a/b ratio, is also confirmed in this research. Blue light has long been considered an important factor in chlorophyll formation and chloroplast development [69]. It is clear that plant chlorophylls absorb mainly in blue (between 400 and 500 nm) and red wavelengths (around 650 to 680 nm) [70,71]. Therefore, a possible reason for reducing chlorophyll and carotenoids under white light is that plants usually adapt to low light conditions by reducing the chlorophyll concentration per unit leaf area [71]. Also, the decrease in chlorophyll content may be due to a change in nitrogen metabolism in the production of compounds such as proline, which is used for osmotic regulation [72]. On the other side, the reason for the accumulation of chl and car when exposed to blue light is the increased free radical scavenging activity of plant extracts through the enhanced synthesis of secondary metabolites, i.e., their increased accumulation to protect plants from blue light [68]. At the same time, a combination of VAM on bioloth and VAM and Azotobacter chroococum in liquid media caused an increase in the chl a + b/car content under blue light, which determines the negative effects of the stress of the combination of bacteria and fungi on the plant. Otherwise, under stress conditions, the chl and car ratio should be lowered, confirming the initiation of the plants’ photoprotective defence mechanism, which was absent here.

3.3. Antioxidant Activity

In this study, light and biopreparation did not significantly influence DPPH. Also, Kook et al. [73] found no significant difference in activity between lettuce treated with blue and white broad-spectrum LEDs. Despite this, the interaction of light and biopreparation for DPPH proved to be significant, which caused a different pattern of behaviour in the results per treatment. Namely, all biopreparations under blue light increased the value of DPPH, in contrast to biopreparations under white light, where the value increased only in treatment four and decreased in the others. This clearly shows the influence of fungi and bacteria (treatments) between the lights. Blue and white light had the most pronounced effect on the combination of VAM on biolith and VAM and Azotobacter chroococum in liquid media, in which the values of DPPH increased by 56 and 39.2%, respectively, observing the interactions of light and biopreparation. A significant increase in antioxidant activity, based on the DPPH assay, was found in red pak choi and basil under 25 and 33% constant blue light [74]. Also, He et al. [75] have attained a higher DPPH in tomato fruits under blue lighting treatments, which they related to the duration of exposure to blue light. Vaštakaitė et al. [69] reported that the highest DPPH activity could be avowed by light due to the absence or overdosage of blue light. Further, kale microgreen showed strong antioxidant effects when tested under the influence of white, red, and blue LED lights. Kale illuminated by a blue LED had the best antioxidant capacity. It was also proven that the inhibition of DPPH radicals was positively correlated with phenolic components capable of antioxidant activity [76].

Moreover, light and biopreparation did not significantly influence FRAP. The antioxidative activity with the FRAP method resulted in similar average values for the different light sources (0.03 mM FeSO₄) and biopreparation, which shows that the antioxidative activity is quite stable in young hemp leaves. For industrial hemp cultivar Białobrzeskie, Stasiłowicz-Krzemień et al. [77] stated that methanol macerated leaves extract had antioxidant potential of DPPH 5.632 mg trolox/g plant material and FRAP 11.066 mg trolox/g plant material. In this study, only statistical difference was confirmed on treatment four (VAM on biolith and VAM and Azotobacter chroococum in liquid media) in blue light when the value was decreased. Contrary to these results, He et al. [75] found that under blue light, tomato fruits attained higher values of FRAP.

4.2. Sample Preparation

Young fresh hemp leaves were crushed and macerated using liquid nitrogen to obtain sample extracts of plant tissue. Ethanol extracts (70% EtOH) were prepared for the antioxidant activity—DPPH and FRAP methods. Also, the content of phenols, flavonoids, flavanols, and phenolic acids was determined. The content of chloroplast pigments was determined from the acetone extracts. Four replicates per treatment were performed for each analysis.

