Phytochemistry, Pharmacology and Medicinal Uses of Plants of the Genus Salix: An Updated Review

The Willows (genus Salix), with more than 330–500 species and 200 hybrids, are trees, shrubs or prostrate plants that are widely distributed in Africa, North America, Europe, and Asia. The genus is traditionally used in folk medicine and represents a valuable source of biologically active compounds among them salicin, a prodrug for salicylic acid. Altogether, 322 secondary metabolites were characterized in the genus including flavonoids 94) (flavonols, flavones, flavanones, isoflavones, flavan-3-ols (catechins and procyanidins), chalcones, dihydrochalcone, anthocyanins, dihydroflavonols), phenolic glycosides (76), organic acids (28), and non-phenolic glycosides (17), sterols and terpenes (17), simple phenolics 13) and lignans 7) in addition to volatiles and fatty acids (69). Furthermore, willows exert analgesic, anti-inflammatory, antioxidant, anticancer, cytotoxic, antidiabetic, antimicrobial, antiobesity, neuroprotective and hepatoprotective activities. The current review provides an updated summary of the importance of willows, their chemical composition and pharmacological activities.


INTRODUCTION
Salicaceae (the Willow and Poplar family) traditionally includes the genera Populus (poplar) and Salix (willow), which are common in Northern temperate regions, and are amentiferous (bearing catkins) (Isebrands and Richardson, 2014). Presently, the Salicaceae have been enlarged to contain most tropical members of Flacourtiaceae, which do not produce catkin (Thadeo et al., 2014). Thus, the family Salicaceae now comprises about 56 genera and 1,220 species (Christenhusz and Byng, 2016).
The members of Salicaceae are fast growing trees or shrubs (Isebrands and Richardson, 2014). They are used for many economic purposes as production of timber, paper, fences, shelter, snowshoes, arrow shafts, fish traps, whistles, nets, rope, as a biomass fuel (a source of renewable energy), for ornamental, architectural and horticulture uses. Also, they are used for environmental enhancement through soil erosion control (Kuzovkina and Vietto, 2014). Willow twigs are elastic and were used to interweave baskets, for caning, and to manufacture woven fences and other lattices (Isebrands and Richardson, 2014).
The genus Salix (the willow) includes 330-500 species and more than 200 hybrids (Isebrands and Richardson, 2014), which are most widely distributed in the Northern hemisphere with a limited number of species occur in the Southern hemisphere (Zhen-Fu, 1987). Salix species are widely distributed in Africa, North America, Europe, and Asia (Argus, 2007). Salix species are fast growing trees, shrubs or prostrate plants; they can withstand a wide range of different weathers more than Populus species, as they grow in temperate, subtropic and tropic regions (Isebrands and Richardson, 2014).

Taxonomy
General morphological characters of genus Salix were reported (Argus, 2006;Lauron-Moreau, et al., 2015). Willows are 6-10 m high trees or shrubs with spirally arranged, sometimes silvery, oblong leaves. The latter is commonly hairy on the underside and often turn black when drying. Leaves are simple, petiolate showing different shapes of lamina (oblong, linear, ovate, obovate or round), stipulate with linear to rounded stipules and with entire, serrate or dentate margin. Their arrangement is mostly alternate or rarely opposite (Lauron-Moreau, et al., 2015). The flowers are catkins, dioecious, with nectaries (glands) instead of perianth and they have bracts, which are pale or black, pubescent or glabrate, constant in male flowers and deciduous in female ones. The flowers blossom in spring, generally prior the leaves (Mabberley 2008). The male catkins have mostly two stamens, more prominent yellow, with few species having 3-12 stamens while the female catkins are greenish, have single pistil with single ovary, style, two-lobed stigma and 2 to 42 ovules per each ovary (Mabberley 2008). The nectar of flowering Willow is the first food source for bees in spring. The seeds are small, with limited longevity, fine hairy coat enabling their spread by wind and they germinate after few days of exposure to moistured surfaces (Mabberley 2008).
Flacourtiaceae) is difficult, as they show very different morphology and exhibit numerous characteristics in common with several other families. The neotropical Salicaceae and Salicaceae displayed similar characters such as the presence of salicoid leaf teeth, collateral and arch-shaped vascular system at the midrib, abundance of crystals, brachyparacytic stomata, secondary growth of the petiole and sclerenchyma accompanying the bundles (Thadeo et al., 2014).

