1. Introduction

2. Materials and Methods

2.1. Materials

2.1.1. Phytochemistry

There are phytoconstituents or secondary metabolites found in the genus Salix, commonly known as willow trees.

Flavonoids are a diverse group of plant secondary metabolites known for their antioxidant properties and various health benefits. They are commonly found in fruits, vegetables, and beverages such as tea and wine.

Glycosides are compounds where a sugar molecule is attached to a non-sugar moiety. They can be further classified into phenolic glycosides (glycosides containing a phenolic group) and non-phenolic glycosides.

Procyanidins are oligomeric flavonoids, also known as condensed tannins. They are found in various plants and are known for their antioxidant properties.

Organic acids such as citric acid, malic acid, and tartaric acid are commonly found in plants. Their derivatives may include esters, salts, and other compounds formed from organic acids.

Phenolic compounds are characterized by the presence of a phenol group. They have diverse roles in plants, including defense against pathogens and environmental stressors.

Sterols are a type of lipid that play structural roles in cell membranes. Terpenes are a large class of compounds derived from isoprene units, with diverse biological activities.

Lignans are phenolic compounds found in plants, particularly in the cell walls. They have antioxidant and estrogenic properties.

Volatile compounds are organic molecules with low molecular weights that evaporate easily at room temperature. Fatty acids are long-chain carboxylic acids commonly found in fats and oils [9].

2.1.2. Flavonoids

The distribution of various flavonoids across different species of the Salix genus, including their occurrence in different plant parts such as leaves, roots, and bark [10,11].

Salix species contain a wide variety of flavonoids, including flavones, flavonols, flavanones, dihydroflavonols, isoflavones, chalcones, dihydrochalcones, flavan-3-ols, and anthocyanins.The highest diversity of flavonoid classes was detected in leaves with rare occurrence in roots.

Flavones, such as apigenin and its glycosides, are major constituents of S. acutifolia, S. matsudana, and S. babylonica leaves and roots.Chrysoeriol and its derivatives are major constituents of S. babylonica, S. matsudana, and S. subserrata leaves [12].

Acylated luteolin glucosides are characteristic of S. gilgiana leaves.Kaempferol and its derivatives are prominent in S. bordensis, S. babylonica, and S. integra × S. suchowensis.

Angeloxyflavone and isoflavones are markers for S. cheilophila twigs.

Sulfated flavanones and dihydroflavonols accumulate in S. integra × S. suchowensis young stems[.

The bark of willows contains the highest number of chalcones, catechins, procyanidins, and anthocyanins.Chalcones accumulate in the bark of S. daphnoides, S. elbursensis, S. acutifolia, and S. rubra. Catechins, epicatechin, and various procyanidins are major constituents of S. sieboldiana bark.Procyanidins are also significant in S. daphnoides bark.

Anthocyanins are found in the bark of several Salix species, including S. purpurea, S. alba, S. phylicifolia, S. nigricans, S. calodendron, S. viminalis, S. triandra, and S. amygdalina [13] (Figure 1).

2.1.3. Phenolic Glycosides

It’s important the classification and distribution of phenolic glycosides within the Salicaceae family, particularly focusing on the genus Salix [14].

These glycosides play important roles as secondary metabolites and can serve as taxonomic markers for different Salix species. The presence of specific glycosides like salicin, tremuloidin, and tremulacin, as well as others such as acmophyllin A and B, chaenomeloidin, cochinchiside, lasiandrin, leonuriside, and salicin-7-sulfate, can help distinguish between various Salix species [15,16].

Furthermore, the identification of certain glycosides, such as acutifoliside and 1,2-cyclohexanediol glycosides, serves as chemical markers for specific parts of Salix plants, like juvenile stems or twigs, aiding in their characterization and classification [17,18].

Overall, the presence and distribution of these phenolic glycosides provide valuable insights into the taxonomy and phytochemistry of Salix species [19] (Figure 2).

2.1.4. Non-Phenolic Glycosides

In S. triandra L. x dasyclados Wimmer Wood, non-phenolic glycosides (compounds 172, 173, 174, 175, 176, 182–188) were identified as major constituents [19].

In S. arbusculoides Andersson twigs, compounds 170 and 171 are reported as major constituents [20].Certain Salix species are characterized by the accumulation of 1,2-cyclohexanediol glycosides.

