Barker College
The medicinal benefits of garlic have been extensively studied and attributed to the medicinally active thiosulfinate, Allicin. This report outlines an investigation into whether temperature can be used to maximize the concentration of allicin in garlic, potentially maximising the medicinal benefits an individual can receive from consuming raw garlic. This investigation observed the effect of temperature on the concentration of allicin in raw garlic cloves, by using a spectrophotometric method that is sensitive enough to measure concentration of allicin in the micromolar range. The garlic cloves were subjected to different temperatures before the garlic was dehydrated and crushed into a fine powder and placed in solution. The data was recorded by first reacting the garlic extract with excess Lcysteine and then 5,5’-dithiobis-2-nitrobenzoic acid which measured the decrease in cysteine concentration. The results of the experiment found that temperature did not have a significant effect on the concentration of allicin measured when garlic cloves were stored over a period of four days.
Garlic (Allium sativum L.) has been applied to culinary and medicinal purposes in modern and ancient practices (Peyman et al. 2013; Gaber et al., 2020; Azene, 2015). Garlic contains a rich source of organosulfur compounds responsible for its flavour and aroma as well as its health benefits (Oregon State University, 1985). Specifically, allicin, an organosulfur compound in garlic is responsible for the majority of the pharmacological activity of crushed raw garlic cloves (Lawson and Hunsaker, 2018), as it is the most biologically active compound in garlic (Rahman, 2007). Allicin, most commonly found in raw garlic, is known to reduce inflammation and offer antioxidant benefits (Bahare, 2019). Additionally, research has focused on allicin’s antimicrobial properties, which has found that allicin in its pure form exhibits antibacterial activity against a wide range of Gramnegative and Gram-positive bacteria, antifungal activity and antiparasitic activity against major human intestinal protozoan parasites such as Entamoeba histolytica and Giardia lamblia and antiviral activity (Ankri and Mirelman, 1999).
Allicin is formed immediately in raw garlic as a self defence mechanism when the clove is damaged by worms, fungi, bacteria, or by physically crushing the clove (Leontiev et al. 2014). Allicin is produced by the precursor molecule, alliin (an amino acid) being converted into allicin by action of the alliinase enzyme (Figure 1) (Chhabria and Desai, 2018). The alliinase enzyme is located in the space between garlic cells, whilst alliin is located within the garlic cells themselves. This means that alliin and the alliinase enzyme can only interact to form allicin once the cell walls of the garlic have ruptured (Janská et al., 2021).
Figure 1: Formation of allicin from alliin catalysed by the alliinase enzyme.
Allicin is known to be a highly unstable and volatile organosulfur compound due to the presence of the thiosulfinate functional group on the molecule (Abe, Hori and Myoda, 2019). This makes allicin heat sensitive, as it rapidly decomposes in the presence of air and water into an abundance of volatile thiosulfinate derivatives (mainly vinyl dithiines, ajoenes and allyl sulfides) (Figure 2) (Cheewinworasak et al., 2018). This is why there is an inability to ensure a certain abundance of allicin is present within a garlic clove.
Research into the factors that influence the concentration of allicin have been vital in understanding the unstable nature of the allicin compound. A study by Wang et al. (2015) as well as a study by Lawson and Hughes (1992) investigated the influence of pH, concentration and light on the stability of allicin in garlic after crushing. Both
Figure 2: Degradation of allicin (After: Oregon State University, 2021)
studies found that at room temperature, allicin in aqueous extract was most stable at pH 4.5-6, however, allicin exposed to pH levels outside this range began to degrade within 30 minutes and were undetectable within 2 hours when the pH was higher than 11 and lower than 3.5. Additionally, both studies found that allicin extract was sensitive to pH and temperature but not to visible light. The paper by Wang et al. (2015) observed that at these temperatures, higher concentrations of allicin in water could be kept for up to five days without obvious degradation.
Research has also looked at the thermal degradation of allicin and the implications on its bacteriostatic properties. Canizares and co-workers (2004) established a link between effective inhibition of the bacterium, Helicobacter pylori (Hp), and the temperature the aqueous garlic extracts were stored at (6 °C, 19°C, 21°C and 26°C) after crushing of the raw garlic material. The research concluded that allicin extract stored at lower temperatures (6 °C) showed the greatest inhibition of the in-vitro growth of Hp and the bacteriostatic properties remained active for up to 10 months of storage at this temperature.
Moreover, research by Mansor et al. (2016) has looked at the effect of different storage temperatures of garlic powder on the degradation of allicin within the range of 30°C - 85°C. This paper has shown that allicin is most stable at temperatures around 30 °C, only having a slight reduction over time. However, at higher temperatures around 70°C-85°C, the organic compound allicin decomposes rapidly (as seen in Figure 3).
