Water Extracts from Industrial Hemp Waste Inhibit the Adhesion and Development of Candida Biofilm and Showed Antioxidant Activity on HT-29 Colon Cancer Cells

2.2. HWEs Chemical NMR Characterization

All samples were prepared from lyophilized powder and dissolved in D₂O. A comparison of the spectra recorded in D₂O using the ZG pulse program (Figure S1) and the AU_WATERSC pulse program (Figure S2) clearly demonstrated that the latter allows for a clearer interpretation of the spectral region close to 4.8 ppm without any evident perturbation of the nearby signals. Hence, for the subsequent discussion of results, only the data obtained under water suppression conditions will be taken into consideration and some assignments were attempted by the comparison with the resonance data reported in the literature and in some public databases.

In order to investigate the presence of cannabinoids (that are not soluble in water), a defined volume of CDCl₃ was added to the previously prepared sample, and the proton NMR spectra were recorded also in this solvent. Data collected in Figure S3 shows that even in an organic solution, the presence of cannabinoids is not detectable by NMR, as expected.

The five HWEs that were characterized underwent three different extraction methods: S2 and 8 were subjected to mechanical extraction, followed by microwave treatment, S14 to mechanical extraction and protein precipitation, and S9 and S21 were obtained only by long maceration and mechanical extraction. In the region from 4,5 ppm to 9,5 ppm (Figure 1), the samples treated with microwave energy (MWE) (S2 and S8) are unique because beta-glucose (11) [34] is present, probably arising from MW-assisted hydrolysis of sucrose (12) [34], which is present in S9 and slightly in S21 because the extracts were subjected only to microbial degradation. Sucrose is also present in extract S14, which underwent mechanical extraction and protein precipitation.

Among these two groups of samples, there is also another difference in the aromatic region. The MWE-treated group shows the presence of not fully identified benzoyl derivatives (14) (e.g., benzoyl amides) [34], while in the group treated with microbial degradation, there is no evidence of benzoic acid and its derivatives; but other different aromatic compounds can be identified at lower chemical shifts. In HWE S2 is evident the presence of benzoic acid (13) [36], which is not present in HWE S8 which belongs to the same group of treatment but differs for the raw material used.

The presence of trigonelline (15) is noticeable in S2, S9, and S14, while formic acid (16) is present in S2, S9, S14, and S21 (while in S8 it was only present in traces).

At low-field frequencies, S14, the only HWE treated with protein precipitation, shows a profile similar to that observed for S9 (Figure 2). In the spectral region between 0 ppm to 4.5 ppm, high-field resonances can detect the same clusters of similarities/differences. S2, S9, and S21 (but not S8 and S14) showed coupling patterns associable to choline (9). In S9, S21, S8, and S14 are evident signals that can be assigned to valine (2), isoleucine (3), leucine (4), and alanine (6).

S14 was the only one that showed a minor content of malic acid (8) and lactate (5), while GABA (7) was observed only in S9 and S14. A non-clear distribution was observed for glycerol/glycerol derivatives (10) (detected in samples S8 and S14), while fatty acids (1) [37] were detected in all the samples.

2.3. Antifungal Susceptibility Testing

The impact of all HWEs on the growth of eight strains of C. albicans, C. parapsilosis, and C. tropicalis (CA1, CA2, CT1, CT2, CT3, CP1, CP2, and CP3) was investigated across a range of concentrations from 2 mg mL⁻¹ to 0.0035 mg mL⁻¹. The results, expressed as the percentage change in growth compared to the control, are summarized in Figure 3 and Supplementary Table S1.

The addition of the HWEs stimulated the growth of the eight strains tested with a dose-dependent response. Overall, the lower concentration boosted the growth of planktonic cells to a lesser extent than their higher counterpart.

Extracts S2, S8 (from short-term macerates, Figure 3a,b), and S9 (from one month macerate, Figure 3e) mostly exhibited their enhancing effects on both strains of C. albicans, as well as on strain C. parapsilosis (CP3) (S8 and S9). Conversely, S21 (from two months macerate (Figure 3d) consistently enhanced the growth of all strains, reaching its peak influence on strain CP3 (C. parapsilosis), where a 413.6% increase was observed at a 2 mg mL⁻¹ concentration.

Interestingly, only S14 (Figure 3c) exhibited a significant inhibitory effect on the growth of the CA1 C. albicans strain across all tested concentrations, ranging from −5% at 0.007 mg mL⁻¹ to −37% at 2 mg mL⁻¹. Also, the C. tropicalis (CT3) strain was inhibited at low concentrations of this extract (ranging from 0.0035 to 0.032 mg mL⁻¹), while the C. parapsilosis strain (CP2) displayed reduced growth across all concentrations except those within the range of 0.065 to 0.25 mg mL⁻¹. Finally, an inverse growth trend was observed for strains CA2 and CT1, reaching their highest increase at 0.25 mg mL⁻¹ and decreasing to a minimum at the highest concentration of 2 mg mL⁻¹.