Figure 3. Structures of reported non-phenolic glycosides from genus Salix.

Figure 3. Structures of reported non-phenolic glycosides from genus Salix.

2.1.5. Organic Acids

Phenolic acids are in various Salix species, which can occur in either free or esterified forms, such as benzyl, cinnamyl, or phenyl ethyl esters. These aromatic acids can be derivatives of benzoic or cinnamic acid.

Benzoic acid derivatives: examples include p-hydroxybenzoic, p-anisic, gallic, salicylic, gentisic, vanillic, 2-amino-3-methoxy benzoic, and protocatechuic acids.

Hydroxycinnamic acid derivatives: these include p-coumaric, caffeic, isoferuolic, and feruolic acids.

Specific compounds and their occurrences in different Salix species are mentioned:

S. purpurea L. and S. alba L. bark were found to contain the highest number of organic acids, including compounds 192–194, 198–200, and 214 [21].

S. tetrasperma Roxb. flowers and bark contain compounds 197, 202, 203, 204, 205–206, 208, 209, and 215 [22].

Figure 4. Structures of reported organic acids from genus Salix.

Figure 4. Structures of reported organic acids from genus Salix.

2.1.6. Simple Phenolics

There are a variety of simple phenolic compounds found within the genus Salix, including phenolic acids and their derivatives.

Accumulation of salicyl alcohol (compound 228):this compound serves as the basic nucleus for salicinoids,identified in: S. capensis Thunb. Bark [23],S. acutifolia Willd. Bark [24],S. subserrata Willd. Bark [25],S. caprea L. inflorescence [26].

Accumulation of different simple phenolics in S. caprea L. wood:aucuparin (compound 218),methoxyaucuparin (compound 219),coniferyl alcohol (compound 221),p-coumaryl alcohol (compound 222),4,2′-dihydroxy-3,5-dimethoxybiphenyl (compound 223),sinapylaldehyde (compound 229),identified in S. caprea L. wood [27].

Figure 5. Structures of reported simple phenolics from genus Salix.

Figure 5. Structures of reported simple phenolics from genus Salix.

2.1.7. Sterols and Terpenes

Highest number of sterols and triterpenes are detected in:

S. cheilophila C. K. Schneid. twigs: this species exhibited the highest number of sterols and triterpenes [28].

S. tetrasperma Roxb. bark, leaves, and flowers:significant amounts of sterols and triterpenes were found in different parts of this species [29].

S. subserrata Willd. leaves: this species also showed the presence of sterols and triterpenes [30].

S. denticulate aerial parts: detection of sterols and triterpenes was reported in the aerial parts of this species [31].

S. babylonica L. roots: sterols and triterpenes were found in the roots of this species [32].

S. subserrata Willd. bark and leaves: both bark and leaves of this species contained sterols and triterpenes [33].

Specific constituents found in S. cheilophila C. K. Schneid. twigs:

Phytane and pimarane diterpene were identified as major constituents in the twigs of this species [28].

Figure 6. Structures of reported sterols and terpenes from genus Salix.

Figure 6. Structures of reported sterols and terpenes from genus Salix.

2.1.8. Lignans

The isolation of lignan derivatives from Salix alba L. bark, as well as the detection of various lignans in the biomass of five willow species cultivated in Quebec, Canada.

Isolation from S. alba L. bark:sisymbrifolin, a lignan derivative (compound 247), was isolated from the bark of Salix alba L.[34].

Detection in the biomass of five willow species in Quebec, Canada:pinoresinol (compound 248),lariciresinol (compound 249),secoisolariciresinol (compound 250),7-hydroxymatairesinol (compound 251),medioresinol (compound 252),lariciresinol-sesquilignan (compound 253).

These lignans were detected in the biomass of five willow species cultivated in Quebec, Canada [35].

Figure 7. Structures of reported lignans from genus Salix.

Figure 7. Structures of reported lignans from genus Salix.

2.1.9. Volatiles

Terpenes (hemi-, mono-, and sesqui-terpenes) and non-terpene compounds (aliphatic and aromatic acids, their esters, carbonyl compounds, and hydrocarbons), in the genus Salix identified in the genus Salix:hemi-, mono-, and sesqui-terpenes,aliphatic and aromatic acids,esters,carbonyl compounds,hydrocarbons.

