“Optimization of Microwave-Assisted Extraction of Olea europaea using response surface methodology”

 “Optimization of Microwave-Assisted Extraction of Olea europaea using response surface methodology”

Komal Nawaz (Botany)

Laureate Folks International

laureatefolks@gmail.com, +923334446261

ABSTRACT:

Plants contain numerous compounds that are responsible for their different properties like color, fragrance, and medicinal characteristics. Compounds are extracted and human beings used these compounds for different purposes. Determination of these chemicals depends upon the quantity, and quality of these extracted secondary metabolites from plants after the method of extraction. Microwave-assisted extraction is one of the methods for plant-based chemicals, which is fast, requires fewer amounts of solvent, and time. In the present study, phenolic and flavonoids will be extracted from fruits of Olea europaea that are being cultivated in Barani Agriculture and Research Institute Chakwal, Pakistan. Extraction will be done by using the Microwave-assisted extraction method. Independent variables will be selected as power level, time, and solvent type (polar to non-polar) for extraction. Phytochemical content will be measured as a response factor and results will be statistically analyzed using (RSM) Response surface methodology. “RSM is a combination of statistical and mathematical techniques, which is more efficient than traditional methods for gathering and construing research results”. The optimized MAE procedure may be used in the future to analyze phenolic and flavonoid content of other parts of the plant for a quality check of olive crop production.

Introduction:

Many methods for extracting these chemicals from plants have been devised, including Hydro- distillation, Soxhlet extraction, Steam distillation, maceration, and others; however, these methods are time-intensive and require a huge volume of solvents. Microwave-assisted extraction is green technology, as an alternative to traditional procedures of extraction for the recovery of natural compounds since it offers important benefits in place of use of the low quantity of solvent, less time, and a greater extraction rate (Akhtar et al., 2020). In the Mediterranean region, The Olea europaea tree is an ancient cultivated crop in the world.  The English name is Olive, and Zaitoon is the Arabic name (Farhangi et al., 2014). The growth of the olive tree is slow but long-lived and it’s well known due to its edible fruit, which is used to make olive oil. Oil of olives is consumed as a primary source of lipid, as well as a therapeutic agent (Schwingshackl et al., 2017). Olive plantations have multiple benefits, comprising the contribution to the economy, effectively modifying climate changes, local edible oil demand, and managing water security concerns. In the country, over 10 million acres have already been identified for olive cultivation. In terms of religion, olive leaves, seeds, and fruits are very important. The Holy Quran also mentions the olive is a wonderful tree and fruit (Quran, Chapter 24 Al-Noor, and Verse 35). Because of their bitter flavor, olives are not eaten as fruit, but rather as olive oil or table olives. O. europaea has the potential to lower uric acid levels, blood sugar levels, and cholesterol levels. It is used to treat inflammation, hypertension, diabetes, diarrhea, urinary tract infections and respiratory, intestinal, and stomach illnesses, hemorrhoids, asthma, as a laxative, rheumatism, mouth cleaner, and vasodilator. Olive oil has biological effects on health through different pathways, including antioxidant, antibacterial, anti-tumor, and gene function modification (Rahmani et al., 2014). The major phytochemical phenolic complex is present low in quantities in olive oil than in Olive leaves (Rivas et al., 2000). Olives also have antioxidant and antimicrobial properties against bacteria, mycoplasma, and fungi (Khayyal et al., 2002). Phenolics which are extracted from fruits of olive, prevent the growth of Escherichia .coli, Staphylococcus aureus, and Klebsiella sp. (Yigit et al., 2001). Researchers studied the effects of olive on Aspergillus parasiticus aflatoxin generation and growth, finding that oleuropein decreased aflatoxin synthesis and increased mold growth. Markin et al. (2003) tested Candida albicans was destroyed in 24 hours when an olive leaf extract in water is used.

The response surface methodology is a set of statistical and mathematical tools for optimizing and analyzing the response of interest. RSM is more effective than traditional approaches for gathering and interpreting research results, to create statistical data for lowering the number of experiments (Chan et al., 2018; Zhao and Zhang 2014).

LITERATURE REVIEW:      

To make a solid base for this research proposal, a detailed online survey was made on reputed scientific journals and a few articles are presented below.