In addition, a study by Mathialagan et al. (2017) ‘obtained result(s) in accordance with the findings from Mansor et al. (2016) on the thermal stability of Allicin’ (Mathialagan et al. 2017 p.g.1750). Both found that after raising the extraction temperature over 35 °C there was a significant amount of allicin deteoration. In another study by Fujisawa et al. (2008) allicin in an aqueous extract degraded stoichiometrically in proportion to its temperature over the range 4° C - 42° C, and from this the half-lives were estimated to be a year at 4°C (degrading from 1.8 mg/ml to 0.9 mg/ml), 32 days at 15°C and 1 day at 37°C (degrading from 2.0 mg/ml to 1.0 mg/ml).
Figure 3: Allicin content vs. time at various temperatures (Source: Mansor et al., 2016)
There have been no previous reports investigating how a change in the conditions the cloves are exposed to before crushing affects the concentration of allicin after the garlic is crushed. This paper investigates whether a change in storage temperature of the garlic cloves would affect the concentration of alliin precursor, which would result in a change in allicin concentration after crushing. This would aid in understanding how simple adjustments to the storage conditions the garlic cloves are subjected to can enhance the allicin concentration. Thus, maximising the medicinal benefits an individual can receive from consuming garlic. Therefore, this paper investigates the effect of temperatures achievable in a domestic setting on the concentration of allicin.
Literature methods for the analysis of allicin content in garlic rely on preparing a stable powder and then extracting the allicin for analysis. Commonly, convective hot air-drying is used to dehydrate garlic. By dehydrating garlic, allicin stability is improved as the reduction in water content considerably minimizes physical, chemical and microbiological degradation during storage which allows for the preservation of allicin content once the garlic has been crushed to form the allicin (Papu et al., 2014). There are several methods reported in the literature for quantifying the allicin content in garlic powder (Ranitha, 2016; Bernhard et al. 1990; Bose, 2014). The simplest method, first reported by Han et al. (1995) is the spectrophotometric method, where allicin is converted into a coloured compound and subsequently analysed using a colorimeter to determine the absorbance and hence the concentration in solution. A benefit of this method is that it does not require an allicin standard to quantitate allicin, instead it relies on the Beer-Lambert law which states that there is a linear relationship between the absorbance and concentration for substances which are able to absorb within the UV/visible region of light (Figure 4). The relationship is expressed as A = εlc, where A is the absorbance, ε is the molar absorptivity (M-1cm -1), l is path length (cm) of the cuvette and c is concentration (mol/L) (Figure 5).
Methods which make use of High Performance Liquid Chromatography (HPLC) (Ranitha, 2016) and Gas Chromatography (GC)(Koichi, 1989) for analysis are also commonly reported in the literature, however, we do not have access to the required equipment to carry out this analysis.
How does the storage temperature of garlic effect the concentration of alliin, and hence the concentration of allicin present in garlic?
Figure 3: Calibration curve which relies on the Beer Lambert law demonstrating the relationship between concentration and absorbance (After: UNC Eshelman School of Pharmacy, 2021)
Figure 4: Principles of the Beer-Lambert Law (Source: Brian McNamara, 2018)
That temperature will have an effect on the concentration of allicin extracted after storage at different temperatures.
5,5’-dithiobis-2-nitrobenzoic acid (DTNB), 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and L-cysteine were purchased from Sigma Aldrich. Five bulbs of garlic and five small plastic bags were purchased from The Veggie Patch (local grocery store). The papery skin of the garlic bulbs was peeled and the cloves were separated from the bulbs. The cloves were placed in a bowl and mixed together. Ten cloves were randomly selected and placed in each of the five plastic bags (50 cloves in total). Each bag was assigned a label: Freezer, Fridge and Room temperature. These bags were then stored at their respective temperatures and were exposed to the same light conditions for four days.
Both ends of the garlic cloves were chopped. The cloves were peeled and the peels were discarded. The garlic cloves were then sliced into uniform widths of 2mm. The sliced garlic was then spread onto a lined tray (aiming for as close to a single layer as possible), with sliced garlic from each assigned bag being kept separate. The tray was placed in the oven at 50 °C for 4 hours. The dehydrated garlic was then removed from the tray and allowed to cool at 25 °C for 10 minutes, then pulverized in a mortar and pestle until it was a fine powder.
Preparation of 5.0 mM DTNB solution 0.404g of DTNB powder was weighed using a mass balance. The weighed powder was transferred into a 200 mL glass bottle and dimethyl sulfoxide (DMSO) (200 mL) was added. The container was wrapped in foil and put into a dark cupboard.