2.4. Influence of HWEs on Biofilm Adhesion and Biofilm Development

All strains employed to evaluate the impact of the HWEs on planktonic growth were able to form biofilm in vitro [45]. The HWEs were tested for their potential to influence both biofilm adhesion and biofilm development (Supplementary Tables S2 and S3, respectively) at the same concentrations tested for the assays of planktonic growth.

The efficacy of S14 HWE in inhibiting biofilm adhesion and formation was comparatively minimal (Figure 4). The strains exhibited a limited correlation between the biofilm produced when the extract was applied during the adhesion phase and when it was exclusively used in the biofilm development phase. Specifically, when S14 was employed during the adhesion phase of biofilm formation, it demonstrated a modest negative impact on the biofilm developed after 24 h (CA1, CA2, CT1, and CT2, Figure 4a,c) predominantly at lower concentrations (0.0039–0.125 mg mL⁻¹). When S14 was exclusively applied during the biofilm development phase, it showed a negative impact, reaching a maximum value of –20% and particularly affecting two strains (CA2 and CT1, Figure 4b,d) across almost all tested concentrations with varying degrees of decrease.

We examined the impact of HWE S14 on the growth of the Candida strains’ biofilm at ten different concentrations, comparing it to the control. Notably, the HWE was administered exclusively during either the priming phase of biofilm development or the subsequent development phase. HWEs S2 and S8, obtained using the same extraction methods but from distinct raw materials (dry and fresh plants), exhibited varying effects on biofilm adhesion and development (Figure 5).

Interestingly, both HWEs demonstrated more favourable outcomes when applied during the adhesion phase rather than the development phase (Figure 5a,e,j for S2 and Figure 5c,g,k for S8). When applied exclusively during the adhesion phase, HWE S8 exhibited a reduction in the quantity of biofilm produced by five strains (CA2, CT3, CP1, CP2, and CP3, Figure 5c,g,k), showing varying degrees of decrease. The observed effects were consistently influenced by concentration, ranging from an average of −2% at 0.003 mg mL⁻¹ to an average of −37% at 2 mg mL⁻¹. The remaining three strains were either unaffected by the HWE (CT1) or experienced negative effects but in a non-concentration-dependent manner. When administered during the biofilm development phase, extract S8 exhibited a considerably milder effect at low concentrations, demonstrating a boosting effect on biofilm development instead (Figure 5d,h,i). Notably, only one strain, CT2, experienced consistent inhibition across all concentrations, while the others showed a consistent decrease in biofilm formation from 0.25 to 2 mg mL⁻¹. The impact of extract S2 on biofilm adhesion was notably less pronounced compared to extract S8 as it strongly inhibited the adhesion of only two strains (CT3 and CA2). The remaining strains were either minimally affected (CP1 and CA1) or experienced a boost. Strain CA2 was the only one influenced by the application of S2 during the biofilm development phase, with the other strains experiencing a concentration-dependent enhancement (Figure 5d). Conversely from the previously mentioned HWEs, S9 and S21 (derived from long maceration and mechanical extraction only) exhibited a more pronounced average inhibition when applied during the biofilm development phase rather than the biofilm adhesion phase. Both HWEs demonstrated a distinct consistent overall trend when considering all the strains together. Specifically, biofilm formation was inhibited by S9 (Figure 6b,d,f), averaging at −10% from 0.003 to 0.03 mg mL⁻¹, with a decreasing impact that reached its minimum at 0.25 mg mL-1 and increasing again from 0.5 to 2 mg mL⁻¹.

A comparable effect was observed with HWE S21 (Figure 7b,d,f), displaying a similar trend of inhibiting biofilm formation when applied during the biofilm development phase. When applied during biofilm adhesion, HWE S9 (Figure 7) inhibited cell adhesion for all strains but CP3, CP2, and CA1, while HWE S21 exerted a less coherent action on the strains used.

The reduction of cell adhesion was null for strains CT1 and CT2 which were boosted at higher concentrations. On the other hand, strains CA2, CT3, CP1, and CP2 were inhibited, resulting in a negative impact on the biofilm adhesion phase with varying degrees of reduction in a concentration-dependent manner. Finally, CT2 was boosted at the highest concentration tested, and CP3 was boosted at the central concentrations and inhibited at 2 mg mL⁻¹.

4.2. Extraction Procedure

Wastes were gently crushed into a small size to facilitate the extraction procedure. To evaluate whether, and to what extent, the time of the maceration process influences the chemical, physical, and biological properties of the Hemp Water Extracts (HWEs) obtained, two different protocols (short- and long-term maceration processes) were optimized.

4.3. Hemp Water Extracts (HWEs)

At the end of the short- and long-term extraction processes, five different HWEs were obtained, as detailed in Table 1.

