Phytochemical and Functional Diversity of Enzyme-Assisted Extracts from Hippophae rhamnoides L., Aralia cordata Thunb., and Cannabis sativa L.

Plant leaves are a source of essential phenolic compounds, which have numerous health benefits and can be used in multiple applications. While various techniques are available for recovering bioactive compounds from by-products, more data are needed on enzyme-assisted extraction (EAE). The aim of this study was to compare EAE and solid–liquid extraction (SLE), to evaluate the impact on bioactive compounds’ extraction yield, phytochemical composition, and the antioxidant, antimicrobial, and antidiabetic properties of Aralia cordata leaves and roots, sea buckthorn Hippophae rhamnoides, and hemp Cannabis sativa leaves. The results indicate that EAE with Viscozyme L enzyme (EAE_Visc) extracts of the tested plant leaves possess the highest yield, antioxidant activity, and total phenolic content. Moreover, the EAE_Visc extract increased by 40% the total sugar content compared to the control extract of A. cordata root. Interestingly, the sea buckthorn leaf extracts exhibited α-glucosidase inhibitory activity, which reached an almost 99% inhibition in all extracts. Furthermore, the sea buckthorn leaves SLE and EAE_Visc extracts possess antibacterial activity against Staphylococcus aureus. Additionally, scanning electron microscopy was used to examine changes in cell wall morphology after EAE. Overall, this study shows that EAE can be a promising method for increasing the yield and improving the functional properties of the resulting extracts in a fast and sustainable way compared to SLE.


Introduction
Growing interest has been observed in exploring natural sources' phytochemical composition and functional properties in recent years [1].The by-products of agro-industries, such as plant leaves, roots, and stems, are emerging as promising renewable resources rich in cellulose, hemicellulose, and lignin [2].Their complex matrix traps and binds bioactive substances such as phenolic compounds, amino acids, proteins, minerals, and lipids, which offer numerous health benefits and have potential applications in various industries, in particular in the food and food supplement industries [3].Moreover, consumers' awareness of diet and health correlations is rising steadily [4].If prepared appropriately, plant by-products can deliver prebiotic, anti-diabetic, and antioxidant functions.For example, hydrolytic treatment has enabled the extraction of previously non-extractable polyphenols, resulting in higher phenolic recovery in plant-by product extracts [5].However, different plant materials and their morphological parts possess miscellaneous compositions, which can fluctuate positively and negatively, accentuating the need for a fundamental analysis.Furthermore, the rising interest in by-product development is fully aligned with the United Nations Sustainable Development Goals (SDGs)-in particular, SDG No. 12, Responsible Consumption and Production, which aims to cut food loss and waste in food production by up to 50%, and SDG, No. 2, End Hunger, achieving food security and improved nutrition, and promoting sustainable agriculture [6].Following these rationales, investigating the possible functionality of plants is crucial, and sea buckthorn (Hippophae rhamnoides L.), Aralia cordata Thunb.(A. cordata), and industrial hemp (Cannabis sativa L.) are plant species that have gained attention due to their rich phytochemical composition and promising functional properties [7][8][9][10][11].
The sea buckthorn (H.rhamnoides) is a valuable plant known for its rich phytochemical composition in both its berries and leaves.The berries contain essential compounds such as carotenoids, flavonoids, phenolic acids, fatty acids, ascorbic acid, and tocopherols, contributing to their powerful antioxidant, anti-inflammatory, and anti-cancer properties.Additionally, the leaves are rich in flavonols (rutin, Q-3-O-galactoside, I-3-O-glucoside, K-3-O-glucoside, K-3-O-rutinoside, quercetin, and kaempferol), phenolic acids (gallic acid, caffeic acid, p-coumaric acid, ferulic acid) vitamins, proteins, and minerals [12,13], which have been found to have anti-obesity, hypoglycaemic, antioxidant, antibacterial, antiinflammatory, antidiabetic and anti-cardiovascular disease properties [14,15].While the berries are commonly used for their nutritional and medicinal benefits, the leaves, utilised in herbal preparations, also possess significant therapeutic potential.
Meanwhile, A. cordata is a plant that is highly valuable due to its rich content of bioactive compounds such as polyphenols, anthocyanins, carbohydrates, and vitamins [7,16].These compounds have been found to possess various pharmacological properties, including anti-inflammatory, cardiovascular, and nervous system protection, compound metabolism regulation, and antibacterial, antiviral, and antioxidant properties.Moreover, the plant has been traditionally used to treat ailments such as hepatitis and rheumatoid arthritis.In a previous study, A. cordata roots and leaves were found to possess significant antioxidant activity and total phenolic content [7].
Hemp (C.sativa L.) is a widespread plant species of the Cannabaceae family found in different habitats.The interest in hemp has remarkably increased due to specific phytochemicals in its leafy anatomical parts.More than 70 biologically active and unique to Cannabis terpenophenolic compounds, phytocannabinoids, have been found [17].Several research studies showed the health-promoting and medicinal properties of phytocannabinoids.Among them, ∆ 9 -tetrahydrocannabinol (∆ 9 -THC) is a well-known natural psychotropic compound; for this reason, only the approved cultivars of C. sativa accumulating less than 0.2-0.3% of ∆ 9 -THC are officially allowed in Canada, the USA, and many European countries.Hemp leaves are rich in phytochemicals, like cannabinoids, terpenes, phenolic compounds, and their biosynthetic routes.Cannabinoids represent the most studied group of compounds, mainly due to their wide range of pharmaceutical effects in humans, including psychotropic activities [18].
Human health, from a holistic viewpoint, requires a comprehensive approach.A highly balanced and functional nutrition has to deliver a whole spectrum of particular properties such as antioxidant, antidiabetic, and antimicrobial ones, among others, together with the nutritional value of the food.Hence, products with a high nutritional value can be enriched with plant by-products [19].Recently, the antidiabetic activity of extracts has been garnering attention.This activity can also be associated with obesity prevention.More than 650 million adults and 340 million children suffer from obesity, and food is one of the key factors in preventing it [20].For example, the study of berry pomace extracts showed a high α-glucosidase and pancreatic lipase inhibition capacity, which are referred to as antidiabetic properties [21].
In order to isolate bioactive compounds and guarantee product quality, efficacy, and safety, the extraction process is essential in the food and pharmaceutical industries.Conventional extraction methods, such as soxhlet extraction, solid-liquid extraction, and maceration, involve subjecting the raw material to high temperatures with chemical solvents.While this practice enhances the yield of compounds, it concurrently diminishes the quality and degrades valuable compounds, due to prolonged exposure and considerable energy consumption [22,23].Lately, innovative technologies, also known as "green technologies", have been developed for extracting bioactive compounds without using toxic chemicals [24].These eco-friendly techniques are directly related to the environment and consumer health [25].Notable innovative methods for extracting bioactive compounds include supercritical fluid, microwave-assisted, enzyme-assisted, ultrasound-assisted, pressurised liquid, and pulsed electric field techniques [26].Enzyme-assisted extraction has been observed to offer multiple benefits compared to conventional extraction techniques, due to its eco-friendly nature and improved ability to extract specific compounds under gentle processing conditions, shorter extraction times, and reduced solvent, as well as energy, usage [27].Additionally, various factors, including the type and concentration of enzymes, extraction duration, substrate-to-solvent ratio, pH, and temperature, should be considered to obtain extracts with higher yields and improved properties [28].
The aim of this research is to evaluate and compare sea buckthorn leaf, industrial hemp leaf, and A. cordata leaf and root extracts obtained by different methods, namely, enzyme-assisted extraction (EAE) and solid-liquid extraction (SLE).This study covers the selection of the raw material and the preparation, extraction, and evaluation of phytochemical composition to achieve a high yield of biologically active substances and effective antioxidant, antidiabetic, and antimicrobial activities.Figure 1 provides an overview of the overall experimental design for the production and characterization of a total of 14 extracts.

