Witch Hazel Bark Hydrosol, Hamamelis virginiana
Kaitlyn Jang Honors Paper 2023.
Abstract
Witch hazel is a flowering shrub native to Canada. Commonly known as a natural astringent due to its ability to soothe inflammation and irritation of the skin, the bark, leaves, and twigs of the plant are extracted and widely used in the cosmetic industry as topical treatments to soothe common skin irritations such as sunburns and eczema. Previous efforts have also been undertaken to study the anti-inflammatory and anti-proliferative effects of witch hazel; some of these studies investigated the inhibitory effect of witch hazel bark extracts on the proliferation of tumoral cells.
This project aims to characterize the chemical components responsible for the anti-inflammatory character of witch hazel through the analysis of witch hazel bark hydrosols using gas chromatography mass spectrometry (GC-MS). Witch hazel bark was extracted by microwave distillation and hydro-distillation. Samples were prepared by solid phase extraction to substitute the aqueous solvent using methanol, before being analysed by GC-MS. To account for variability in injection volumes, an internal standard was added to the samples at six different known concentrations to calibrate and determine chemical concentrations. Three trials of witch hazel bark sample analysis were conducted to ensure the standardization, accuracy, and reproducibility of results. Compounds were then identified using the Kovats retention index to normalize corresponding peak retention times.
The main components of witch hazel bark were identified to be hexanoic acid, alpha-bisabolol, and cadinol.
Additionally, through minimum inhibitory concentration assays, it was found that witch hazel hydrosols were unable to inhibit the growth of both gram-negative and gram-positive bacteria.
My name is Kaitlyn and I am in my final year of the biopharmaceutical sciences program, with a specialization in medicinal chemistry.
For my honours project I was studying the hydrosols of witch hazel bark.
I was already familiar with witch hazel, particularly I have used skin care products containing witch hazel.
During my project I characterized the compounds in witch hazel responsible for its anti-inflammatory effects, and its other various benefits.
In my free time, I enjoy working out , hiking, and baking sweets with friends and family.
In recent years, more interest and research efforts have been emphasized on natural products, mainly due to their numerous properties. Natural products are currently being applied in various areas such as the cosmetic, food, and pharmaceutical industries.
Aromatherapy is a holistic therapy that uses essential oils that are extracted from plants, flowers, and spices as a form of a natural therapeutic agent to solve many physical and mental health illnesses1. These essential oils are linked to possessing many antibacterial, antiviral, and antioxidant benefits, thus making them a promising natural remedy to combat various diseases2. Essential oils are primarily extracted using distillation methods. During the distillation, only a small amount of essential oil is created, while an aqueous by-product of the plant extraction remains. This by-product is called hydrosol, also known as an herbal distillate or essential water.
Since it is the leftover product from the distillation of plant material, it is said to contain trace amounts of terpenes and terpenoids found in the oil, as well as other hydrophilic compounds3. Terpenes are a class of compounds that give most plants their aromas and flavours. They are classified into mono, tri, di, and sesquiterpenes by the head-to-tail linkage of isoprene units.
1. Thomas, D. V. Aromatherapy: Mythical, Magical, or Medicinal? Holist Nurs Pract 17, (2002).
2. Sharma, M. et al. Essential oils as anticancer agents: Potential role in malignancies, drug delivery mechanisms, and immune system enhancement. Biomedicine & Pharmacotherapy 146, 112514 (2022).
3. Lante, A. & Tinello, F. Citrus hydrosols as useful by-products for tyrosinase inhibition. Innovative Food Science & Emerging Technologies 27, 154–159 (2015).
Contribution of Hydrosols
Essential oils have been extensively used in aromatherapy. During the distillation process to produce essential oils, an immense amount of hydrosol is produced and commonly discarded.
Since they are easily produced and safe for human use, in recent years more research has been conducted to see if they can produce similar effects as their oils.4
Previous studies suggest that hydrosols can be used as antimicrobials to prevent microorganism in the foods. These results demonstrated that common hydrosols, such as carrot, basil, rosemary, and cumin possess antifungal, antibacterial, and antioxidant activity5. In addition, sideritis, bay leaf, thyme, summer savory, oregano, rosemary, and salvia hydrosols showed ability in decontaminating potential pathogens in fresh iceberg lettuce4.
Hence, these results further suggest hydrosols can have more in-vitro industrial applications.
Overview of Witch Hazel
Witch hazel is a flowering shrub in the plant family of Hamamelidaceae.
Hamamelis virginiana, also known as American witch hazel, is native to Canada, and can be found in the woods of Nova-Scotia, Quebec, and Ontario. Historically, witch hazel was used by early settlers and Indigenous peoples as a natural remedy since it was said to possess medicinal properties. The twigs, leaves, and bark of the plant were used to make teas to soothe common colds, fevers, and were used as a topical treatment to treat skin conditions6.
Today, witch hazel is commonly used as a natural astringent and is widely used in skincare products to treat acne, sunburns, and irritated skin. The presence of tannins in witch hazel distillates is what mainly contributes to its astringent properties7. Tannins are polymeric phenolic compounds, found in either condensed or hydrolysable forms in plant metabolites8. The quantity and type of tannins present can depend on the part of the witch hazel being extracted; this variability can affect the compounds present in the extract as does the extraction method.
We are extracting using distillation which will only extract the volatile molecules, namely the terpenes and terpenoids.