Preparation of HEPES buffer To make the 1.0 M HEPES buffer at pH 7.6, a pH-meter was calibrated by placing it into a pH 7.0 buffer solution. Then 23.8g of HEPES buffer powder was weighed into a clean, dry beaker using an electronic balance. Deionised water was used to dissolve the HEPES and the solution was transferred to a 100 mL volumetric flask with the final desired volume made up to 100mL with distilled water. A magnetic stirrer was used for approximately 10 minutes to dissolve the HEPES, with sodium hydroxide slowly added to the solution via a glass pipette whilst constantly measuring the pH using the pH meter until 7.6 was measured. Preparation of 50 mM HEPES buffer 5.0 mL of 1.0 M HEPES buffer is introduced into an empty 100 mL volumetric flask and the final volume was made up to 100 mL with deionized water.
Preparation of 2.0 mM L-cysteine 0.096 g of L-cysteine was weighed using an electronic balance. The crystals were then transferred into a 500 mL glass bottle and 400 mL of deionized water was added into the flask. The mixture was stirred until a solution was obtained.
10 g of garlic powder was dissolved in 300 mL of deionized water. The mixture was stirred at room temperature using a magnetic stirrer for 1 hour. Finally, insoluble solids were filtered under vacuum using a sintered glass funnel to collect the dissolved allicin in the filtrate.
Then 0.5 mL of garlic extract was added to 1.2 mL of 2 mM L-cysteine and allowed to sit at room temperature for 10 minutes. Then, 3 mL of 50 mM HEPES buffer (pH 7.6) and 1 mL DTNB was added to the solution where it was then stirred and allowed to sit for 2 minutes at room temperature. Absorbance of the samples were read at 450nm with a colourimeter to obtain the concentration of excess L-cysteine remaining in the sample (seen in Figure 6).
Figure 6: Allicin reacting with L-cysteine and DTNB to form a yellow compound
Since the absorbance of the solute varies due to its concentration the relationship is expressed as:
A = εlc,
where for this specific experiment - A = absorbance measured by the colourimeter at 450nm (maximum absorbance wavelength for the yellow compound) - ε = molar absorptivity which is 14150 M-1cm -1 (The constant value specific for absorption of yellow compound) for this experiment - l = path length of the cuvette (1 cm therefore equals to 1 in the experiment) - c = concentration in mol/L
Since the concentration of the excess L-cysteine is twice the concentration of allicin (allicin reacts with L-cysteine in a 1:2 ratio).Concentration of allicin is therefore defined as <the concentration of yellow compound> subtracted from <the initial concentration of L-cysteince>all divided by two.
Table 1: Mean and standard deviation for each group temperature
Control (25°C) 2.2925x10-4 1.1274 0.0012 Room Temp (25°C) 2.2946x10-4 1.1284 0.0011 Fridge (3°C) 2.2960x10-4 1.1291 0.0014 Freezer (-18°C) 2.2948x10-4 1.1285 0.0003
The mean concentration allicin (mg/g) was calculated by multiplying the mean concentration (mol/L) by the molecular mass of allicin (162.28 g/mol) and by a factor of 1000 to get mg/L. This value given in mg/L was then divided by 33g (as there was 33g of powder in each litre of water) to give a value in mg/g.
Table 2: ANOVA test output for comparing the concentration of allicin after being stored at selected temperatures
H0= There is not a statistically significant difference between the concentration of allicin extracted from garlic cloves stored at different temperatures
HA= There is a statistically significant difference between the concentration of allicin extracted from garlic cloves stored at different temperatures
F-stat 2.1135 α 0.05
P-value 0.1387
Analysis The result is not significant as p > 0.05
The ANOVA statistical analysis (Table 2) has a p-value of 0.1387, since the p > 0.05, the null hypothesis (H0= There is not a statistically significant difference between the concentration of allicin extracted from garlic cloves stored at different temperatures) was accepted. This indicates that temperature of garlic cloves does not play a major role in influencing allicin concentration, in contrast to the effect of temperature on the degradation of allicin. The standard deviation for each of the four groups is small, indicating that there is not a large amount of variation from the mean in each group, indicating the high reliability of the data.
In the original method (Han et al, 1995) 0.5 mL of garlic extract is added to 1.2 mL of 2 mM L- cysteine and allowed to sit at 30 °C. However, in my experiment it was not kept at 30°C but instead at room temperature (approx. 25°C). This slightly lower temperature may have slowed down the reaction rate. Additionally, my analysis took place over two days, with the garlic extract (garlic powder in solution) being kept in a cupboard overnight. This may have impacted the results as the second day displayed more stable absorbance readings. This is likely due to the additional storage time facilitating further settling of any fine suspension present in the extract. Samples with fine suspension will likely affect the absorbance readings obtained by the colourimeter due to light scattering. This suggests that garlic extract should be left for a period of time to allow all debris to collect on the bottom of the container to gain a better set of results. Due to time constraints, we couldn’t repeat this part of the experiment. Additionally, there was no access to a centrifuge which would be another way of mitigating the presence of any fine suspension on the extract.