Plant Material
Sea buckthorn (Hippophae rhamnoides L.) and Aralia cordata plants were grown at the Institute of Horticulture, Lithuanian Research Centre of Agriculture and Forestry experimental fields (55 • 08 ′ N, 23 • 80 ′ E).Industrial hemp (Canabis sativa L.) leaves were obtained from a local food factory, "Allive Europe" (Voskoniai, Lithuania).Fresh A. cordata, sea buckthorn, and industrial hemp leaves were harvested in 2021.The collected leaves and roots were instantly frozen at −35 • C before freeze-drying.The samples were lyophilised in Zirbus lyophiliser (Zirbus Technology GmbH, Bad Grund, Germany) at 0.01 mbar pressure and −85 • C condenser temperature.The freeze-dried samples were ground to a powder (particle size 0.2 mm) using a Retsch 200 knife mill (Haan, Germany) and stored in a sealed container before the analysis.

Enzyme Products
Cellulase and Viscozyme L were purchased from Sigma-Aldrich (Steinheim, Germany).Cellulase was produced from Trichoderma reesei and declared to have ≥700 units/g.Viscozyme L is a cellulolytic enzyme complex from Aspergillus aculeatus.Furthermore, the manufacturer states that the enzyme mixture contains a wide range of arabanase, Cellulase, β-glucanase, hemicellulase, and xylanase.The product is declared to have ≥100 FBGU/g.

