Willow bark has been used in medicine for centuries, most notably by Hippocrates (460 B.C. to 377 B.C.), who is now known as the ‘father of modern medicine’. Today the painkiller industry is much more advanced, and aspirin, whose origins began with willow bark, is now a common medication used to treat a variety of symptoms. However, willow bark extracts are still used, and research has been done to determine their effectiveness.
This study investigates the extraction of the active ingredient salicin from willow samples and aims to determine how the age and region of willow plant used may affect the quantity of salicin produced. The results showed that although all forms of the willow (fresh, old, bark, and twig) contained salicin, the aged brown bark produced the highest yield.
From Willow Bark to Aspirin
Hippocrates’ use of willow bark in treatments has been well documented, from chewing on willow bark to help relieve fever to making a brew from willow bark to ease labour pains. Although willow bark had been used since ancient civilisations, the active ingredient responsible for its pain-relieving effects was not identified until 1828, when Joseph Buchner, a German pharmacologist, extracted bitter crystalline structures from the plant. He named the new compound salicin, from which salicylic and acetylsalicylic acid were later derived and buffered against.[2,3] After multiple experiments, Felix Hoffmann, a German chemist working for the pharmaceutical company Bayer, successfully synthesised acetylsalicylic acid in a pure and stable form, which was registered as aspirin in 1899 and patented in the US a year later.
Medicinal Properties of Willow Bark
Although aspirin is now a more common way to deal with pain, many people around the world continue to substitute it with willow bark. The plant now comes in tablet, capsule, powder, and liquid forms, and the recommended extract dose is 120 – 240 mg salicin per day for the treatment of pain.[4,5] One of the reasons why people use willow bark as an alternative is because it causes fewer adverse effects than other non-steroidal anti-inflammatory drugs like aspirin. For example, unlike synthetic aspirin, willow bark does not damage the gastrointestinal mucosa, and the highest recommended dose does not have any major impacts on clotting. One explanation may result from willow bark having a different metabolic pathway than aspirin has. Aspirin is mainly metabolised into salicylic acid, whereas willow bark enters the body as inactive salicin which is then metabolised to salicylate derivatives. When calculated as having the same molecular weight as salicylic acid, the salicylate derivatives from a 240 mg salicin dose corresponded to 100 mg of aspirin, a fifth of the typical dose. Therefore, the lower concentration may have reduced adverse effects on the body.[5,6] In addition, the extract also contains other compounds, like polyphenols and flavonoids, which not only reduce the overall concentration of salicin but also contribute to analgesic and anti-inflammatory effects, providing a broader mechanism of action and therefore a reduced risk of serious adverse events, as well as a reduced impact on blood clotting.
White willow bark is commonly given for conditions associated with pain, inflammation, or fever, such as joint or knee pain, acute back pain, osteoarthritis (OA), headaches, menstrual cramps, tendonitis, and flu symptoms. Regenerative, non-surgical treatments by have also been shown to be effective in the treatment of various chronic pains, learn more on a Regenerative Medicine Clinic site. In vitro studies and animal models have shown that anti-inflammatory activity is linked to downregulation of inflammatory mediators in the body such as tumour necrosis factor-alpha (TNFɑ) and nuclear factor-kappa-beta (NF-κβ); therefore, willow bark may function by reducing the effectiveness of these factors to subsequently lower the inflammatory response. Willow bark extract contains in total 16 different compounds with possible therapeutic effects. Catechin and amelopsin, in particular, are known to have high antioxidant and free radical-scavenging activity; other compounds have also been implicated to possess antiseptic and immune-enhancing properties. These anti-inflammatory and analgesic effects may potentially explain why willow bark extracts are now also used in sports performance and weight loss products; however, there is still a lack of supporting evidence to confirm their effectiveness.