There are highest percent of volatiles and fatty acids reported in certain parts of specific Salix species:

S. caprea L. inflorescence: the highest percentage of volatiles and fatty acids was reported in the inflorescence of this species [26].

Leaves of S. egyptiaca L., S. babylonica L., and S. alba L.: high percentages of volatiles and fatty acids were reported in the leaves of these species [36].

Figure 8. Structures of reported volatiles and fatty acids from genus Salix.

Figure 8. Structures of reported volatiles and fatty acids from genus Salix.

2.1.10. Pharmacological Activity

Are well know the traditional use of various Salix species and their isolated compounds, such as salicylic acid and salicin, in folk medicine to treat various ailments including rheumatic diseases, back pain, toothache, headache, and menstrual cramps. Additionally, it mentions the diverse range of biological activities exhibited by Salix species and their compounds, including analgesic, anti-inflammatory, antioxidant, anticancer, cytotoxic, antidiabetic, antimicrobial, anti-obesity, neuroprotective, and hepatoprotective activities.

Specifically, salicylic acid is noted for its effects on cyclooxygenases (COX I, II), which are enzymes crucial for the synthesis of prostaglandins, molecules involved in inflammation and pain regulation [37].

2.1.11. Anti-Inflammatory Activity

Inflammation and its consequences: Inflammation is a common response to various stimuli such as microbial infection and injury. While inflammation is essential for controlling infection and promoting tissue repair, unresolved inflammation can contribute to the pathogenesis of diseases like atherosclerosis, obesity, cancer, and inflammatory bowel disease [38].

Anti-inflammatory effects of Salix extracts:

S. tetrasperma Roxb.: hydroalcoholic extract of S. tetrasperma Roxb. demonstrated anti-inflammatory effects in a rat paw edema model induced by carrageenan. It inhibited cyclooxygenases (COX-1, COX-2), lipoxygenase (LOX), and reduced levels of tumor necrosis factor-alpha (TNF-a) and nuclear factor kappa B (NF-κB)[39].

S. canariensis: oral administration of S. canariensis extract showed dose-dependent anti-inflammatory activities, attributed to pentacyclic triterpenes and polyphenolics [40].

S. caprea L.: identified as a potent cyclooxygenase inhibitor [41].

S. subserrata Willd. and S. tetrasperma Roxb.: exhibited anti-inflammatory effects against carrageenan-induced hind paw edema due to the presence of phenolic glycosides, especially salicin, as well as flavonoids like luteolin, quercetin, and rutin [42].

S. matsudana Koidz.: Methanol extract of S. matsudana Koidz. leaves showed inhibitory activities against cyclooxygenases (COX-1 and COX-2) due to the presence of matsudone, luteolin 7-O-glucoside, and 4′,7-dihydroxyflavone [43].

These findings suggest that Salix extracts possess significant anti-inflammatory properties, potentially attributed to various bioactive compounds present in different species.

2.1.12. HPLC Method

Salicin, found in willow bark, has garnered attention for its therapeutic properties, particularly its anti-inflammatory effects.

Single drug therapyis focusing on the use of a single medication for treating a particular condition, has a long history dating back to ancient times [44,45]. It’s widely accepted by physicians today for various diseases.

With the increased use of herbal remedies alongside conventional medications, understanding interactions between herbs and drugs has become crucial in clinical practice to ensure safety and efficacy.

Developing a marker profile is vital for quality control and scientific validation of single drugs. This ensures consistency in composition and potency, aiding in reliable therapeutic outcomes.

It’s described as the metabolic precursor of salicylic acid, possessing anti-inflammatory properties. Salicin is an alcoholic beta-glycoside containing D-glucose, found in willow barks, particularly in Salix alba L (White Willow). It’s noted as a weaker forerunner of aspirin [46].

Willow bark contains various chemical compounds, including glycosides (such as salicin), tannins, aromatic aldehydes and acids, salicyl alcohol (saligenin), and flavonoids [47,48,49,50,51]. These constituents contribute to its pharmacological effects.

Salicin exhibits antipyretic and analgesic effects, making it useful in treating fever and conditions like arthritis [46]. Its similarity to aspirin in action (Figure 9).