Enzyme-Assisted Extraction (EAE) and Solid-Liquid Extraction (SLE)
Enzyme-assisted extraction (EAE) was carried out as outlined by Puzeryte et al. under optimal conditions [8].Aralia cordata leaves and roots, sea buckthorn leaves, and industrial hemp leaves were extracted using EAE at a 1:20 (w/v) ratio and mixed to obtain a homogeneous suspension.The suspension pH, 4.9, was adjusted using 6 M HCl and 0.5 M NaOH, and the enzymes Viscozyme L and Cellulase 1% (v/w of dry matter) were added.EAE was carried out at 45 • C in an incubator for 3:15 h.After extraction, the enzyme was deactivated by heating the hydrolysed material at 95 • C for 10 min.Then, the separation of the suspension into liquid and solid fractions was accomplished using a filter (200 mesh).Sample extracts (liquid fraction) were frozen and stored at −35 • C before analysis.Furthermore, the extract to be used as a control was prepared under optimal conditions without enzyme addition.SLE of the tested plant sources was performed in a ratio of 1:20 (w/v) with H 2 O for 24 h in the dark.All the experiments were performed in triplicate.

Protein Content by Lowry Assay
The protein content of the sample extracts was determined using the colorimetric method described by Lowry et al. [29].

Determination of Sugars and Organic Acids
Sugars and organic acid concentrations were determined through high-performance liquid chromatography (HPLC).Briefly, the samples were centrifuged and the supernatants were collected, filtered through 0.22 µm filters, and injected into the HPLC system for analysis.The HPLC system consisted of an Aminex HPX-87H cation exchange column (300 × 7.8 mm) (Bio Rad Laboratories Pty Ltd., CA, USA), coupled to refractive index and ultra-violet detectors.Throughout the analysis, the column was maintained at 40 • C, and the mobile phase utilised was 5 mM H 2 SO 4 at a flow rate of 0.7 mL min −1 .The compounds were identified by the retention time and quantified through the area of the corresponding peak by interpolation of calibration curves determined from the respective standards.

Determination of Total Phenolic Content
The total polyphenol content (TPC) in the extracts was determined according to the Folin-Ciocalteu method [30], using gallic acid (GA) as the standard, according to the method of Bobinaite et al. [31].The reagent was prepared by diluting a stock solution with ultra-pure distilled water (1/10, v/v).The samples (1.0 mL, three replicates) were introduced into test cuvettes, followed by 5.0 mL of Folin-Ciocalteu's phenol reagent and 4.0 mL of Na 2 CO 3 (7.5%).The system was then placed at ambient temperature for 1 h.The absorbance of all the samples was measured at 765 nm using a Cintra 202 ("GBC Scientific Equipment", Knox, Australia) spectrophotometer.The total concentration of phenolic compounds was determined from the calibration curve and expressed in mg of gallic acid equivalents in 100 mL of extract.

Evaluation of Phenolic Compounds in Extracts Using the UHPLC-ESI-MS/MS Technique
The qualitative and quantitative content of phenolic compounds in the tested A. cordata leaf and root, sea buckthorn leaf, and industrial hemp leaf samples extract were evaluated using ultra-high-performance liquid chromatography (UHPLC) coupled to a mass spectrometer.The method employed was described and validated in an article by Gonzalez-Burgos et al. [32].The phenolic content of the sample extract was analysed using a liquid chromatography system, "Waters ACQUITY UPLC ® H-Class", with a tandem quadrupole mass detector, "Xevo TQD" (Waters, Milford, MA, USA).The compounds were separated using a "YMC Triart C18" (100 Å, 100 × 2.0 mm; particle size 1.9 µm) with a pre-column.Both qualitative and quantitative analyses were performed.The mass spectrometry parameters for the phenolic compound analysis are presented in Table 1.

Determination of Antioxidant Activity
An ABTS •+ radical cation decolourization assay was applied according to the methodology described by Re et al. [33].An amount of 2 mL of ABTS •+ (2,2 ′ -azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)) solution, with an absorbance of 0.800 ± 0.02, was mixed with 20 µL of the samples.After 30 min, the absorbance of each sample was measured at 734 nm using a Cintra 202 spectrophotometer (GBC Scientific Equipment, Knox, Braeside, VIC, Australia) to determine the decrease in absorbance.
A ferric ion reducing antioxidant power (FRAP) assay was conducted following the method described by Benzie and Strain [34], with some modifications.The FRAP solution was made by combining TPTZ (0.01 M dissolved in 0.04 M HCl), FeCl 3 × 6H 2 O (0.02 M in water), and acetate buffer (0.3 M, pH 3.6) at a ratio of 1:1:10.A quantity of 2 mL of the freshly prepared FRAP reagent was mixed with 20 µL of the samples.After 30 min, the absorbance increase was determined at 593 nm using a Cintra 202 (GBC Scientific Equipment, Knox, Australia) spectrophotometer.
All antioxidant activity assays were calculated using Trolox calibration curves and expressed as the µmol of the Trolox equivalent (TE) per one millilitre of extract (µmol TE/mL of extract).