On the other hand, it must be kept in mind that willow bark, like any drug, is not completely safe, and various cautions and warnings have been issued regarding its use. It is suggested that people with gastritis, stomach ulcers, diabetes, asthma, or haemophilia should avoid taking willow bark extract. Drug interactions are also theoretically possible if willow bark is taken simultaneously with other drugs. For example, there may be an increased risk of bleeding with anticoagulants, an increased risk of stomach bleeding with NSAIDs, and a reduced effectiveness of beta blockers and diuretics. It is also advised that children under the age of 16 should not take the extract because of the potential of developing Reye’s syndrome, a disorder that can cause serious damage to the liver and brain. Although there has been no evidence of willow bark extract leading to such adverse side-effects as of yet, there is still the possibility of an allergic reaction in salicylate-sensitive individuals. Therefore, willow bark products are contraindicated in aspirin allergic patients; a case of severe allergic reaction was reported in the study by Chrubasik et al., and the authors suggested that the bark extract may have caused the reaction.[5,8]
Studies Involving Willow Bark
Numerous studies have explored the medicinal properties of willow bark. For example, Chrubasik et al. carried out a randomized four-week double-blind study with 210 patients, where 39% of those who took a high dose of oral willow bark extract containing 240 mg of salicin found relief from their lower back pain exacerbations, as did 21% of those who took a 120 mg dose. Although willow bark appears effective, less than half of the patients in the high-dose group experienced pain relief, which puts the feasibility of the treatment into question. However, it seems to have its effect only in high doses on a selected group of patients. The non-surgical treatment offered by qckinetix.com/greensboro/ has better and immediate effect on painful areas. In addition, Schmid et al. compared willow bark extract at its highest recommended dosage to a placebo in a two-week double-blind randomized controlled trial and found that willow bark could reduce pain scores by 14% as compared to the 2% increase seen in the placebo group. However, a different study conducted by Biegert et al. of the effects of willow bark on patients with rheumatoid arthritis and osteoarthritis determined that unlike using aspirin, there is no evidence that taking the highest recommended dosage of willow bark could result in significant statistical differences in the mean reduction of pain between the willow bark and placebo groups. Both Schmid et al. and Biegert et al. used the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC index) as a measure of pain for OA, which allows for a valid basis of comparison between the studies on the measurement of pain level. In addition, both have similar sized experimental groups (Schmid et al.: 39 patients, Biegert et al.: 43 patients) which again makes direct comparison between the two studies easier. Since the study of Biegert et al. lasted for longer (six weeks), this may suggest that the effects of willow bark decrease in the long term. It is important to note that although all studies use willow bark extract, there is no specification of age or region of bark used, which may affect the results.
This study aims to determine the type of willow that produces the highest yield of the active ingredient salicin. Willow bark of different ages were initially compared: fresh (green) willow bark and old (brown) bark. The sample that produced the highest yield was then used to determine whether there were any statistically significant differences in the yields of salicin from different parts of the branch: bark and twig.
Extracting salicylic acid from willow bark
The method is based on the protocol detailed in activity WM2.1 ‘Extraction of an active chemical from willow bark’ from the A2 Salters Chemistry scheme of work. The investigation consisted of two parts: the first was to see which age of bark produced the highest yield of salicin, and then using this age, the bark and twig of a willow branch were compared to see which contained more of the active ingredient. The plant species used in this investigation was the tortuosa variant of the Salix matsudana twisted willow.
The bark was prepared by initially prying it away from the branch using a knife, cutting as thinly as possible so that only the bark was collected rather than the twig. Next, the pieces were finely cut into thin strips in order to maximise the surface area available for reaction. A 2.3 g sample of the aged brown bark was placed into a 50 cm3 pear-shaped flask which was then filled with a 30 cm3 mixture containing 15 cm3 of 2.0 moldm-3 sulphuric(VI) acid and 15 cm3 of 0.2 moldm-3 potassium manganate(VII) solution. This was done to mimic processes in the body; willow extract is initially hydrolysed by enzymes in the stomach, and the degraded products are then oxidised in the bloodstream to form active salicylic acid from salicin. In this experiment, the sulphuric acid causes the hydrolysis, while the potassium manganate(VII) solution causes the oxidation; the reduction of manganate ions results in a colour change as purple MnO4– ions are converted to colourless Mn2+ ions. The flask was set up with a reflux condenser as shown in Figure 1, and the mixture was refluxed for 15 minutes. The colour changes in the solution were noted before the flask was allowed to cool and the concentration of salicylic acid produced was measured. The process was repeated for the fresh willow bark.
Once it was established that aged willow bark produced a greater concentration of salicin, the second part of the experiment repeated the procedure but with the twig instead. When preparing the sample, it was ensured that the exterior bark was completely removed to avoid contamination.
Figure 1: Diagram of the reflux condenser and flask apparatus used. Modified from protocol.
Before the concentration of salicylic acid in the reaction solution could be determined, a calibration curve needed to be created to estimate the concentration produced depending on the absorbance.
0.2 g of salicylic acid was dissolved in a 100 cm3 solution of ethanol and water in a 50:50 ratio. This produced a stock solution, and six different concentrations of salicylic acid were produced using the same ethanol and water solution as the diluting agent as shown in Table 1:
Table 1: Concentrations of solutions used to construct the absorbance calibration curve.
