Effect of temperature on antioxidation capacity of aloe vera 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