Oxygen Radical Absorbance Capacity Assay (ORAC)
The ORAC assay was conducted following the procedure established by Dávalos, Gómez-Cordovés, and Bartolomé [35] with some modifications.Firstly, a phosphate buffer (75 mM, pH 7.4) was used, and 20 µL of the sample (after dilution), along with 120 µL of fluorescein (1166.1 µM), was added to a black microplate (Nunc, Denmark).The mixture was pre-incubated at 37 • C for 15 min.After that, 60 µL of 2,2 ′ -azobis-(2-methylpropionamidine)-dihydrochloride (AAPH) (46.6 mM) was added rapidly and then incubated at 37 • C for 140 min.The microplate was read at 1 min intervals using a Multidetection plate reader (Synergy H1, Vermont, USA) at 458 nm and 528 nm.The software used was the Fluostar Control 1.32 R2 version.The calibration curve was made using Trolox (0.0002 to 0.0016 µmol TE/mL) as an antioxidant standard, and the results were expressed in mmol TE/mL of extract.A blank was prepared by using the phosphate buffer instead of a sample.All analyses were carried out in triplicate.
Normalised antioxidant curves were obtained by multiplying the original data by fluorescence blank,t=0 /fluorescence sample,t=0 , and dividing by the blank curve corresponding to the same assay.

α-Glucosidase Inhibition Assay
The α-glucosidase inhibitory activity was measured to assess antidiabetic potential according to the procedure described by Kwon et al. [36], with slight modifications.To begin, 50 µL of the sample was mixed with 100 µL of 0.1 M phosphate buffer (pH = 6.9) containing α-glucosidase solution (1.0 U/mL) in each well.The mixture was then incubated at 25 • C for 10 min.Afterwards, 50 µL of 5 mM p-nitrophenyl-a-D-glucopyranoside solution in 0.1 M phosphate buffer (pH = 6.9) was added to each well.Subsequently, the absorbance measurements of the reaction mixtures were taken at 405 nm using a multi-detection plate reader (Synergy H1, VT, USA), after a further 5 min of incubation at 25 • C.
For the experiment, 50 µL of buffer solution was used as the negative control, and 50 µL of acarbose at a concentration of 10 mg/mL was used as the positive control.All assays were performed in triplicate.The inhibition of α-glucosidase was calculated as follows:

Antibacterial Analysis
In order to determine the antimicrobial activity of A. cordata leaves and roots, as well as the leaves of sea buckthorn and industrial hemp extracts (SLE and EAE_Visc), a well diffusion test method was used based on Kaewchomphunuch et al. [37] with minor modifications.A wide range of pathogenic bacteria were tested, which are listed in Table 2.Each microorganism was grown on Tryptic Soy Agar plates (TSA) at 37 • C for 24 h prior to analysis.The extracts were freeze-dried and then resuspended in 2 mL of water before testing.Agar well diffusion assays were performed in 90 mm diameter Petri dishes containing Mueller Hinton Agar to a depth of 4 ± 0.5 mm.Bacterial suspensions were obtained with a cell concentration of 1 McFarland (around 3 × 10 8 CFU/mL) in saline solution (0.9% NaCl, Honeywell, Fluka, Cambridge, UK).This suspension was spread uniformly using a sterile cotton swab.Six wells with a 5 mm diameter were used in the agar plates.Then, 50 µL of the test extracts and 10 µL of the positive control, ampicillin, at a concentration of 100 µg/mL was placed into each well using a sterile pipette.The plates were then incubated for 24 h at 37 • C.After incubation, the diameters of the growth inhibition zones were measured in millimetres to the nearest 0.1 mm.Each experiment was repeated three times, and the means and standard deviations were calculated.

Scanning Electron Microscopy (SEM)
This study examined the structure and changes in the samples that were hydrolysed by the Viscozyme L enzyme, using scanning electron microscopy (SEM).The samples, both before and after EAE_Visc, were freeze-dried and mounted on SEM pins using doublesided adhesive carbon tape (NEM tape; Nisshin, Tokyo, Japan).They were then coated with gold/palladium and visualised in a PhenomTM XL G2 (Thermo Fisher Scientific, Eindhoven, The Netherlands) SEM.These analyses were conducted at an accelerating voltage of 5 kV using the secondary electron detector (SED).

Statistical Analysis
All analyses were performed in triplicate.MS Excel 2020 (Redmond, WA, USA) calculated the mean values and standard deviations.Afterwards, a one-way ANOVA was performed, followed by Tukey's HSD test to compare the means that showed significant variation (p < 0.05).These calculations were performed using GraphPad Prism 8.0 software (GraphPad, San Diego, CA, USA).

