Salix spp. Bark Hot Water Extracts Show Antiviral, Antibacterial, and Antioxidant Activities-The Bioactive Properties of 16 Clones

Abstract

Earlier studies have shown that the bark of Salix L. species (Salicaceae family) is rich in extractives, such as diverse bioactive phenolic compounds. However, we lack knowledge on the bioactive properties of the bark of willow species and clones adapted to the harsh climate conditions of the cool temperate zone. Therefore, the present study aimed to obtain information on the functional profiles of northern willow clones for the use of value-added bioactive solutions. Of the 16 willow clones studied here, 12 were examples of widely distributed native Finnish willow species, including dark-leaved willow (S. myrsinifolia Salisb.) and tea-leaved willow (S. phylicifolia L.) (3 + 4 clones, respectively) and their natural and artificial hybrids (3 + 2 clones, respectively). The four remaining clones were commercial willow varieties from the Swedish willow breeding program. Hot water extraction of bark under mild conditions was carried out. Bioactivity assays were used to screen antiviral, antibacterial, antifungal, yeasticidal, and antioxidant activities, as well as the total phenolic content of the extracts. Additionally, we introduce a fast and less labor-intensive steam-debarking method for Salix spp. feedstocks. Clonal variation was observed in the antioxidant properties of the bark extracts of the 16 Salix spp. clones. High antiviral activity against a non-enveloped enterovirus, coxsackievirus A9, was found, with no marked differences in efficacy between the native clones. All the clones also showed antibacterial activity against Staphylococcus aureus and Escherichia coli, whereas no antifungal (Aspergillus brasiliensis) or yeasticidal (Candida albicans) efficacy was detected. When grouping the clone extract results into Salix myrsinifolia, Salix phylicifolia, native hybrid, artificial hybrid, and commercial clones, there was a significant difference in the activities between S. phylicifolia clone extracts and commercial clone extracts in the favor of S. phylicifolia in the antibacterial and antioxidant tests. In some antioxidant tests, S. phylicifolia clone extracts were also significantly more active than artificial clone extracts. Additionally, S. myrsinifolia clone extracts showed significantly higher activities in some antioxidant tests than commercial clone extracts and artificial clone extracts. Nevertheless, the bark extracts of native Finnish willow clones showed high bioactivity. The obtained knowledge paves the way towards developing high value-added biochemicals and other functional solutions based on willow biorefinery approaches.

Keywords: Salix spp.; antimicrobial; antioxidant; antiviral; bark; debarking; water-extracts.

GRAPHICAL ABSTRACT

FIGURE 1

Original willow samples (A), combined willow sticks in reactor (B), and steam-treated bark and woody material (C).

FIGURE 2

Willow steam treatment, debarking, and extractions. Willow steam treatment condensate, original willow bark ASE-350 extract, and steam-treated bark ASE-350 extract contained polyphenols. Carbohydrates were extracted from woody samples at temperatures from 150 to 180°C after debarking. Collected samples are indicated with blue color.

FIGURE 3

ORAC (A) and FRAP (B) activities, and total phenolic content (Folin-Ciocalteu) (C) of willow wood after debarking extracted at different temperatures (150, 160, 170, and 180°C) and bark extracts (original bark extract, steam-treated bark extract, and steam condensate). Error bars present the standard deviations of the sample triplicates in a microplate. ORAC test results are expressed as Trolox equivalents (TE), FRAP results are expressed as ferrous ion equivalents (Fe(II) eq.), and Folin-Ciocalteu test results are expressed as gallic acid equivalents (GAE).

FIGURE 4

ORAC (A), FRAP (B), and H₂O₂ scavenging (D) activities, as well as total phenolic content (Folin-Ciocalteu) (C) of the Salix spp. clone extracts. Error bars present the standard deviations of the sample triplicates in a microplate. The clones are numbered 1–16 (see Table 1) and 13–16 are commercial clones. ORAC test results are expressed as Trolox equivalents (TE), FRAP results are expressed as ferrous ion equivalents (Fe(II) eq.), Folin-Ciocalteu test results are expressed as gallic acid equivalents (GAE), and hydrogen peroxide scavenging test results are expressed as the percentage of H₂O₂ inhibition.

FIGURE 5

Grouped ORAC (A), FRAP (B), and H₂O₂ scavenging (D) activities, as well as total phenolic content (Folin-Ciocalteu) (C) of the Salix spp. clone extracts. Error bars show the standard deviation between the grouped clones. Significant differences are indicated with a blue asterisk. ORAC test results are expressed as Trolox equivalents (TE), FRAP results are expressed as ferrous ion equivalents (Fe(II) eq.), Folin-Ciocalteu test results are expressed as gallic acid equivalents (GAE), and hydrogen peroxide scavenging test results are expressed as the percentage of H₂O₂ inhibition.

FIGURE 6

The bacterial biosensor results. Efficacy against E. coli (A) and S. aureus (B) after 40 min incubation time. The Salix spp. clones (Table 1) of 5% v/v concentration per a microplate well are indicated with numbers 1–16. P-16 = 2-L scale clone 16; commercial substances Salixin P = Salixin Organic Powder 48^TM (250 μg/ml) and Salixin E = Salixin Organic Extract 800NP^TM (1.25% v/v); SA = salicylic acid (250 μg/ml). Results obtained for salicin, triandrin, and picein are also shown at the concentration of 250 μg/ml per microplate well. Error bars indicate the standard deviation between the sample triplicates in the microplate. Lower RLU values indicate stronger antibacterial activity.

FIGURE 7

The grouped antibacterial results against E. coli (A) and S. aureus (B). Error bars show the standard deviation between the grouped clones. Significant differences are indicated with a blue asterisk.

FIGURE 8

Testing the antiviral activity of (A) Salix spp. extracts (0.1% v/v) and (B) reference compounds (salicin, salicylic acid, picein, and triandrin) and Salixin Organic Powder and Extract against CVA9 using CPE inhibition assay. Virus control and test samples are normalized against a mock infection. The results are mean of two independent experiments with n = 3. The average values + standard errors of mean (SEM) are shown. P-16 = 2-L scale clone 16; Salixin P = Salixin Organic Powder 48^TM; Salixin E = Salixin Organic Extract 800NP^TM; SA = salicylic acid.

FIGURE 9

Effect of time and temperature on the antiviral activity of selected Salix spp. extracts and reference compounds using CPE inhibition assay. Salix spp.-virus mix was incubated at 37°C and 21°C for (A, B) 5 min and (C, D) 45 s. Virus control and test samples are normalized against a mock infection. The results are mean of two independent experiments, with n = 3. The average values + standard errors of mean (SEM) are shown. P-16 = 2-L scale clone 16; SA = salicylic acid.