Application of high-performance liquid chromatography for research of salicin in bark of different varieties of Salix

Willow (Salix L.) species are widely spread in Lithuanian natural dendroflora. Willow bark contains active substances known for anti-inflammatory properties and is known as a phytotherapeutic precursor of aspirin. Bark extracts are components of analgesic and antirheumatic preparations. Therapeutic effectiveness is associated with salicin (2-(hydroxymethyl)phenyl-β-D-glucopyranoside), which turns into salicylic acid. Increasing attention to natural preparations gives primary importance to research of plants. This study focused on 12 willow taxa and employed routine pharmacopoeia methods. High-performance liquid chromatography method was applied for the analysis of bark extractions. The investigation revealed that not all willow species accumulated a therapeutically sufficient amount of salicin. Bark samples were investigated after 1and 2-year growth in autumn and spring. Salicin content ranged from 0.08 to 12.6%. Higher contents of active materials were determined in autumn and in 2-year-old willows. Certain willow taxa (Salix alba L., Salix mollissima L., Salix triandra L., Salix viminalis “Americana”, Salix dasyclados L.) possessed extremely low salicin amounts. In the second year, analysis covered 32 willow species. Results indicated striking differences in salicin amounts (from 0.04% in Salix viminalis “Americana” to 12.06% in Salix acutifolia). Willow species, plant age, and season should be considered when collecting medicinal plant material. The amount of salicylates in 2-year-old willow bark collected in autumn exceeded by 25% that in 1-year-old willow bark collected in spring. Bark of some analyzed willow species contained the amount of salicylates too low for using as anti-inflammatory or antipyretic remedy.


Introduction
Willow is a deciduous shrub native to Britain, central and southern Europe (1), Asia, and North America (2).The material of commerce comes mainly from southeastern European countries, including Bulgaria, Hungary, Romania, the former Yugoslavia (2,3).
There are approximately 400 species worldwide, including 150 in the western hemisphere.Salix species are chiefly located in North Temperate and Arctic regions, growing primarily in moist habitats, especially along stream banks.The Salicaceae (includes 2 genera, Salix and Populus) are differentiated from other catkin-bearing families by the presence of only one bract subtending each flower.Members of willow species are difficult to conclusively identify due to high variability and hybridization among the genera.Important diagnostic characters include habitat; leaf position, shape, margin, and pubescence; the timing of catkin emergence; bract morphology; number of stamens per flower; shape and pubescence of the ovary (4)(5)(6)(7)(8).
Some of the modern therapeutic applications for willow bark originate from its early uses in ancient Greek medicine, which were first reported by Dioscorides in his herbal De Materia Medica written in the first century CE  (9).During the Middle Ages, willow bark was used in Europe to reduce fever and relieve pain.Anti-inflammatory and fever-reducing actions of willow are attributed to salicylates, contained in the bark.These glycosidic constituents were first isolated by a French chemist in 1829 and were synthesized six years later (10).Salicylic acid, first isolated from meadowsweet leaf (Filipendula ulmaria; syn.Spiraea ulmaria), is the phytotherapeutic precursor of acetylsalicylic acid, the analgesic drug commonly known as aspirin (2,11,12).
Willow bark comprises the bark of young two-to three-year-old branches of S. alba L., S. purpurea L., S. fragilis L., and other Salix species (Salicaceae) harvested during early spring and their preparations in effective dosage.The bark contains at least 1% of total salicin derivatives, calculated as salicin relative to the dried herb.As the Commission E allows for several different Salix species, the quantities of salicin may vary significantly depending on the species used (13)(14)(15).
The British Herbal Compendium indicates the use of willow bark for rheumatic and arthritic conditions and feverish conditions such as common cold or influenza (11).ESCOP indicates its application for treatment of feverish conditions, symptomatic treatment of mild rheumatic complaints, and relief of headache (16).In France, willow bark preparations are allowed for use as an analgesic to treat headache and toothache as well as painful articular conditions, tendinitis, and sprains (11,17).
The main objective of this study was to investigate characteristics of the willow bark collected from different Salix species growing in Lithuania.The assay of salicin in the willow bark was done by high-performance liquid chromatography (HPLC) method according to the European Pharmacopoeia.