Extraction Yield and Concentration
The extraction yields and concentrations of A. cordata leaf and root, sea buckthorn leaf, and industrial hemp leaf extracts obtained by solid-liquid extraction (SLE) and enzymeassisted extraction (EAE) with Viscozyme L (Visc), Cellulase (Cell), and without an enzyme (Control) are presented in Table 3.It can be seen that the extraction methods, as well as the tested plant by-products sources, had significant differences in extraction yield (p < 0.05).The extraction of compounds from plant by-products can be challenging due to the complex and diverse polysaccharides present in their cell walls, which can reduce extraction efficiency using classical methods [38].Degrading the structure of these cell walls is essential to release the compounds.Hydrolytic enzymes were found to increase the extraction yield in plant leaves [39], and this effect was confirmed in the study conducted on all three plant by-products.Regarding the extraction methods and plant by-products, the highest yield was obtained in EAE_Visc extracts in the following order: A. cordata leaves, roots, sea buckthorn leaves, and industrial hemp leaves, respectively.
Meanwhile, there was no significant difference in yield between SLE, EAE_Control, and EAE_Cell in A. cordata and industrial hemp leaves (p > 0.05, Table 3).However, the highest yield was achieved with EAE_Visc extracts in A. cordata leaves, which were 46%, 41%, and 57% higher compared to the SLE, EAE_Control, and EAE_Cell extracts, respectively.In the case of A. cordata roots, EAE_Visc extracts delivered a yield 44% higher than EAE_Control extracts.Similarly, the highest yield was obtained in EAE_Visc extracts of industrial hemp leaves and sea buckthorn leaves, which delivered a yield 25%, 44%, and 30% higher in industrial hemp leaves and 46%, 48%, and 77% higher in sea buckthorn leaves compared with the SLE, EAE_Control, and EAE_Cell extracts, respectively.In terms of concentration, a similar correlation can be observed (Table 3).To the best of our knowledge, no previous studies have performed an extraction comparison including the EAE of functional properties from A. cordata leaves and roots, sea buckthorn leaves, and industrial hemp leaves.The lack of research on this topic limits the ability to make further comparisons beyond what has been achieved in this study.Generally, these results are compatible with previous reports, indicating that EAE significantly increases the soluble yield in a shorter time frame [40,41].

Characterisation of the Extracts
The composition of total protein, fructose, glucose, sucrose, total sugars, and total phenolics content in different extracts of A. cordata leaves and roots, sea buckthorn leaves, and industrial hemp leaves are presented in Table 4.As expected, a higher total protein content was obtained in all tested plant EAE extracts with Viscozyme L and Cellulase enzymes (p < 0.05).At the same time, the total protein content did not show a significant difference in the root extract of A. cordata in comparison to the other tested plants.Meanwhile, the highest protein content was determined in industrial hemp EAE_Visc extract (11.00 mg/mL of extract), which is almost 2 times higher than EAE_Cell extract and 1.7 times higher than what was obtained in SLE.The total protein content was increasingly higher in sea buckthorn leaves compared with A. cordata leaves and roots and industrial hemp leaves.The highest total protein content was detected in sea buckthorn leaves EAE_Cell extract (24.23 mg/mL of extract), 6% higher than in EAE_Visc extract and 30.7% higher than what was obtained in SLE extract.Previous studies have reported that the protein content ranged from 14.90% to 18.60% in sea buckthorn leaf water extract [42], while Ghabru et al. (2023) reported a total protein content of 22.09% in sea buckthorn leaves [43].Plant cell walls consist of glycoproteins, oligosaccharides, polysaccharides, and other complex carbohydrates that challenge protein extraction [44].Therefore, enzyme-based technologies allow an increased protein content from plants by degrading the complex carbohydrate-rich plant cell wall.The selection of enzymes is crucial in this process [44,45].
To determine the impact of Viscozyme L, Cellulase, and naturally occurring enzymes on the release and content of simple carbohydrates, the concentrations of mono-and disaccharides in the EAE and SLE extracts were measured using HPLC under optimal conditions.There were no significant differences in total sugar content between the A. cordata leaf extracts (p > 0.05).Meanwhile, the EAE_Visc extract (10.74 mg/mL of extract) demonstrated a 40% increase in total sugar content compared to the EAE_Control extract (7.77 mg/mL of extract) extracted from A. cordata roots.The EAE_Visc (5.05 mg/mL of extract) and EAE_Cell (4.06 mg/mL of extract) extracts of sea buckthorn leaves were shown to have a higher total sugar content, while in the industrial hemp leaves, the EAE_Visc (3.71 mg/mL of extract) and EAE_Control (3.58 mg/mL of extract) showed a higher amount of total sugar content (p < 0.05).As expected, the enzyme's hydrolytic activity significantly increased the saccharide content, particularly glucose and fructose (Table 4).In this case, the results indicate that there was no significant difference between the A. cordata leaf extracts, correlating with observations concerning the total sugar content.However, the A. cordata root EAE_Visc extract demonstrated a significant increase in glucose and fructose content by 3.8 and 2 times, respectively.Furthermore, a significant difference in glucose value was observed in the EAE_Visc extracts of sea buckthorn and industrial hemp leaves.The sea buckthorn leaves did not show an increase in fructose content, whereas the industrial hemp leaves showed an increase in fructose content in both the EAE_Visc and EAE_Control extracts (p < 0.05).The total phenolic content varied significantly based on the extraction methods and plant sources (p < 0.05).Sea buckthorn extracts had the highest total phenolic content, ranging from 200.80 to 285.57mg/100 mL of extract (Table 4).EAE_Visc extract had significantly the highest value (285.57mg/100 mL of extract), which was 85%, 75%, and 46% higher compared with the sea buckthorn leaf EAE_Control and EAE_Cell extracts, respectively.A similar tendency was established among the A. cordata and industrial hemp leaf extraction methods.Meanwhile, there was no significant difference between the A. cordata root extracts.Phenolic compounds in plants exist in soluble and insoluble forms.Soluble phenolics can be extracted easily, while insoluble-bound phenolic compounds are challenging to extract.Insoluble-bound phenolic compounds are covalently bound to plant cell wall structural elements such as cellulose, hemicellulose, structural protein, or polysaccharides [46].The present study confirmed that using the Viscozyme L cellulolytic enzyme complex resulted in a higher total phenolic content compared to the SLE, EAE_Control, and EAE_Cell methods due to the release of insoluble-bound compounds.The results are consistent with Wang et al. (2017), who conducted a study on enhancing the bioavailability of insoluble-bound phenolics from guava leaves.The study investigated the ability of enzyme-assisted extraction to improve the release of insoluble-bound phenolics and found that complex enzyme-assisted extraction greatly improved the soluble phenolics content [47].In a study conducted by Habeebullah et al. (2020), the Flavourzyme and Viscozyme L complexes were found to be the most effective enzymes in increasing the total phenolic content of brown seaweed varieties [48].