Ozturk, I., Tornuk, F., Caliskan-Aydogan, O., Durak, M. Z. & Sagdic, O. Decontamination of iceberg lettuce by some plant hydrosols. LWT 74, 48–54 (2016).
5. D’Amato, S., Serio, A., López, C. C. & Paparella, A. Hydrosols: Biological activity and potential as antimicrobials for food applications. Food Control 86, 126–137 (2018).
6. Fulling, E. H. American witch hazel—History, nomenclature and modern utilization. Econ Bot 7, 359–381 (1953).
7. Wang, H., Provan, G. J. & Helliwell, K. Determination of hamamelitannin, catechins and gallic acid in witch hazel bark, twig and leaf by HPLC. J Pharm Biomed Anal 33, 539–544 (2003).
8. Dable-Tupas, G. et al. 11 - Bioactive compounds of nutrigenomic importance. in Role of Nutrigenomics in Modern-day Healthcare and Drug Discovery (eds. Dable-Tupas, G. & Egbuna, C.) 301–342 (Elsevier, 2023). doi:https://doi.org/10.1016/B978-0-12-824412-8.00003-5.
Microwave extraction is an extraction technique that utilizes the rapid heat conducted from a microwave to create steam. An ice cone is added to the top of the vessel to condense the steam and a beaker is placed in the vessel to collect the hydrosol.
This extraction technique uses about 200 grams of plant witch hazel powder that was soaked in 800 ml of deionized (DI) water before being extracted. The microwave extraction method was altered due to the dry woody nature of the bark. Soaking the bark over night, seemed to help soften the wood and leech the chemicals more readily.
Each extraction can be heated in the microwave for about 10-11 min at high power, with a total of 3 extractions per 200 g of witch hazel plant material.
Hydro distillation is a traditional extraction method commonly used to extract essential oils.
The witch hazel bark was soaked overnight in 1000ml of water, to soften and aid in the leaching of the chemical compounds within the wood.
The plant product is immersed in water and brought to a boil. To extract the bioactive compounds in the plant material, the water vapour from the plant mixture is condensed within the copper tubing on top of the vessel. The condenser was kept cold with a water/ice slurry. The vapour is condensed into a receiving flask at the end of the copper tubing.
The extracts were processed through the SPE cartridges , which were purchased from Chromatographic Specialities https://chromspec.com/. Clean-UP C18 extraction columns (CEC18123) , encapped. 5ml of hydrosol was passed through each cartridge, an initial test of 1ml, proved too dilute.
4 x 250ul methanol/ACN was used to extract the chemicals.
This was used in the GC-MS to quantify and identify the compounds present in witch hazel.
To calculate how much actual chemical we can extract in 1L, 250ml of hydrosol was also passed through larger 6ml cartridge, (C612053) and extracted with 4 x 0.5ml of methanol/ACN. The Methanol/ACN was dried off in a 40C oven over night and the contents weighed.
Internal Calibration.
A number of external calibrations have been collected on the instrument.
This thesis outlines a new internal calibration method.
To ensure precision of results and to control GC-MS variability, all batches were calibrated using an internal standard. The internal standard used in this project was 4-ethylguaiacol, supplied from Sigma-Aldrich (Oakville, ON, Canada). This compound does not interfere with the sample and elutes from the column at a known retention time (RT) of 9.7 minutes. It is similar to eugenol but is not a compound found in essential oils to our knowledge.
Batches were calibrated using six different concentrations from 0.025 ml/ml to 1.0 ml/ml of internal standard. This was added at the same time as the hydrosol to the SPE cartridge and would therefore experience the same process as the hydrosol.
Calibration curves were prepared by plotting each concentration as a function of the area under the curve of the produced chromatogram. A trendline was formatted to produce an equation of the line and the concentration of the compounds were calculated using this equation.
This method was tested with 3 different batches of hazel hydrosol and three repeats in the GC-MS.
The picture shows two of those calibrations curves generated from batches one and two.
The repetition of the results allowed for confirmation of the instrument precision, the number of batches tested the hydrosol uniformity and the sum of all the compounds allowed for the averaging of all the areas adding more points to the calibration too.
RESULTS
Batches 1 and 2 were very similar having a number of small acids and a couple of oxygenated sesquiterpenes. They were both at very low concentrations.
With Batch 3, it was decided to soak the bark over night in boiling water. This produced a much more concentrated hydrosol with more chemicals present in the GC-MS.
Batch 3, was significantly different than batches #1 and 2. Common monoterpenoids, such as alpha-terpineol and eucalyptol were found in this batch. Additionally, sesquiterpenoids such as β-Nerolidol and spathulenol were found as well. This result could suggest that soaking the witch hazel bark in boiling water before being extracted using hydro distillation produces more terpene and terpenoids. Since the plant product is a bark and not a leaf there is the possibility it is not as concentrated as the leaf. Alpha-terpineol and eucalyptol are both very common terpenoids found in many essential oils, such as black cardamom, lavender, and rosemary.
The disappearance of the small alcohols is strange and needs further investigation.
After extracting three batches of witch hazel and having two different sets of results, alpha-bisabolol and cadinol were the only two compounds found in all three batches. In batch 1 and 2, higher average peak areas under the curve for alpha bisabolol than cadinol was present as seen in the picture. However, batch 3 resulted in the reverse, and more cadinol was found present than alpha-bisabolol.
Sales, A., Felipe, L. de O. & Bicas, J. L. Production, Properties, and Applications of α-Terpineol. Food Bioproc Tech 13, 1261–1279 (2020).