Phytochemistry
Different phytoconstituents or secondary metabolites of the genus Salix as flavonoids, glycosides (phenolic and nonphenolic glycosides), procyanidins, organic acids and their derivatives, simple phenolics, sterols and terpenes, lignans, volatiles and fatty acids were reported (Supplementary Tables S1-S7, included in Supplementary materials). Salix leaves mainly contain flavonoids, phenolic acids, their derivatives, and phenolic glycosides, while stem bark mainly contains procyanidins.
They are classified into two main classes: Salicin derived glycosides (salicinoids) and other phenolic glycosides as glycosylated phenylpropanoids, phenylethanoids and benzenoids and glycosylated salicylic acid derivatives. Salicinoids, which are considered as taxonomic markers for genus Salix, are derivatives of salicin, produced by esterification of one or more hydroxyl groups of salicyl alcohol or glucose moieties, mainly 2ˋand/or 6′ of glucose, with organic acids as acetic, benzoic and 1-hydroxy-6oxocyclohex-2-en-1-carboxylic (HCH) acids. The phenolic glycosides isolated and/or identified from genus Salix are presented in Supplementary Table S2 and Figure 2.

Volatiles
Terpenes (hemi-, mono-and sesqui-terpenes) and non-terpene (aliphatic, aromatic acids, their esters, carbonyl compounds and hydrocarbons) volatiles were identified in the genus Salix. The highest percent of volatiles and fatty acids was reported in S. caprea L. inflorescence (Ahmed et al., 2017), and the leaves of S. egyptiaca  Table S7and Figure 8).

Traditional Uses
Salix plants have been used medicinally since antiquity and have been linked to the discovery of acetylsalicylic acid and aspirin. These plants had been traditionally used to treat painful musculoskeletal joint pain conditions, inflammation, and fever. Salicin is a major pharmacologically active metabolite in Salix and hydrolyzes in the gastrointestinal tract to confer salicyl alcohol and D-glucose. The latter is oxidized, upon absorption, into salicylic acid, the active drug which inhibits cyclooxygenases (COX I, II) (Mahdi, 2010). S. egyptiaca L (Musk Willow) was important in the Middle East, especially in Iran, as it has been traditionally used to treat anemia and vertigo, as a cardiotonic agent, and also in the preparation of local candies as a fragrance additive (Asgarpanah, 2012). S. alba L (white willow), had used in folk medicine to treat fever, chronic and acute inflammation, pain and infection (Zengion and Yarnell, 2011;Maistro et al., 2019). S. tetrasperma Roxb. had been used to treat diseases such as epilepsy, diabetes, fever, rheumatism, piles, swellings, stones in bladder, dysentery, wound, ear pain, cough and cold (Prashith Kekuda et al., 2017). S. alba L. bark is traditionally used for treatment of flu, rheumatism, fever and headache (Van Wyk and Wink, 2018).

Pharmacological Activity
Different Salix species and the isolated compounds as salicylic acid and salicin have been utilized in folk medicine to treat rheumatic diseases, back pain, toothache, headache, and menstrual cramps (Highfield and Kemper, 1999). They exert analgesic, anti-inflammatory, antioxidant, anticancer, cytotoxic, antidiabetic, antimicrobial, anti-obesity, neuroprotective and hepatoprotective activities. The main targets of salicylic acid are cyclooxygenases (COX I, II) which are key enzymes of pathway to prostaglandins which control inflammation and pain. The available scientifically based reports on biological activities of genus Salix are summarized in Tables 1-8.

Antimicrobial Effects of Salix
Multidrug-resistant bacteria are widely spread, and natural resources have been used as a means of discovering novel antibacterial compounds as they offer limitless opportunities for the discovery of new agents, particularly against multidrug resistant bacteria.
The main methods used to evaluate the antimicrobial activity of Salix extracts are disc diffusion assays, agar well diffusion, broth microdilution methods and the assessment of antibiofilm function (Masika et al., 2005;Fayaz and Sivakumaar, 2014;  Table 1, microbial growth inhibition zones and percentages along with minimum inhibitory concentrations (MICs) displayed the potential of Salix species as substantial antimicrobials and predict their efficacy as functional foods .