Kirbaslar and Sahin (2021) had reported that sustainability and valorizations of feedstock based on biomass have become an important necessity. Evaluations of agricultural wastes and food have little economic value and may pose a hazard to the environment, which is unavoidable. They use olive tree by-products, such as a leaf. Microwave-assisted extraction was utilized for total phenolics (TPI), antioxidant activity (AA), and flavonoids (TFI) extraction using a variety of common solvents (ethanol, acetone, methanol, acetonitrile solutions). RSM was used for the establishment of parameters that were operative in a minor number of tests using a central composite design (CCD). In general, the utmost effective characteristic for each solvent system was solvent volume. Microwave power (150–250 W), solvent (50–100 mL), and time (0.5–1.5m) were chosen as process parameters. “The best outcomes for AA, TPI, and TFI were found 96.34%, 10.45-mg GAE/g DL, and 9.69- mg CE/g DL under optimal conditions (230 W, 1.5 m, and 63.16 mL of 30% acetonitrile solution)”.

Zin et al. (2020) used electromagnetic irradiation to recover the phytochemicals (Microwave Assisted-Extraction). They talked about whether dielectric heating (microwave irradiation) may be used to recover phenolic chemicals from vegetable and fruit wastes. They concluded that a combination of high power, temperature, and a short irradiation period is beneficial for the MAE of plant materials. For simplicity, they believe that the extraction of bioactive chemicals through a microwave extractor is the ideal solution.

Da Rosa et al. (2019) studied altered methods for extraction and their effects on total phenolic content and antioxidant activity from leaves of olive. These methods include Ultrasound-assisted extraction, Maceration, and Microwave-assisted extraction. Due to phenolic compounds, Olive leaves were found to be high in antioxidant activity. MAE at a shorter extraction time (3m) and  higher temperature (86 °C) was more efficient in terms of TP yield. By using MAE olive leaf cells were effectively destroyed with water as a solvent, allowing the compounds to be released. MAE increased the TP yield by 82%. Microwave-assisted extraction is a technique that is used for extracting bioactive chemicals after heating. The potential benefit of using MAE is the time reduction of extraction and improvement of bioactive chemicals.

Nicoli et al. (2019) investigated profiles of antioxidant activity and phytochemicals from Italian Olea europaea L. leaves. They are grown in similar climatic conditions to determine if this waste may be used for health. By using HPLC ESI/MS-TOF profiling of phenolics and their amounts of seven components were examined. “Antioxidant activity was measured using three different antioxidant tests that are superoxide anion scavenging assay, ORAC, and DPPH”. Antioxidant activity and TPC varied greatly across the cultivars. Total phenolics and antioxidant activity were found to be high in the cultivars Strana, Apollo, and Maurino, indicating that they are a good source of biological compounds for health benefits.

Nile et al. (2017) investigated anticancer, antioxidant, total phenolic content, and enzyme inhibitory properties in Indian aromatic and medicinal plants utilizing various extraction methods, including microwave aided extraction. They discovered that these plants had a high relationship between phenolic content, enzyme inhibitory activity, and antioxidant activity showing that phenolics are the primary molecules implicated in these biological processes. In this study, they proved the plants as inhibitors of antitumor cell growth, and oxidative stress, which also correlates in the Unani system of medicine to ethnobotanical evidence.

Kadi et al. (2016) used MAE to extract oil from pomace olive using acidic hexane. When increased acetic acid concentration, time, and radiation power, according to the findings oil extraction yield increases. For both radiation powers ranging from optimal extraction, time is 1.5m, 170, and 510W, and concentration of acetic acid in hexane is 5.0%. The oil yields obtained at 510 watts were somewhat higher than those at 170 watts were. When compared to pure hexane data, yield gains were 6% at 510 W and 8.4% at 170 W. On the other hand, oil obtained from pomace olive after increasing acetic acid was of bad quality.

Brahmi et al. (2013) investigated compounds of phenolic in olive fruits and leaves to see how they are effective at scavenging free radicals. Analysis of olive leaves and fruits by HPLC, time of harvest has a significant impact. Total polyphenols, total flavonoids, and total o-diphenols decrease when the fruits are ripe and the leaves are growing and vice versa. Total tannins and carotenoids in the leaves fluctuated much more than polyphenols, flavonoids, and o-diphenols during the olive's vegetative cycle. They concluded that unripe and mature fruits from the two types that ripened exhibited less antioxidant activity than leaves obtained at two altered stages of the olive vegetative cycle.