Chemicals and reagents
Salicin, resorcinol, and picein standards were purchased from Sigma (St. Louis, MO, USA).The standards were stored in the room protected from light, at room temperature (20°C).HPLC-grade tetrahydrofuran and phosphoric acid were obtained from Roth GmbH (Germany).Sodium hydroxide and hydrochloric acid were purchased from Fluka.Purified water was generated by a Milli-Q reagent water system (Millipore Corporation, 106 Bedford, MA).
The samples of willow bark were collected in Miroslavas region, Alytus.Internal standard solution was prepared by dissolving 50 mg of resorcinol in 10.0 mL of methanol.
Reference solution (a) was prepared by dissolving 18.5 mg of salicin in 10.0 mL of the mixture containing 20 volumes of water, 80 volumes of methanol and added 1.0 mL of the internal standard solution.
Reference solution (b) was prepared by dissolving 1.0 mg of picein R in 1.0 mL of reference solution (a).
Test solution preparation was prepared as follows: 50 mL of methanol were added to 0.5 g of the powdered bark, and this mixture was heated under a reflux condenser for 30 min, then cooled, and filtered.The residues were taken up with 50 mL of methanol and proceeded as above.The filtrates were combined and evaporated under reduced pressure.The residues were taken up with 5.0 mL of methanol; 5.0 mL of 0.1 M sodium hydroxide were added and then heated in a water-bath at 60°C under a reflux condenser, with frequent shaking for about 1 h.After cooling, 0.5 mL of 1 M hydrochloric acid was poured.The solution was diluted to 20.0 mL with the mixture containing 50 volumes of methanol and 50 volumes of water.Then 1.0 mL of the internal standard solution was added to 10.0 mL of this solution and filtered through a membrane filter.

Discussion
Willow bark is a perspective herbal remedy, which possesses a lot of therapical effects, such as antipyretic, anti-inflammatory, analgesic, antineoplastic, and antifungal.As the effect is based on salicin content, this investigation was conducted in order to compare the amounts of salicin in different species of Salix.The results of this study show striking differences in amounts of salicylic derivatives among different Salix species (Fig. 3-5).According to the European Pharmacopoeia, minimal amount of 1.5% of salicin is  1  and 2).According to the results of this study, medical usage of the species mentioned above is recommended.However, many species were noticed to possess very low amounts of salicin: Salix dasyclados (0.15%-0.39%),Salix mollisima (0.25%-0.84%),Salix triandra (0.73%-0.98%), and all clones of Salix viminalis (0.038%-0.98%).Hybrids of Salix viminalis (the lowest amount of salicin) and Salix purpurea (the highest amount of salicin) possess the required amount of salicin (4.61%-7.26%).
The first step of the study focused on three samples of each plant, collected at different growing period (first-year autumn, second-year autumn, and secondyear spring).Comparison of the quantities of the salicylic derivatives, depending on age of the plant (Fig. 4) and time of collection (Fig. 5), showed that two-year-old plants and the ones collected in the autumn accumulated the greatest amounts of active substances (approximately a 25.0% increase) when compared with one-year-old plants and the material collected in spring season.These findings agree with data reported by other authors (13)(14)(15).
Salix alba is a common species in Lithuania, and it is described in foreign literature as herbal remedy as well.According to this study, it possessed only 1.29% of salicin in the spring of the first year, but in autumn of the second year, it accumulated an appropriate amount of salicin (1.87%).Therefore, time of collection of willow bark is important to consider.The amount of 1.87% is not very high, and according to literature, it would be necessary to use 35 g of white willow bark to achieve the effect of one tablet of aspirin.Such high amount of bark can be toxic due to effect of tannins.The usual dosage is 1-3 g of willow bark.In this case, the effect of herbal remedies cannot be based on the effect of salicin only.It is proposed to evaluate other components (tannins, flavonoids), which can contribute to overall effect.

Conclusions
1.The results of this study show striking differences in amount of salicylic derivatives among different Salix species from 0.04% (Salix viminalis "Americana") to 12.06% (Salix acutifolia).
2. The amount of salicin in the bark of some analyzed species is lower than it is required according to the European Pharmacopoeia (1.5%).These species are not recommended to be used for treatment due to very low amount of salicin.
3. It is important to consider season of the year and age of the plant during collection of willow bark.The comparison of the quantities of the salicylic derivatives depending on age of the plant showed that two-year-old plants and the ones collected in the autumn accumulated the greatest amounts of active substances (approximately a 25.0% increase) when compared with one-year-old plants and the material collected in the spring.
4. Further studies are required to evaluate amounts of other compounds (flavonoids, tannins, or salicin esters), which may contribute to the overall effect while salicylate levels are too low to explain therapeutic activity.

Fig. 3 .
Fig. 3. Dependence of the salicylic derivatives quantity on the age of the plant

Fig. 5 .
Fig. 5. Variation of the amount of salicylates among different Salix species

Quantity of salicylates in different species of willow bark Taxa % required
in willow bark.Considering this requirement, it is possible to divide species analyzed in this study to the ones that can be used in preparation of qualitative herbal remedies and to the ones, inappropriate for medical usage.Salix integra × Salix kochiana