Antibacterial Activity
Many previous studies evaluated the antibacterial activity of Salix plants and active constituents of their extracts against different types of bacteria such as Pseudomonas eruginosa, Escherichia coli, Staphylococcus aureus and Bacillus subtilis, dental biofilm forming bacteria (Streptococcus mutans and Lactobacillus), and Salmonella enterica ( Table 1). Catechol and 2-hydroxybenzyl alcohol derived from the bark of S. capensis Thunb. were previously tested for their antibacterial activity. Both compounds exhibited similar antibacterial activity against P. eruginosa (Masika et al., 2005). Moreover, Salix alba L. bark extract demonstrated antimicrobial activity against the dental biofilm forming bacteria with MIC of 125 μg/ml. Furthermore, it also exhibited a moderate potential against the Staphylococcus aureus but the least activity was observed against E. coli (Fayaz and Sivakumaar, 2014). Previous studies also showed that the twigs aqueous extract with leaves of S. babylonica L. exhibited potent antimicrobial properties against Gram-negative bacteria (E. coli, Salmonella enterica, MIC 50 is 70.4 ± 17.41 mg/ml) with a comparable activities to thiamphenicol (The broad spectrum antibiotic). Its effects cover Gram-positive bacteria such as S. aureus (Popova and Kaleva, 2015). A recent study performed in our laboratories tested the extracts of both stem bark and flowers of S. tetrasperma Roxb. for anti-quorum sensing activity against Pseudomonas eruginosa. Both extracts inhibited P. eruginosa bacterial growth at 40 mg/ml; however, the bacterial viability was not affected by 1/4 and 1/8 MIC concentrations. When the extracts were tested as antiquorum sensing agents, they impaired virulence of P. eruginosa by declining its swimming and swarming motilities and reducing its hemolytic and proteolytic properties .

Antifungal Activity
Poisoned food technique, broth microdilution method, filter disc assay and growth curve study methods were used to determine the antifungal properties of Salix extracts ( Table 2). The antifungal activity was evaluated against Candida guilliermondii, C. glabrata, C. parapsilosis and Fusarium oxysporum.

Anthelmintic Activity
The anthelmintic potential of Salix species to inhibit gastrointestinal and pulmonary parasites in animals was studied. The anthelmintic activity was evaluated against Ostertagia, Moniezia, Dictyocaulus, Eimeria, Chabertia, Cooperia, and Hemonchus contortus ( Table 2). It was reported Salix babylonica L (at dose of 20 ml, weekly) was effective against the main parasite species detected in sheep (Eimeriaspp., Dictyocaulus spp., and Chabertia spp.) more than the most

Anti -HIV Activity
Human immunodeficiency virus (HIV) infection that causes acquired immunodeficiency syndrome (AIDs) represents a major health problem worldwide. Chemical anti-retroviral agents are usually used to treat AIDs patients. However, they possess many adverse effects and resistance emerged for many of them. Recently, novel anti-retroviral agents isolated from medicinal plants, played an essential role to replace synthetic drugs. One study investigated the anti-retroviral effects of S. egyptiaca L. extract. Results of this study and bioinformatics analyses suggested that the plant had anti-HIV properties and might be a substantial candidate for AIDS patients ( Table 2) (Eftekhari et al., 2014).

Anti-Inflammatory Activity
Inflammation is a frequent condition because of exposure to different stimuli including microbial infection and wounding. It decreases the spread of infection, followed by resolution and the restoration of normal structural and functional of affected tissues (Nathan and Ding, 2010). However, non-resolving inflammation contributes significantly to the pathogenesis of many diseases such as atherosclerosis, obesity, cancer, and inflammatory bowel disease. Salix extracts exert potent anti-inflammatory effects that are responsible for many biological effects. The hydroalcoholic extract of S. tetrasperma Roxb. in two dose levels (100 and 200 mg/kg) demonstrated anti-inflammatory effects in carrageenan induced rat paw edema model (Kishore et al., 2014). We showed previously that the flower extract of S. tetrasperma Roxb. has analgesic, antipyretic, and antiinflammatory effects against carrageenan induced vascular permeability and carrageenan induced hind paw edema. It inhibited COX-1, COX-2 and LOX and suppressed elevated levels of TNF-a and NF-κB in chronic neuropathic pain model . Oral administration of S. canariensis extract significantly decreased writhing, moderately reduced formalininduced pain and showed a promising dose-dependent antiinflammatory activities. These effects were attributed to the presence of pentacyclic triterpenes and polyphenolics (Gutiérrez et al., 2017). An early study showed that S. caprea L. is a potent cyclooxygenase inhibitor (Tunon et al., 1995). Another study showed that S. subserrata Willd. and S. tetrasperma Roxb. showed anti-inflammatory effects against carrageenan induced hind paw edema due to the presence of phenolic glycosides mainly salicin as well as the flavonoids luteolin, quercetin and rutin (Karawya et al., 2010). S. matsudana Koidz. leaves methanol extract also showed Frontiers in Pharmacology | www.frontiersin.org February 2021 | Volume 12 | Article 593856 19 significant inhibitory activities against cyclooxygenases (COX-1 and COX-2) due to the presence of matsudone, luteolin 7-Oglucoside and 4′,7-dihydroxyflavone (Li et al., 2008).

