Effect Of Temperature On Antioxidation Capacity Of Aloevera Juice

My main reason for opting Biology in my DP program was to understand the impact of biological phenomenon and bio-chemical components in human life. In Topic-2, while studying the section of enzyme and its actions, I came across a lot of phytochemicals which behaves as potential biological catalysts and has a major impact on food and drug industry. While researching more on this, I came across the use of aloevera juice in various industries like cosmetics, drugs, therapeutic and food. One of the factors that strike me was the sunscreen that I use. Incidentally, the sunscreen contained aloevera as a major ingredient and it was mentioned in the packet was – ‘Store under cool conditions’. This gave rise to various inquiries in me. Is it written because the compound would degrade or lose its ability to act as a UV protector if stored at high temperatures. Further research led me to know that aloevera juice has various phytochemicals which behaves as antioxidants and thus constitute to be a major ingredient of not only sunscreen but many other cosmetic product as well. Thus, I wanted to explore how various physicochemical factors like temperature, pH, storage conditions would affect the antioxidation capacity of these compounds.

Aloevera is a ‘perennial succulent xerophyte’ (Rahmani et al.). They have the ability to store water inside their leaf in ‘storage tissues’ (Rahmani et al., “Aloe Vera”)which allows them to store water and thus grow in areas with deficiency in water. The inner layer of the leaf contains – ‘thin walled parenchyma cells’ (Manvitha and Bidya) that contains water, carbohydrates in the cell wall and also many other phytochemicals. The phytochemicals mainly found in the soft and dense juice which is biologically coined as – ‘viscous mucilage’ (Manvitha and Bidya) are polyphenols, alkaloids, and flavonoids. These phytochemicals especially the polyphenol is mainly responsible for the presence of antioxidant behavior of aloevera juice.

How does the percentage activity of antioxidants in aloe vera (Aloe barbadensis miller)extract depends on the temperature at which it is stored, determined using DPPH assay?

The phytochemicals in aloevera juice interact with each other to initiate certain biochemical reactions which causes these molecules to change their structures and consequently modify their properties (Nejatzadeh- Barandozi). As temperature rises, these molecules begin to react with each other at a faster rate and thus they lose their ability to act as antioxidants.

Thus, a negative correlation is predicted between temperature and the antioxidant capacity of the phytochemicals present in aloevera juice.

The prediction is also supported by a literature reference. The paper is titled as- ‘ Evaluation of Antioxidant Potential of Aloe vera (Aloe barbadensis Miller) Extracts’. It was published in the journal ‘Agricultural Food Chemistry’ by ACS Publications and the author was Yun Hu and Juan Xu. In this article, the antioxidant capacity of aloevera juice was studied as a function of age of the plant and temperature. The study has revealed that for a 3 year old alovera plant the antioxidant capacity was reduced from 72.19 % to 65.20 % as the temperature was increased from 40.00 C to 90.00 C. The method used to determine the antioxidation capacity was DPPH radical scavenging activity in ethanolic medium.

The temperature at which the aloe-vera juice is heated.

A water bath is used to heat the aloevera juice. The temperature chosen are - 30.0⁰ C (room temperature; water bath was not used), 35.0⁰ C, 40.0⁰ C, 45.0⁰ C, 50.0⁰ C and 55.0⁰ C. The purpose of the investigation is to study the effect of temperature or precisely the room temperature at which the aloevera juice is stored before it is consumed. Usually, in a tropical country like India it would range from 25.00C to 40.00C. Often it may rise above 40.00C in some parts of the country.

The percentage activity of the antioxidants in the aloevera juice is the dependent variable. DPPH assay is used as the method to determine this. DPPH is a violet colored dye showing maximum absorbance at 517nm. Antioxidants provides hydrogen ions which can reacts with DPPH molecules and reduce them. As the DPPH molecules are reduced, the color changes to yellow. Thus, more the amount of antioxidants, more the number of DPPH molecules reduced and less the intensity of the violet color of the oxidized form. Thus, monitoring the absorbance at 517 nm (Kedare and Singh) is an effective way to estimate the antioxidant activity. For this, the DPPH solution is added to the sample and the absorbance is measured instantly. The absorbance of the same solution is measured after a definite time interval. The reduction in the value of absorbance shows the amount of DPPH molecules reduced by the antioxidants in the sample. Thus, more the reduction of absorbance at 517 nm, more the amount of antioxidants present in the sample.