Volume of diluting agent (cm3)
Volume of stock solution (cm3)
Concentration of salicylic acid solution (g/100cm3)
Absorbance measured at 520nm
The colorimeter was set to a 520 nm filter, and 4 cm3 of distilled water in a cuvette was used to zero the reading. The other cuvettes were filled with 2 cm3 of the remaining solutions (solutions 2-6), and 2 cm3 of iron (III) chloride solution was added to each cuvette. The addition of iron (III) chloride is a test for the phenol group present in salicylic acid but not in salicin (Figures 2 and 3). If the group is present, a purple, blue or green colour change will confirm this. The intensity of the colour produced then affects the absorbance values measured and therefore is responsible for the differing results of each concentration of salicylic acid produced.
Figure 2: Chemical structure of salicylic acid with the phenol group circled in blue. Modified from PubChem.
Figure 3: Chemical structure of salicin. As can be seen from Figure 3, the -OH group is attached to a methyl group, so the phenol is not present. Therefore, salicin will result in a negative test upon treatment with iron chloride. Modified from PubChem.
The recorded absorbance for each concentration was used to plot a calibration curve (Figure 4).
Figure 4: Graph of colorimetry results showing the concentration curve (absorbance vs concentration)
Measuring the concentration of salicylic acid produced
After the reaction solution cooled, a new cuvette was filled with 2 cm3 of the reaction solution and 2 cm3 of iron (III) chloride solution as before. The colorimeter was adjusted to the same settings and zeroed with 4 cm3 of distilled water before measuring the absorbance of the reaction solution. The concentration of salicylic acid was then calculated using the line of best fit from the concentration curve.
Two measurements were taken: the first was when the solution was not agitated after refluxing, and the second was after the mixture was agitated. In order to avoid contamination of the different concentrations of salicylic acid solutions which would affect the results, fresh cuvettes and plastic pipettes were used for the different solutions.
Throughout this experiment all variables were controlled apart from the independent variables, which were the age and region of willow sampled for investigation. The dependent variable was the concentration of salicylic acid, and the control variables were the mass of willow used, time for reflux, wavelength of the filter used in the colorimeter, and the volumes and concentrations of the solutions, both used in the reflux reaction and in the construction of the calibration curve.
Calculating the concentration of salicylic acid
The equation, y=9.9734x, generated by the concentration curve (Figure 4) was used to calculate the concentration of salicylic acid in the samples. An example is outlined below using the absorbance value of 0.9 from one of the non-agitated samples of aged willow bark.
y = 9.9734x
0.9 = 9.9743x
x = 0.9 / 9.9734
x = 0.09 g/100 cm3 salicylic acid
Age of the bark
The results show that aged S. matsudana bark contained a higher concentration of salicin than fresh bark. When the mixture was not agitated, old willow bark produced a concentration of salicylic acid 0.045 g/100cm3 higher than that of fresh willow bark; this was an 81.8% increase in salicylic acid. When the mixture was agitated, the concentration produced by old willow bark was 0.043 g/100cm3 higher than that of fresh willow bark; here there was a 66.2% increase in salicylic acid. All samples of willow bark had a greater measurement of salicylic acid after the mixture was agitated.
The results show that after agitation of the mixture, the percent increase of salicylic acid produced was lower than that of the non-agitated mixture. This may be because initially after the reaction goes to completion, the salicylic acid collects at the bottom of the flask. This means that after the mixture is agitated by gentle shaking of the pear-shaped flask, the concentration becomes more uniform, so the difference between the concentrations is less. The results can be seen in Figures 5 and 6.
Figure 6: Graph to show the concentration of salicylic acid in differently aged willow bark. The solution was agitated before the measurement was taken.
Part of willow branch
Old willow was used to measure the difference of salicin content in the bark and twig. The results show that the bark contained more salicin than the twig did. The bark had a higher concentration of salicylic acid by 0.065g/100 cm3 when the mixture was not agitated; this was 185.7% more salicylic acid than that of the twig. The bark also had a higher concentration of salicylic acid than the twig had by 0.058g/100 cm3 when the mixture was agitated; this was a 116.0% increase in salicylic acid. This massive percent increase shows that bark produced remarkably more salicylic acid than twig regions did; this can be verified with further statistical testing. Additionally, both the bark and the twig samples had a greater concentration of salicylic acid after the mixture was agitated, which highlights the importance of creating uniform concentration. The results can be seen in Figures 7 and 8.
Figure 7: Graph to show the concentration of salicylic acid in different parts of a willow branch. The solution was not agitated prior to measurement.
Figure 8: Graph to show the concentration of salicylic acid in different parts of a willow branch. The mixture was agitated before the measurement was taken.
There are a number of possible explanations for the results obtained in this investigation.