2.2. Methods

HPLC was performed using an Agilent EZChrom Elite system equipped with a DAD detector and a HiCHROM LiChrosorb 100 RP-18 column, 10 μm particle (4.6 x 250 mm).

Extraction of salicylic derivatives: for the analysis of the samples represented by branches of willow

The sample (1 g) was extracted with 8 mL of a solution consisting of a part of 4.2 g/L NaOH solution and a part of methanol. The mixture was stirred at 60°C at reflux for 60 min. After cooling, 0.4 mL of 10 g/L HCl solution was added and the mixture was centrifuged at 9000 rpm for 5 minutes. The supernatant was diluted to 10 mL with a mixture of equal volumes of methanol and high purity water. Before HPLC analysis, the samples were filtered with a PA filters.

Figure 10. Concentration of Salicin from Salix alba bark from. Olt district(Romania).

Figure 10. Concentration of Salicin from Salix alba bark from. Olt district(Romania).

Figure 11. Concentration of Salicin from Salix babylonica bark from. Olt district(Romania).

Figure 11. Concentration of Salicin from Salix babylonica bark from. Olt district(Romania).

Salicin derivatives are the main constituents of willow bark and can be quantified as salicin equivalents. According to the Monograph of the European Pharmacopoeia [3] and the Evaluation Report of the European Medicines Agency [52], the content of salicin derivatives in the bark of different species of willow varies from 0.5 to 10%. However, quality standards involve at least 1.5%.

Although willow bark remains the main source of salicin.

3.2.1. Extraction Method for a Aqueous Extracts

We prepare the aqueous extracts from Salix alba bark.

Preparation of stock aqueous extracts:10 grams of air-dried and milled plant material are soaked in 100 ml of distilled water, resulting in a concentration of 10% (w/v).

The soaking occurs at room temperature (°C) for 24 hours with occasional shaking to facilitate the extraction process.

Filtration and centrifugation:after 24 hours of soaking, the mixtures are filtered through two layers of cheesecloth to remove solid particulate materials.

The filtered mixtures are then centrifuged for 20 minutes at 10,000 rpm. This step helps to further remove any remaining particulate matter and debris, resulting in purified extracts.

Adjustment of pH:the purified extracts are adjusted to a pH of 6.8 using 1.0 M HCl (hydrochloric acid). pH adjustment is often done to optimize the stability and solubility of bioactive compounds present in the extract.

Storage:finally, the adjusted extracts are stored in the refrigerator at 4°C for future use. Storing the extracts at low temperature helps to preserve the stability and integrity of the bioactive compounds present in the extract.

This method ensures the extraction of water-soluble compounds from the plant material and results in purified aqueous extracts suitable for further analysis or use in various applications such as pharmaceuticals, cosmetics, or food products.

3.2.2. Antimicrobial Assay

The Kirby-Bauer diffusometric method suitable for natural plant extracts was used to analyse the antimicrobial activity of the willow extract. This method is qualitative and allows the evaluation of the antimicrobial activity of the tested products based on the diameter of the inhibition surfaces for microbial growth. The antimicrobial method is based on the following principles: Stainless steel cylinders are used, which are placed on the surface of a specific growth medium inoculated with the microbial suspension to be tested (medium CaSoA - for the activation of bacterial strains). The tested extract is used in the cylinders. After incubation, the appearance of an inhibition zone of microbial growth was observed, which proves the sensitivity of the tested culture and the special properties of diffusion in the environment of the tested substance.

The level of microbial activity was classified according to the diameter values of the inhibition zones according to the European Pharmacopoeia. The microbial strains selected for the test were Escherichia coli ATCC 8739 and Staphylococcus aureus ATCC 6538. The antimicrobial activity test was performed in triplicate.

3. Results

3.1. Experimental Antimicrobial Activity Results

Figure 12. Zone of inhibition for the extract tested on Escherichia coli ATCC 8739 (right - top). Zone of inhibition for the extract tested on Staphylococcus aureus ATCC 6538 (right - top).

Figure 12. Zone of inhibition for the extract tested on Escherichia coli ATCC 8739 (right - top). Zone of inhibition for the extract tested on Staphylococcus aureus ATCC 6538 (right - top).

4. Discussion

5. Conclusions

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