Quantitative Composition of Phenolic Compounds of Extracts
Phenolic compounds in A. cordata leaf and root, sea buckthorn leaf, and industrial hemp leaf extracts were identified and quantified by UHPLC-ESI-MS/MS analysis, as presented in Tables 5-7.As shown in Table 5, four flavonols and six phenolic acids were identified in A. cordata leaf and root extracts.Chlorogenic, p-coumaric, and 3,4-dihydroxyphenylacetic acids were found in all tested extracts of A. cordata leaves, and neochlorogenic acid was only present in the EAE_Visc extract (Table 5).Neochlorogenic, chlorogenic, ferulic, caffeic, pcoumaric, and 3,4-dihydroxyphenylacetic acids were found in all tested extracts of A. cordata root.The amount of identified phenolic acids varied significantly (p < 0.05).Chlorogenic acid was found to be most abundant in EAE_Visc extract, with the highest amounts of 95.53 µg/mL of extract and 160.23 µg/mL of extract present in A. cordata leaves and roots, respectively.In addition, Matsuo et al. [49] also reported studies that A. cordata roots have a high concentration of chlorogenic acid, which is essential in regulating glucose and lipid metabolism and managing related disorders such as diabetes, obesity, cardiovascular disease, and cancer [50,51].
Regarding the group of flavones, they were only present in A. cordata leaves (isoquercitrin, isorhamnetin, isorhamnetin-3-O-glucoside, and keampferol-3-O-glucoside) (Table 5).Only isorhamnetin was obtained in all extracts, and the amount did not vary significantly.The two most abundant flavonols found in A. cordata leaf EAE_Visc extract were isoquercitrin (9.99 µg/mL of extract) and kaempferol-3-O-glucoside (8.46 µg/mL of extract).Studies have reported that isoquercitrin can be obtained by the enzymatic hydrolysis of rutin, which, due to its antioxidant activity, scavenges ROS and RNS, including superoxide anion radicals, hydroxyl radicals, peroxyl radicals, and peroxynitrite [52].In addition, it was reported that kaempferol 3-O-glucoside had a hepatoprotective effect on tacrine-induced cytotoxicity in HepG2 cells derived from the human liver [53].
As demonstrated in Table 7, eight phenolic compounds, including four phenolic acids and four flavonols, were identified in industrial hemp leaves.Among these, neochlorogenic, chlorogenic, p-coumaric, and 3,4-dihydroxyphenylacetic acids were found only in the EAE_visc extracts of industrial hemp leaves.Additionally, only p-coumaric acid was found in all of the extracts.The amount of identified phenolic acids varied significantly (p < 0.05).The most abundant phenolic acid in industrial hemp leaf EAE_Visc extract was chlorogenic acid (91.19 µg/mL of extract), which is similar to that found in A. cordata leaves (95.53 µg/mL of extract).However, three flavonols, including isoquercitrin, isorhamnetin-3-O-glucoside, and kaempferol-3-O-glucoside, were obtained in EAE_Visc, while vitexin-2rhamnoside was established in the SLE, EAE_Control, and EAE_Cell extracts of industrial hemp leaves.