Anticancer Activity
There are several risk factors that can increase the development of cancer that have a basis of low-grade inflammation and oxidative stress. Therefore, targeting inflammatory pathways and suppressing oxidative stress may contribute to inhibition of initiation, proliferation and even cancer metastasis and subside resistance to chemotherapy and radiation. Salix extracts, by possessing both antiinflammatory and potent antioxidant potential, are promising natural sources in fighting cancer. The antiproliferative activities of Salix extracts were determined by cell viability percentages and IC 50 values using several in vitro assays. The most commonly utilized cancer cell lines were human acute lymphoblastic leukemia (ALL cells), human acute myeloid leukemia cells (AML cells), PC3 cells (Prostate cancer cells), Hep G2 cells (Liver cancer cells), HCT116 (Colorectal cancer cells), MCF7 (Breast cancer cells), HT-29 and HCT 116 (human colon COX-2 positive and negative cells respectively), A549, SW2 cells, and human lung cancer cell line (H1299).
It was observed that a fraction of Salix extracted by non-polar solvents such as (petroleum ether, ether, and chloroform) has the minimum killing potential against AML cells while fraction Frontiers in Pharmacology | www.frontiersin.org February 2021 | Volume 12 | Article 593856 20 extracted by polar solvents such as 70% ethanol and water has major destructive effect on AML cells (El-Shemy et al., 2003). Thus, Salix cytotoxic activity could be attributed to the polyphenolics, tannins, and glycosides, that are commonly dissolved in water or ethanol solutions including salicin and saligenin. When salicin is tested against leukemic cells it caused destruction of myeloblasts by 70-75%. Eight compounds isolated from S. hulteni Flod (1-p-coumaroyl-β-D-glucoside, aromadendrin, catechin, 4-hydroxyacetophenone, picein, sachaliside 1, naringenin and dihydromyricetin) were tested for their cytotoxic potential against brine shrimp and a human lung cancer cell line (H1299). Naringenin, aromadendrin, catechin, and 1-p-coumaroyl-β-D-glucoside showed mild cytotoxic activity, with dihydromyricetin showing the strongest cytotoxic effects. 4-Hydroxyacetophenone, picein, and sachaliside one did not show a significant cytotoxic activity indicating that flavonoid compounds are responsible for the cytotoxic effects of S. hulteni Flod. (Jeon et al., 2008). Brine shrimp lethality test is commonly used to test cytotoxic effects of natural products. The methanol extract of S. nigra exerted concentration dependent cytotoxic effects against brine shrimp indicating promising cytotoxic effects . Willow bark extract (A pharmaceutically used extract BNO 1455) and its fractions (flavonoids, proanthocyanidins, salicyl alcohol derivatives) showed dose dependent cytotoxic effects against human colon and lung cancer irrespective of their COX-2 selectivity (Hostanska et al., 2007). S. caprea L. exerted a protective effect against phorbol ester induced skin tumor promotion when applied to the skin of mice prior to the application of phorbol ester. Anti-tumor activity of S. caprea L. may be attributed to potent antioxidants constituents of S. caprea L. such as luteolin, dihydrokaempferol and quercetin (Sultana and Saleem, 2004).

Neuroprotective Effect
Only few studies investigated the effect of Salix species on central and peripheral nervous system. Virupaksha et al. (2016) investigated the effects of S. tetrasperma Roxb. leaf extract on locomotor activity and muscle relaxant activity. They demonstrated that the extract decreased locomotor activity indicating central nervous system (CNS) depressant activity and induced a decrease in fall off time due to loss of muscle grip implying skeletal relaxation (Virupaksha  Candida guilliermondii, C. glabrata and C. parapsilosis Methanol extract of S. alba L. leaves Broth microdilution method, filter disc assay and growth curve study MIC 800 μg/ml, 800 μg/ml and 1,600 μg/ml respectively. Inhibition i.e. 12 mm for C. glabrata followed by 11 mm measured in C. parapsilosis. C. guilliermondii inhibition was 10 mm Zarger et al.