Since there is not a certain abundance of garlic in each clove, this was accounted for by mixing the garlic cloves together and then randomly selecting 10 to place into each bag. In the original method the absorbance readings were read at 412 nm, however my absorbance readings (Appendix 1.1) were read at 450 nm as this was the closest wavelength the colourimeter could be set to.
Previous studies have used a much larger range of temperatures when investigating allicin concentration. However, these values were not practical for this project due to the incubation conditions available in school. Moreover, temperatures which would be more applicable to household settings were chosen to enhance the applicability of any results obtained. The effect of higher or lower garlic clove storage temperatures cannot be ruled out and may be a subject for future investigations.
These results cannot be compared to other results in the literature as there are no literature reports on the effect of temperature on alliin concentration, or the effect of temperature before the extraction of allicin. It can be speculated that a similar experiment has been conducted and not reported in the literature because it was found that temperature did not have an effect.
There are not many reports on the stability of alliin, however it can be speculated that the sulfoxide functional group on the alliin molecule allows for its relatively high stability compared to allicin, as the sulfoxide functional group is extremely stable (Figure 7) (ScienceDirect Topics, 2021). This may be why temperature did not have an effect on the allicin concentration obtained after altering the storage temperature of garlic cloves, and hence the concentration of alliin before crushing. Conversely, allicin is heat sensitive due to its thiosulfinate functional group located on the molecule, since the disulfide bond typically has a bond dissociation energy of 250 kJ/mol it can be easily broken (Figure 7). In addition, since the disulfide bond is 40% weaker than C – C and C – H bonds (Mansor et al., 2016), it is the most susceptible to cleavage at higher temperatures and is therefore the weakest link in the structure of allicin. This is why temperature has a substantial impact on allicin concentration but not alliin concentration.
The alliinase enzyme is usually 80% stable over the pH range 6-8. Additionally, alliinase is 80% stable at temperatures below 40°C and has a sharp decrease in enzyme activity above this temperature. This indicates that alliinase is relatively stable at the temperatures that were tested in this paper (Chhabria and Desai, 2018). This further supports the experimental results as the temperatures investigated in this study are likely not extreme enough to affect alliin or alliinase concentration, hence explaining why no significant difference in concentration was measured for allicin extracted after storage of the garlic (Appendix 2.1). Carbon sulfoxide functional group
Thiosulfinate functional group
Disulfide bond
Figure 5: The structure of alliin compared to the structure of allicin
This research project explored the effect of storage temperature before extraction on the concentration of allicin extracted from garlic, to investigate whether small changes in storage conditions would allow for the maximization of allicin concentration and hence the medicinal benefits pf garlic. The garlic cloves were stored at selected temperatures: -18°C, 3°C or 25°C before the garlic cloves were dehydrated to produce a garlic powder. Data was then collected by first reacting the powder with excess L-cysteine and then DTNB, which measured the decrease in L-cysteine concentration. The data analysis involved an ANOVA statistical test to compare if there was a statistically significant difference between the means of the four different groups. The results of my analysis showed that temperature did not have a statistically significant effect on the concentration of allicin in garlic, possibly due to the stability of both alliin and alliinase present in the garlic cloves before crushing.
I would like to acknowledge and thank Dr Katie Terrett for her continuous supervisory support. I would also like to acknowledge Caitlin Tedesco who helped me develop potential research ideas and understand key concepts.
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Table 1: The effect of temperature on Absorbance
Control (25°C) 0.044 (90.4%) Room Temperature (25°C) 0.032 (92.9%) Fridge (3°C) 0.022 (95%) Freezer (-18°C) 0.034 (92.5%) 0.053 (88.5%) 0.036 (92.1%) 0.042 (90.75%) 0.038 (91.6%) 0.044 (90.3%) 0.044 (90.3%) 0.029 (93.5%) 0.038 (91.6%) 0.035 (92.5%) 0.044 (90.4%) 0.038 (91.7%) 0.035 (92.2%) 0.039 (91.5%) 0.030 (93%) 0.035 (92.3%) 0.037 (91.9%)
Table 2: Absorbance and concentration of Allicin for Control (25°C)
0.044 0.053 0.044 0.035 0.039
0.000229214 0.000228896 0.000229214 0.000229532 0.000229391
Table 3: Absorbance and concentration of Allicin for Room Temperature (25°C)
0.032 0.036 0.044 0.044 0.03
0.000229638 0.000229497 0.000229214 0.000229214 0.000229709
Table 4: Absorbance and concentration of Allicin for Fridge (3°C)
0.022 0.042 0.029 0.038 0.035
0.000229992 0.000229285 0.000229744 0.000229426 0.000229532
Table 5: Absorbance and concentration of Allicin for Freezer (-18°C)
0.034 0.038 0.038 0.035 0.037
0.000229568 0.000229426 0.000229426 0.000229532 0.000229462