One explanation for the result of aged willow bark containing more salicin is that fresh willow bark is still growing and therefore requires a greater supply of water from the xylem vessels. This means that when 2.3g of each willow bark sample was used in the experiment, a greater proportion of the mass of the fresh bark consisted of water. As a consequence, there was less salicin available for reaction, and so in comparison, more salicylic acid was produced by aged bark. It may be worthwhile in the future to repeat the experiment using only the dry mass of willow bark in order to determine whether water content is a significant factor and to control any possible confounding effects.
Another explanation may be related to secondary metabolite production. Secondary metabolites are organic compounds such as terpenes and glycosides, that are often regarded as not having direct roles in the maintenance of fundamental processes carried out by the plant, but instead play an important part in interacting with the plant’s environment as well as in adaptation and defence. There is evidence to show that abiotic stress factors strongly influence secondary metabolite production as well as growth in plants. Salicylic acid is one of the secondary metabolites involved in plant stress responses, which may explain the results, as aged willow bark would have had more exposure to environmental stress factors and therefore more salicin may have accumulated over time. In addition, nutrient stress and potassium, sulphur, and magnesium deficiencies all have an effect on phenolic levels which may be linked to salicin, as salicylic acid is a phenolic phytohormone. Furthermore, the older the willow bark, the higher the chance that it has been subject to pathogenic attack. Salicylic acid is known to be involved in the process of systemic acquired resistance; thus, over time it may build up in the plant tissue. The defensive response is initiated by salicylic acid which then also has a role in inhibiting viral replication, interfering with viral spread, and triggering the production of pathogenesis-related (PR) proteins that provide protection against viruses. Moreover, as mentioned earlier, salicylic acid also acts as a hormone and in some cases infections in one part of the plant cause the delivery of salicylic acid to other regions of the plant to promote PR protein synthesis before the infection can spread.
However, another source details a methodology to detect salicylic acid in willow bark and provides a suggestion to use young willow branches which contrasts the findings that this study implies. The authors of the article published in the Journal of Chemical Education specify to use “only freshly fallen branches having a green inner bark”, and they add details about a fixed length and branch diameter to control the mass of willow used for extraction. The authors do not include a reason for this, but there is the possibility that they may have previously seen fresh branches generate a greater yield of salicylic acid, which differs from the findings of this study. It may be worthwhile to repeat this investigation to collect more data in order to form stronger conclusions about which age of bark produces more salicylic acid.
In all graphs produced (Figures 5 to 8), the error bars for the concentration produced from old bark samples were greater than that of the young bark and twig samples. However, this is because repeats were not carried out for the fresh bark and old twig samples, so error bars are not applicable in this aspect of analysis. The error bars of the old bark samples do not overlap with the values for fresh bark and old twig samples, which indicates that the differences in concentration produced from the different samples of willow may be statistically significant. In the context of the aim, this suggests that old willow consistently contains more salicin than young willow does, with old willow bark containing the most out of all samples. However, this can only be confirmed with more statistical testing (for example, student’s t test) to consolidate conclusions, as well as further experiments using samples of specific known age instead of basing age on physical appearance. For further developments, it is necessary to include repeats for all types of samples in the experiment and compare data to assess for anomalous results, thus allowing more reliable means to be calculated and used in statistical tests.
This study shows that the old bark of twisted willow produces a higher yield of salicylic acid than fresh bark and old twig regions do. Although this wasn’t measured, it is likely that fresh twig contains lower concentrations of salicylic acid as well. Explanations for this may involve the additional water content in fresh bark and the production of secondary metabolites, which potentially accumulate in the bark. However, other methods suggest using young willow branches for acid extraction, so it may be useful to alter the investigation in further studies to gather more data and calculate mean values for a more concrete evaluation of the differences between young and old willow. This investigation highlighted the first step in distinguishing how age affects salicin yield, which may prove useful in the making of willow-based home remedies. There are a number of sources dedicated to using willow bark in such a way, so this experiment will help inform about which part and age of willow plant has the highest yield of active ingredient.[18,19] Future work may also include investigating whether there is a significant difference between salicin concentration in the bark in different seasons as well as comparing different species of willow; this would help discern the best time in which to harvest the bark and the best species to use. Although more work is required to refine the results, this study has explored which part of willow contains the most active ingredient and therefore would be most useful in preparing home remedies or performing classroom experiments to extract salicylic acid, the basis of aspirin.
I would like to thank Dr Paine who supervised this investigation.
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About the Author
Anna has been interested in science for a long time and now she is studying Medicine at University. Although she particularly enjoys reading accounts written by doctors, Anna also likes reading a wide variety of fiction, from historical novels to light-hearted romance. Her other hobbies include badminton and playing the clarinet.