4.3. Determination of Chlorophyll and Carotenoid Content

In a 15 mL test tube, 0.05 g of powder sample extract was weighed, and 10 mL of acetone was added. After mixing on a vortex mixer, the samples were centrifuged for 10 min at 4000 RPM at 4 °C. 2 mL of supernatant was used to determine the absorbance at wavelengths 662, 644, and 440 nm, whose values were included in the Holm-Wettstein equations [84,85] for calculating the concentration of chlorophyll a, chlorophyll b, total chlorophyll and carotenoid content, in mg dm⁻³. Final concentrations of pigments are expressed as mg g⁻¹FW (fresh weight).

4.4. Determination of Antioxidant Activity with DPPH Method

The total antioxidant activity was determined using the DPPH reagent, according to the Brand-Willams method [86]. Increasing concentrations of ascorbic acid were used to create the standard curve. The antioxidant activity of the standards and ethanol extracts of the samples was determined by measuring the absorbance at 520 nm after adding the DPPH reagent and incubating for 30 min in the dark. The results are expressed as the volume of extract required for 50% IC.

4.5. Determination of Antioxidant Capacity with FRAP Method

The FRAP method determined the antioxidant capacity, according to Keutgen and Pawelzik [87]. The FRAP reagent was added to the ethanol extract of the hemp sample and the reaction mixture was incubated for 4 min at 37 °C, after which the absorbance was measured at a wavelength of 593 nm. Increasing concentrations of FeSO₄ were used to create the base diagram, and the results were expressed as mM FeSO₄ g⁻¹FW.

4.6. Determination of Total Phenols and Flavonoids

Total phenols were determined by the Folin–Ciocalteau method [88] in a reaction mixture consisting of industrial hemp sample extract, distilled water, Folin–Ciocalteau reagent, and Na₂CO₃. After incubation for 60 min at 37 °C, the absorbance at 765 nm was determined. The concentration of total phenols was calculated from the equation of the direction of the Bazdar diagram, obtained by measuring the absorbance of increasing concentrations of gallic acid, and the results were expressed as gallic acid equivalent in mg GAE g⁻¹FW.

The content of flavonoids in the ethanol extract was determined after the addition of AlCl₃ and ethanol (96%), homogenization and incubation for 60 min at room temperature. Absorbances were measured at 415 nm, and the concentration of total flavonoids was calculated from the equation of the direction of the Bazdar diagram, obtained by measuring the absorbance of increasing concentrations of quercetin. The results were expressed as quercetin equivalent in mg QC g⁻¹FW.

4.7. Determination of Total Flavanols and Phenolic Acids

The content of total flavanols was determined from the ethanolic extracts after the addition of dDMACA (p-Dimetihylaminocinnamaaldehyde) reagent and incubation for 10 min. Absorbances were measured at 640 nm, and the concentration of total flavanols was calculated from the equation of the direction of the Bazdar diagram, and was obtained by measuring the absorbance of increasing concentrations of catechins. The results were expressed as catechin equivalent in mg CTH g⁻¹FW [75].

The content of phenolic acids was determined in the reaction mixture containing ethanol extract, water, 0.5 M HCl, Arnow reagent and 1 M NaOH (European Pharmacopoeia, 2004). Absorbances were measured at 490 nm, and the concentration of phenolic acids was calculated from the equation of the direction of the Bajdar diagram, obtained by measuring the absorbance of increasing concentrations of kava acid. The results were expressed as kava acid equivalent in mg CFA g⁻¹FW.

4.8. Data Analysis

The content of chlorophyll, carotenoids, phenols, flavonoids, and the antioxidant activities of DPPH and FRAP were determined spectrophotometrically. The content of chloroplast pigments and antioxidant activity using the DPPH method were measured on a Varian Cary 50 UV-VIS Spectrophotometer with Cary WinUV software (3.00(339)). The content of phenols and flavonoids, flavanols, phenolic acids, and antioxidant activity using the FRAP method were determined on a TECAN microtiter plate reader with SPARK CONTROL software (Spark V 3.1. SP1).