The antioxidant capacity is expressed in percentage according to the equation written below:

Percentage antioxidation capacity = \(\frac{A\ _{start}-A\ _{end}}{A\ _{start}}\)× 100

A _start = absorbance of the sample solution as soon as DPPH is added in ± 0.001 abs

A _end = absorbance of the sample solution with DPPH after some time in ± 0.001 abs

Time for which DPPH reacts with sample
The amount of DPPH molecules reduced by the antioxidants in the aloevera sample is used as the fundamental basis for the measurement of the antioxidation capacity of the aloevera juice. Thus, more the contact time of the DPPH sample solution and the aloevera sample, more the amount of DPPH molecules reduced and greater the percentage antioxidation capacity. Hence it is inevitable to keep the time of reaction between the sample and the DPPH molecule to be constant throughout. The aloevera sample was in contact with the DPPH solution for 10.00 minutes as monitored by a stop-watch.

Wavelength used in the colorimeter
DPPH is a chromophore. It means that this molecule has specific molecular features which enables it to absorb certain wavelengths of the electromagnetic spectrum and emit the complementary color. Thus, the wavelength used to determine its absorbance must be kept same in all cases and moreover it should be the wavelength where the molecule exhibits maximum absorbance.

As indicated in the figure above, the absorption of DPPH is maximum at 517 nm. So, the wavelength of the colorimeter was fixed at 517 nm for all the trials. This choice of wavelength is scientifically justified as the change in absorbance of this molecule would be definitely best monitored in a wavelength at which it shows maximum absorbance.

Sample type – Same juice used in all cases.
Variation in sample type and sample origin is a common source of error in any biological studies. To minimize this methodological limitation, the aloevera leaf used to extract the juice out of it was the same in all cases and was taken from the same part of the same plant.

• Ethanol
• DPPH solution
• Aloevera

• 150 cc conical flask-1
• Blender
• Colorimeter
• Cuvette
• Water bath
• Test tube
• Graduated pipette – 10 cc

• Personal protective clothing like lab coat, gloves and safety mask was used.
• The knife was used with utmost care.
• The ethanol used was neither inhaled or consumed.
•  All unused materials were returned for re-use.
• Disposal of waste materials was done as per the school’s safety protocol.

A fleshy and healthy aloe vera leaf was cut out from an aloevera plant using a knife. A sharp knife was used to remove the outer thorny layer of the leaf. Once, it is done a thick yellowish layer was observed which is actually the latex of the leaves. The latex was also got rid off by using the same knife. The sharp pointed edge of the knife was utilised to scoop out the thick colorless gel inside the aloevera leaf. This gel collected was then transferred to the blender. 100 cm³ of distilled water was added to the same using a graduated measuring cylinder. The blender was operated at medium speed for around 5.00 minutes to homogenize the gel. The solution thus obtained was aloevera juice which was partially transparent in nature with a tincture of dark white color.

• A 150 cm³ conical flask was taken.
• 10.00 ± 0.01 cm³ of the aloe vera juice was transferred to it using a graduated pipette.
• The flask was set on the water bath and the temperature was adjusted to 35.0⁰ C.
• The aloe vera juice was then heated using the water bath for 10 minutes.
• After 10 minutes, the flask was removed from the water bath.
• 2.00 cm³ of DPPH solution was added to it using a 10.00 cm³ graduated pipette.
• 2.00 cm³ of ethanol was added to the same.
• The colorimeter was calibrated at 517 nm using ethanol as the blank solution.
• The absorbance of the solution in the flask using a cuvette at 517 nm.
• The solution in the flask was kept for 1 hour to allow the antioxidants in the juice to react with DPPH.
• After 1 hour, the absorbance of the solution was recorded again at 517 nm.

The same process was repeated for other temperatures – 30.0⁰ C (room temperature; water bath was not used), 40.0⁰ C, 45.0⁰ C, 50.0⁰ C and 55.0⁰ C. At each temperature, the data was collected in seven trials. Raw data:

Sample calculation

For Trial-1,

Absorbance at start (A start) = 0.678 ± 0.001 abs

Absorbance at end (A end) = 0.543 ± 0.001 abs

Decrease in absorbance (∆ A) = (0.678 ± 0.001) – (0.543 ± 0.001) = 0.135 ± 0.001 abs