4.4. HWEs Chemo-Physical Characterization

4.5. Antimicrobial Activity Assay

With the purpose of evaluating the potential cytotoxic activity against human pathogen Candida, the HWEs were used for an antifungal susceptibility test where the HWEs were tested at ten different concentrations starting from 2mg mL⁻¹ until 0.0325 mg mL⁻¹. The test was performed following the guidelines of EUCAST E.Def 7.3.2 [62]. All the HWEs were prepared at the stock concentration of 40 mg mL⁻¹ using sterile deionized sterile water. The eight Candida strains that were used in the tests (Table 3) were isolated from clinical samples of different hospital structures located in Italy and conserved in a dedicated collection of the laboratory at −80 °C.

Before testing, each strain was cultured in agar plates of YAPD + Chloramphenicol at the concentration of 0.5 g/L and was left incubated at 37 °C for two days. At the end of incubation, one colony from each agar plate was used for pre-inoculum in 10 mL of the same liquid medium (YAPD + Chloramphenicol) and incubated overnight at 37 °C under shaking conditions (120 rpm). At the end of the growing period, the optical density was measured at 600 nm using a spectrophotometer instrument (Jasco V-730, Spectrophotometer, Jasco Europe Srl, Lecco, Italy), and the OD₆₀₀ was standardized at the value of 0.2, two-fold the OD value used for the test. The cells were then resuspended in a liquid RPMI 1640 liquid medium (Sigma-Aldrich, St. Louis, MO, USA) added with glucose and MOPS (Sigma-Aldrich) at the concentrations of 36 g L⁻¹ and 69,06 g L⁻¹, respectively. All HWEs were prepared two-fold to the higher concentration used by diluting the stock solution 1:10 using deionized sterile water. In total, 100 μL of the highest concentration was added in all the wells of column number 1, and 50 μL was placed in all the other wells of the 96 multiwell plate. Using a multichannel pipette, the HWEs were diluted following the principle of microdilution in the plate, relocating 50 μL from column 1 to the others until column number 10. Column 11 was used as a positive control and column number 12 as the control of the medium and HWEs. Subsequently, 50 μL of the standardized cell suspensions were added in all columns but the last. The plates were then sealed with parafilm to avoid excessive evaporation and were incubated for 24 h at 37 °C. The day next, the volume of each well was resuspended and measured at 405 nm with a Sunrise plate reader. Afterwards, 30 μL of resazurin at a concentration of 0.015% was added to each well of the plates and left incubating at room temperature for 2–4 h. Before use, the resazurin solution was sterilized using filtration (0.22 filters) and stocked at 4 °C.

4.6. Biofilm Formation

To determine the potential anti-biofilm activity exerted by the Hemp Water Extracts (HWEs), the biofilm assay was made in two different ways to determine if the HE can interfere with the adhesion or development of the biofilm, using the assay protocol of Christopher G. Pierce et al. [63], with some modifications [64,65]. Briefly, each Candida strain was grown in 10 mL of the appropriate medium (YEPD + Chloramphenicol) in a 100 mL bottle to maintain the appropriate head space for the oxygenation. The strains were left for 24 h at 37 °C in an orbital shaker at 150–180 rpm. After 24 h of incubation, the cells were resuspended in an RPMI-1640 (Sigma-Aldrich, Saint Louis, MO, USA) to obtain a cell density of OD₆₀₀ = 0.2. After that, 50 µL of this standardized cell suspensions were seeded in selected wells of a 96-microtiter plate; cells without treatment were used as a positive control, and unseeded wells were considered as a negative control. To determine the effect of HWEs during the adhesion phase, 50 µL of the hemp HWEs were added in selected wells for a final concentration ranging from 2 mg mL⁻¹ to 0.003 mg mL⁻¹. The plates were incubated for 2 h at 37 °C and subsequently washed three times with a PBS solution. Finally, 100 μL of fresh medium was added to each well.

The second approach was that of testing the HWEs in the phase of biofilm development. The inoculum calibrated at OD₆₀₀=0.1 was added to each well, and the plates were incubated at 37 °C for two hours and washed trice afterwards. Subsequently, 50 µL of fresh medium (2×) was added to each well along with 50 µL of each HE at twice the concentration for each tested concentration.

After 24 h of incubation at 37 °C, the plates were washed thrice with the PBS solution and were stained with 100 µL of Crystal violet solution at 0.1%. The plates were incubated for 15 min and then were washed three times with sterile water and dried at room temperature until measured at 570 nm in a Tecan plate reader (Tecan Infinite Nano+, Tecan Trading AG, Mannedorf, Switzerland).

We thank Davide Barbaro and JJ Farm Società Agricola Semplice (Castiglione del Lago (Pg), Umbria, Italy) who kindly supplied Cannabis waste for all experimentations.