Leaves
Methanol extracts of S. purpurea L., S. cinerea L., Salix×smithiana willd., S. alba L., S. eriocephala michx., Salix×rubra huds DPPH The scavenging effect ranged between 33.6 (S. purpurea L.) and 45.7% (S. cinerea L.), 50.7 (S. purpurea L.) to 56.3% (Salix×rubra huds.) Gą secka et al.  , 2016). The CNS depressant activity of the extract was attributed to binding of flavonoids to gamma-aminobutyric acid (GABA) receptors in the CNS (Hossain et al., 2009). Another study from our laboratory investigated the possible protective effect of S. tetrasperma Roxb. on neuropathic pain model, chronic constriction injury of sciatic nerve model. In this work, we explored the effects of the extract on central and peripheral nervous system in this model. We showed that the extract improved hyperalgesia and allodynia, the major signs of neuropathic pain through inhibition of oxidative stress and inflammation in sciatic nerve and brain stem .

Hepatoprotective Effects
S. subserrata Willd. flower extract showed marked hepatoprotective effects mostly through lowering the elevated liver enzymes and decreasing the protein levels of two inflammatory biomarkers (NF-κB and TNF-α) in carbon tetrachloride (CCl 4 )-induced liver damage model (Wahid et al., 2016). It also presented a remarkable ability to reduce lipid peroxidation and had antioxidant effects related to several active ingredients that include flavonoids such as quercetrin, luteolin-7-glucoside, rutin, and quercetin and phenolic compounds such as salignin and catechins.

Anti-Obesity and Anti-lipidemic Effects
As shown in Table 8, remarkable anti-obesity and anti-lipidemic effects have been attributed to Salix extracts. The reduction of parametrial adipose tissue weight and body weight gain, the reduction of liver total cholesterol contents and inhibition of the elevated blood triacylglycerol are among the most  prominent, directly attributed to its ability to inhibition of intestinal absorption of dietary fat (Liu, 2012). These effects have been mostly attributed to polyphenol fractions (apigenin-7-O-D-glucoside, luteolin-O-D-glucoside and chrysoeriol-7-O-D-glucoside) which inhibited palmitic acid incorporation into small intestinal brush border membrane vesicles (Han et al., 2003). It was reported that methanol extract of S. pseudo-lasiogyne H. Lév. twigs and salicortin derivatives reduced lipid accumulation in a concentration-dependent manner. They inhibited the differentiation of adipocytes in 3T3-L1 cells. The 2′,6′-O-acetylsalicortin exhibited the most potent inhibitory activity with IC 50 11.6 μM. It remarkably downregulated the expressions of sterol regulatory element binding protein Intercept the free radicals and protect cellular macromolecules from oxidant damage. Effectiveness in inhibiting the ornithine decarboxylase activity and maintaining the activity of phase II enzymes after toxicant exposure (Sultana and Saleem (2004)   Decreased hepatic lipid peroxidation, increased hepatic glutathione (GSH) content and the activities of antioxidant enzymes (catalase (CAT), glutathione reductase (GR) and glutathione peroxidase) Alam et al. (2006)  1 (SREBP1c) and CCAAT/enhancer binding protein α (C/ EBPα). Thus, salicortin derivatives possessed antiadipogenic effects via down-regulation of SREBP1c and C/EBPα dependent pathways (Lee et al., 2013).

CONCLUSION AND FUTURE PERSPECTIVES
The current review outlined the complete research progress in the phytochemistry, traditional use and pharmacology of genus Salix plant extracts and constituents. Salix extracts and some of its components exerted potent antioxidant, anti-inflammatory, antiproliferative, and antimicrobial properties confirming the traditional use of willow extracts in folk medicine. They also demonstrated substantial abilities in suppressing inflammatory pathways, both in cancer prevention and treatment, and in other chronic diseases. Thus, as a potential perspective, Salix extracts alone or their isolated active components should be examined more thoroughly, and its anti-HIV, hepatoprotective and neuroprotective therapeutic approach should also be discussed.

AUTHOR CONTRIBUTION
NT retrieved the relevant literature and drafted the manuscript. AME and MW originated the work, led the discussions, provided helpful comments, and revised the manuscript. MF wrote the biological activity part. DH, MS and NF provided helpful comments and revised the manuscript. All authors read and approved the final version of the manuscript.