Percentage activity = \(\frac{0.135}{0.678}\) X 100 = 19.91

For average percentage activity,

Average = \(\frac{19.91+19.50+20.06+20.03+20.21+20.35+20.03}{7}\)= 20.01

Calculation of percentage error

At temperature = 30.0 ± 0.5^o C

Percentage error = \(\frac{0.002}{20.01}\)× 100 = 0.01

The graph clearly shows a decrease in the value of percentage antioxidation capacity with the increase of temperature. With the value of temperature increasing from 30.0 ± 0.5oC to 55.0 ± 0.5oC, the percentage antioxidation capacity decreases from 20.01 to 1.31. The decrease is quite significant. The trendline was plotted to understand the pattern of decrease. Both linear and exponential trend line was plotted and the correlation value obtained for linear and exponential trend line was 0.95 (see the equation written in blue color in the graph) and 0.97 (see the equation written in black color) respectively. This claims that the exponential trend line is a better fit than the linear trend line. Thus, we can arrive at the conclusion that the decrease of percentage antioxidation capacity of aloevera juice with the rise of temperature is exponential in nature. This suggests that as temperature increases, the polyphenolic contents of aloevera juice begins to interact with each other which causes them to undergo biochemical degradation to simple organic compounds like aldehydes, ketones and carboxylic acids or free radicals which do not have any antioxidation capacity. So, the polyphenolic content in the thick tissues of the juice decreases which eventually decreases the antioxidation capacity of the juice.

The difference in the consecutive values suggests that the decrease was less significant as the temperature has increased. For example, as the temperature increases from 30.0 ± 0.5oC to 35.0 ± 0.5oC, the value decreases from 20.01 to 14.91 (decreasing by 20.01-14.91 = 5.1 units) whereas as temperature changes from 45.0 ± 0.5oC to 50.0 ± 0.5oC, the value decreases from 4.70 to 3.20 (decreases by 4.70-3.20 = 1.50 units). Thus, it is observed that the decrease in the percentage antioxidation capacity is less as the temperature reaches 45.0 ± 0.5oC. Thus, in general we can conclude that the decrease in percentage antioxidation capacity becomes less significant as the temperature increases. This is also mathematically supported by the equation obtained for the exponential trendline ( y= 616.59 e^-0.108x). As the power of the exponent is negative and it is an exponential curve, it is supposed to become parallel to the horizontal axes beyond a point. Thus, we can again claim that there must be theoretically some antioxidation capacity of the aloevera juice even if the temperature is too high which is again a subject of inquiry.

This investigation does not fit for descriptive statistical analysis as there are not more than one groups selected for the investigation. So, T test of independence or ANNOVA test cannot be performed. Thus, inferential statistics has been used. To investigate the correlation between the two variables- temperature and percentage antioxidation capacity, a simple test of covariance is conducted as shown below.

Degrees of freedom (n) = 6

Variance of temperature\((S^2_x) =\frac{\sum(X-X_{mean})^2}{(n-1)}\) = \(\frac{437.5}{(6-1)}\) = 87.5

Variance of percentage antioxidation capacity = \((S^2_x) =\frac{\sum(Y-X_{mean})^2}{(n-1)}\)= \(\frac{267.6}{(6-1)}\)= 53.52

Co-variance = \(\frac{\sum(X-X_{mean})(Y-Y_{mean})}{n-1}\) = \(\frac{-333.57}{5}\) = −66.71

Correlation coefficient (r) = \(\frac{covariance}{\sqrt{(s^2_x} \ \times \ s^2_y)}=\frac{66.71}{\sqrt{87.5\times 53.52}}=\frac{-66.71}{68.43}\) = −0.97

The value of correlation coefficient ranges from 0 to 1. The value O indicates that there is no correlation and the value 1 indicates that there is a strong correlation. As the magnitude of correlation coefficient obtained is 0.97, which is close to1, we can claim that there is a strong correlation between percentage antioxidation capacity and temperature. The sign of the correlation coefficient indicates the type of correlation. As the value obtained is negative, it indicates that the correlation is negative.

Thus, a negative strong correlation exists between temperature and percentage antioxidation capacity. This supports the claim that the antioxidation capacity of aloevera juice decreases as the temperature increases.

How does the percentage activity of antioxidants in aloe vera (Aloe barbadensis miller)extract depends on the temperature at which it is stored, determined using DPPH assay?

As temperature increases from 30.0 ± 0.5^o C to 55.0 ± 0.5^o C, the percentage antioxidation capacity decreases from 20.01 to 1.31. the decrease of percentage antioxidation capacity of aloevera juice with the rise of temperature is exponential in nature. This suggests that as temperature increases, the polyphenolic contents of aloevera juice begins to interact with each other which causes them to undergo biochemical degradation to simple organic compounds like aldehydes, ketones and carboxylic acids or free radicals which do not have any antioxidation capacity. So, the polyphenolic content in the thick tissues of the juice decreases which eventually decreases the antioxidation capacity of the juice.

In general we can conclude that the decrease in percentage antioxidation capacity becomes less significant as the temperature increases. There must be theoretically some antioxidation capacity of the aloevera juice even if the temperature is too high which is again a subject of inquiry.