Determination of Antioxidant Activity
Plant extracts' effectiveness as antioxidants depends on their phytochemical composition and bioactive compounds-specifically, the content of phenolic compounds such as flavonoids and phenolic acids [23].Antioxidants have shown promising results in reducing or halting the progression of various chronic diseases [58].Plant extracts comprise various biologically active compounds, each possessing its own mechanism of antioxidant activity.This makes it difficult to assess their antioxidant capacity accurately using only one methodology [59].Multiple methods are recommended to measure the antioxidant capacity of plant extracts accurately [60,61].Different antioxidant assays (ABTS •+ , FRAP, and ORAC) were used to evaluate the antioxidant capacity of different sample extracts.The results of antioxidant activity in the tested samples are presented in Table 8.The EAE_Visc extracts of the tested plant leaves showed stronger antioxidant activity in all the methods used, whereas no significant differences were found between A. cordata root extracts (p > 0.05).On the other hand, the EAE_Visc extract of A. cordata leaves showed 1.5 and 1.2 times higher antioxidant capacity in ABTS •+ compared to the SLE, EAE_Cell, and EAE_Control extracts, respectively.The ABTS •+ antioxidant activity in vitro of industrial hemp extracts varied similarly (p < 0.05).Meanwhile, the EAE_Visc extract (46.11 µmol TE/mL of extract) of sea buckthorn leaves was reported to have the highest antioxidant capacity in ABTS •+ compared with the other investigated plants.For the ferric reducing antioxidant power (FRAP), the EAE_Visc extracts were capable of a 46-50% and 18-62% increase in scavenging activity in A. cordata and sea buckthorn leaves, respectively (Table 8).Meanwhile, the FRAP-reducing activity in vitro in the EAE_Visc extracts of industrial hemp leaves was 1.6-4.2times higher than in the other tested extracts.Furthermore, the antioxidant capacities obtained with the oxygen radical absorbance capacity (ORAC) assay showed a similar correlation with the ABTS •+ and FRAP in the tested extracts.However, the tested extracts obtained higher values when measured by the ORAC method.This can be explained by the difference in the working mechanisms, where the ORAC method uses the hydrogen atom transfer radical-quenching mechanism of peroxyl radicals [62].

Antidiabetic Properties
Type 2 diabetes is a significant public health concern with a growing burden worldwide [63].It affects quality of life and contributes to substantial morbidity and mortality, especially in younger people [64].Unhealthy lifestyles and an aging population are the leading causes [65].Additionally, Chen et al. (2020) [66] and Amaliah et al. (2019) [67] in their studies demonstrated the blood sugar-lowering effects of Moringa oleifera and Siam weed leaves, respectively.The obtained antidiabetic activity of different extracts of A. cordata leaves and roots, sea buckthorn leaves, and industrial hemp leaves are presented in Figure 2. The inhibitory activity of α-glucosidase in extracts from the leaves and roots of A. cordata varied between 41.61% to 45.50% and 30.87% to 55.78%, respectively.The extraction method used for A. cordata leaves did not show any significant differences.However, there were significant differences between the extraction methods used for A. cordata roots (p < 0.05).The EAE_Visc extract in A. cordata roots showed a 25% higher α-glucosidase inhibitory activity compared to the EAE_Control extract.On the other hand, the αglucosidase inhibitory activity in different industrial hemp extracts ranged from 28.8% to 47.1%.Statistical differences were observed between the SLE and EAE_Control extracts, and the EAE_Visc and EAE_Cell extracts, which indicates that enzyme-assisted extraction has a positive effect on increasing α-glucosidase inhibitory activity in industrial hemp leaves (p < 0.05, Figure 2).However, no significant difference between sea buckthorn leaf extracts was indicated.Nevertheless, the highest α-glucosidase inhibitory activity was obtained in sea buckthorn leaves, almost reaching 99% in all extracts (Figure 2).These results are consistent with previous studies demonstrating sea buckthorn leaves' significant antidiabetic potential [14].Research by Bhardwaj et al. (2015) [68] has shown that the methanolic extract of these leaves possesses a potent inhibitory action against α-glucosidase-a crucial enzyme in carbohydrate metabolism.This was further supported by Sharma et al. (2011) [69], who found that the leaves can reduce blood glucose levels and oxidative stress in diabetic rats.These findings collectively suggest that sea buckthorn leaves could be a natural antidiabetic agent.

Antibacterial Properties
It was previously reported that sea buckthorn leaves possess antimicrobial activity against various pathogens and can be promising antimicrobial alternatives in the food, pharmacy, and cosmetic industries [70].In this study, the antimicrobial activity of A. cordata leaf and root, sea buckthorn leaf, and industrial hemp leaf SLE and EAE_Visc extracts were investigated against seven pathogens: Streptococcus intermedius, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Salmonella enterica, Yersinia enterocolitica, and Listeria monocytogenes.However, A. cordata leaves and roots, as well as the industrial hemp leaf tested extracts, did not show inhibition effects against all the tested pathogenic bacteria.Therefore, these results are not presented in Table 9.On the other hand, the water-based SLE and EAE_Visc sea buckthorn extracts showed positive effects against Gram-positive Staphylococcus aureus (S. aureus) bacteria, as presented in Table 9.  aureus, but it also highlights that the effectiveness of the inhibition may vary depending on the type of plant species [11].Different extraction methods and solvents can result in variations in the types and quantities of compounds extracted, thereby influencing their antimicrobial activity.Overall, these studies demonstrate that sea buckthorn leaves can be a valuable natural source of antibacterial agents.

Scanning Electron Microscopy Analysis of Plant Material before and after EAE
The impact of EAE on cell wall degradation was assessed using scanning electron microscopy.Figure 3 depicts the microstructure of the plant material before and after treatment with the Viscozyme L enzyme.The control sample (Figure 3a,c,e,g) showed a smooth surface without any ruptures or significant disruption to the microstructure.However, after using Viscozyme L, a considerable amount of tissue fragments were noticed (see Figure 3b,d,f,h) that were coated with various small particles.Furthermore, partial exfoliation and changes in morphology were detected on the cell surface.After hydrolysis, the surface appeared rough and uneven and was more susceptible to destruction.In Figure 3b,d,f,h, the process of hydrolysis and the resulting changes in the shape of individual particles can be observed.Based on these observations, the alterations in morphology are a result of the hydrolysis process.Our prior research on sea buckthorn leaves had revealed consistent morphological alterations in cell walls exposed after EAE with the Viscozyme L enzyme [8].The structural support and protection of the plant cell wall depends on its complex composition that prevents the release of intracellular components [71,72].It is possible for bioactive compounds to exist in both bound and free states.The extraction of these compounds can be significantly improved by breaking down the cell wall through enzymatic degradation [73].This process results in a significantly higher yield of these compounds and enhances their antioxidant activity [74,75].This fact is supported by the changes in morphology that can be observed (Figure 3).Accordingly, the prior research has revealed that enzymatic hydrolysis results in discernible modifications to cell wall morphology [76,77].

Conclusions
This study has shown that the leaves and roots of A. cordata, and sea buckthorn and industrial hemp leaves, can be a natural and cost-effective source of bioactive compounds.This might be also extendable to other agro-industrial by-products, with various potential applications in different industries.Enzyme-assisted extraction is proposed as a promising method for obtaining extracts with engaging biological properties, especially using the

Figure 1 .
Figure 1.Overview of the experimental design.

Figure 2 .
Figure 2. α-glucosidase inhibitory activity of A. cordata leaves and roots, sea buckthorn leaves, and industrial hemp leaves for all analysed conditions.Values are expressed as mean ± standard deviation (n = 3); different letters indicate statistically significant differences between extraction methods (one-way ANOVA and Tukey's HSD test, p < 0.05).
indicates no detected inhibition zones for tested concentration.Values are expressed as mean ± standard deviation (n = 3); different letters indicate statistically significant differences between extraction methods (one-way ANOVA and Tukey's HSD test, p < 0.05).Upadhyay et al. reported a study on the antibacterial activity of sea buckthorn leaf water and 70% ethanol extracts and found that, depending on the concentration, waterbased extracts can positively inhibit S. aureus and E. coli pathogens [70].Another study conducted by Criste et al. confirms the antibacterial activity of sea buckthorn leaves against S.

Table 1 .
Parameters of mass spectrometry for analysis of phenolic compounds.

Table 2 .
Targeted microorganisms and their sources used for antimicrobial analysis.

Table 3 .
Extraction yields and concentrations of tested sample extracts.
Values are expressed as mean ± standard deviation (n = 3); different letters in columns indicate statistically significant differences between extraction methods (one-way ANOVA and Tukey's HSD test, p < 0.05).

Table 4 .
Contents of protein, sucrose, glucose, fructose, total sugars, and total phenolics in the different extracts of A. cordata leaves and roots, sea buckthorn leaves, and industrial hemp leaves.
Values are expressed as mean ± standard deviation (n = 3); different letters in columns indicate statistically significant differences between extraction methods (one-way ANOVA and Tukey's HSD test, p < 0.05).

Table 5 .
Nature and content of phenolic compounds (µg/mL of extract) in A. cordata leaves and root extracts.
nd: not detected.Values are expressed as mean ± standard deviation (n = 3); different letters in rows indicate statistically significant differences between extraction methods (one-way ANOVA and Tukey's HSD test, p < 0.05).

Table 6 .
Content of phenolic compounds (µg/mL of extract) in sea buckthorn leaves extracts.Values are expressed as mean ± standard deviation (n = 3); different letters in rows indicate statistically significant differences between extraction methods (one-way ANOVA and Tukey's HSD test, p < 0.05).

Table 7 .
Content of phenolic compounds (µg/mL of extract) in industrial leaves extracts.

mL of Extract) Industrial Hemp Leaves SLE EAE_Control EAE_Visc EAE_Cell Phenolic acids
Values are expressed as mean ± standard deviation (n = 3); different letters in rows indicate statistically significant differences between extraction methods (one-way ANOVA and Tukey's HSD test, p < 0.05).

Table 8 .
Analysis of antioxidant activity in different extracts of A. cordata leaves and roots, sea buckthorn leaves, and industrial hemp leaves using ABTS •+ , FRAP, and ORAC assays.

Table 9 .
Antibacterial activity determination of sea buckthorn leaf extract.