The hypothesis predicted is found to be valid. The exponential trend line obtained has a negative power of exponent which proves the claim made above. As the magnitude of correlation coefficient obtained is 0.97, which is close to1, we can claim that there is a strong correlation between percentage antioxidation capacity and temperature. As the value of correlation coefficient obtained is negative, it indicates that the correlation is negative.

• The investigation conducted follows a simple procedure and the results are reproducible. There are various methods of determining antioxidation capacity like using – FRAP (using Iron-III solution as the reagent), using Copper-II ions and using radical scavenging capacity (Prior et al.). DPPH assay method has been used as this method is found to be most accurate analytical method to compare and determine antioxidation capacity especially for biological reference.
• The data collected has been sufficient. Seven trials has been obtained for each values of temperature which reduces the chances of sampling error and other random errors. As the sample size is high and the data obtained are quite close, the collected data can be claimed to be quite precise.
• The percentage error has also been calculated and the uncertainty in absorbance being low as ± 0.002, the value is not high. So, the data set may be claimed to have sufficient accuracy.

• Like any other analytical method, this investigation has also various sources of random error associated with it. For example, the human error in responding to the stop-watch, the uncertainties in the volume measured and so on. An attempt to minimize this can be done by adopting certain changes in the design of the investigation. To reduce the response error due to the use of stop-watch, the sample size must be large and sufficient trials must be done. To improve the uncertainties, apparatus with smaller gradations must be used like a graduated pipette instead of any other apparatus like a measuring cylinder or a beaker to measure the volume.
• As the data was collected using a colorimeter, a systematic error is associated with it. To nullify this, the instrument must be calibrated. This can be done using distilled water or the solvent used (which is ethanol in this case) and adjusting its absorbance to null at the preset of the investigation.
• While preparing the juice, there is a chance that the outer layer of the leaf may come into the solution along with the thick moist juice which has been referred as gel here. Thus, the gel must be removed from the leaf carefully and it must be ensured that no other part of the leaf is collected except the thick moist gel inside it.
• To have a fair result, it is important that the juice used is homogenous and the composition is uniform throughout. This can be done by taking the juice and blending it with water to prepare a homogenous juice.

To extend the investigation further, I would like to extract other juices like that from bell peppers, citrus fruits like tomatoes, apples as they are acclaimed sources of antioxidants. I would like to study the effect of temperature on the percentage antioxidation capacity of these juices to understand if there is a significant difference between the way temperature affects the percentage antioxidation capacity of aloevera juice and others. This can be done by collecting the data in the same process as done here and then performing a T-test or ANNOVA to depict the correlation.

Kedare, Sagar B., and R. P. Singh. “Genesis and Development of DPPH Method of Antioxidant Assay.” Journal of Food Science and Technology, vol. 48, no. 4, Aug. 2011, pp. 412–22. PubMed Central, doi:10.1007/s13197-011-0251-1.

Kleinrichert, Kody, and Bindhu Alappat. “Comparative Analysis of Antioxidant and Anti-Amyloidogenic Properties of Various Polyphenol Rich Phytoceutical Extracts.” Antioxidants, vol. 8, no. 1, Jan. 2019, p. 13. DOI.org (Crossref), doi:10.3390/antiox8010013.

Manvitha, Karkala, and Bhushan Bidya. “Aloe Vera: A Wonder Plant Its History, Cultivation and Medicinal Uses.” Undefined, 2014, https://www.semanticscholar.org/paper/Aloe-vera%3A-a-wonder-plant-its-history%2C-cultivation-Manvitha-Bidya/5936ff5850ffef2cab79876285dd07228cf42ce8

Nejatzadeh-Barandozi, Fatemeh. “Antibacterial Activities and Antioxidant Capacity of Aloe Vera.” Organic and Medicinal Chemistry Letters, vol. 3, July 2013, p. 5. PubMed Central, doi:10.1186/2191-2858-3-5. Prior, Ronald L., et al. “Standardized Methods for the Determination of Antioxidant Capacity and Phenolics in Foods and Dietary Supplements.” Journal of Agricultural and Food Chemistry, vol. 53, no. 10, May 2005, pp. 4290–302. ACS Publications, doi:10.1021/jf0502698.

Rahmani, Arshad H., et al. “Aloe Vera: Potential Candidate in Health Management via Modulation of Biological Activities.” Pharmacognosy Reviews, vol. 9, no. 18, 2015, pp. 120–26. PubMed Central, doi:10.4103/0973- 7847.162118.

“Aloe Vera: Potential Candidate in Health Management via Modulation of Biological Activities.” Pharmacognosy Reviews, vol. 9, no. 18, 2015, pp. 120–26. PubMed Central, doi:10.4103/0973- 7847.162118.