Research Hypothesis:

Using Microwave-assisted extraction and Response surface methodology (RSM), not much work is conducted on the phytochemical analysis of Olea europaea of Olive Valley of Pakistan, up to my knowledge. Also, very little data is available on the comparison of phenolic and flavonoids of varieties of O.europaea of Olive Valley. As a result, the goal of this study is to employ Response surface methodology is used to measure the response of MAE of phytochemicals from O.europaea.

1.      Use of Microwave-assisted extraction method for phenolics and flavonoids from the fruit of O. europaea.

2.      Optimization of MAE parameters using RSM.

 MATERIAL AND METHODS:

The purpose of this study is an analysis of phenolics and flavonoids extracted from Olea europaea by using the Microwave-assisted extraction (MAE) method. For this study, fruits of two varieties will be used and plant samples will be collected from Barani Agriculture and Research Institute Chakwal, Pakistan. It will involve the following procedure:

Plant Material:

Olea europaea will be collected from Barani Agriculture and Research Institute Chakwal, Pakistan. Plant samples of O. europaea will be washed thoroughly in running tap water. Seeds will be separated and fruits will be washed and stored inside the fridge till further use.

Microwave-Assisted Extraction (MAE):

The Microwave Assisted Extractor (Model MDS-6G) will be used for the extraction. Time (1-10 minutes), power level (300-900 W), and solvent type (ethanol, acetone, n-hexane) will be the extraction parameters. The inner main vessel will be placed within the outer protective vessel, and the main vessel will be filled with solvent. In the inner vessel, we will take1g of plant material and mix it with 10 milliliters(ml) of the solvent. The covered vessel will be placed on the frame with care. The sample containers will be placed for the frame to be balanced. The extractor will be started after the time, temperature, and power have been specified. The extracts obtained from plant material will be filtered into labeled vials then dried and kept in the refrigerator at 4^oC for subsequent analysis. Fruit will be used to optimize the MAE of both varieties and the estimation of phenolics and flavonoid content of O. europaea.

Quantification of Phytochemicals:

Quantification of compounds will be done with the help of UV spectrophotometry, Gas chromatography, and Mass Spectrometry as a response factor of MAE.

Total Phenolic Content:

Take 1g of plant material, which will be homogenized in 10 milliliters(mL) acetone and filtered through Whatman filter paper no. 4 According to the protocol, the total phenolic quantity of acetone extracts will be measured using the Folin-Ciocalteu reagent. 500 microliters(µL) of distilled water and 125 microliters(µL) of Folin-Ciocalteu reagent will be used to dissolve 125 microliters(µL) of sample extract. The mixture will be thoroughly mixed before adding 1.25 milliliters(mL) of 7%Na₂CO₃, which will be combined with distilled water to make a total volume of 3 milliliters(mL). The absorbance at 760nm will be measured in comparison to the prepared blank after 90minutes of incubation in the dark. Through a calibration curve with Gallic acid, total phenolic levels will be reported as mg GAE/g (Rebey et al., 2012).

Determination of total flavonoid contents:

The spectrophotometric approach will be used to determine the flavonoid content of the extracts. 1.25 milliliters(mL) distilled water and 75 microliters(µL) of a 5%NaNO₂ solution will be add to the extract (concentration 1 mg/ml). 150 microliters(µL) of 10%AlCl₃ solution will be add after 5m. After 5m, the combination will be prepared using 500 microliters(µL) of 1M NaOH and 275 microliters(µL) of distilled water. The solution will be well mixed, and the absorbance at 510nm will be measured. The standard curve will be calculated using catechin, and the results will be presented in micrograms of catechin equivalents (CEs) per mg of extract.

Statistical analysis:

The results of experimental work will be collected and “the design expert software-version 11” will be used to apply “Response surface methodology”. RSM will be selected to determine the optimized conditions for MAE from plant extract. The effect of the independent variables like microwave power, solvent (Ethanol, Acetone, and n-hexane), and extraction time will be investigated using a central composite design (CCD).

Conclusion:

It will be expected that in the microwave-assisted extraction method, the polar solvent extract will have a higher yield of phenolic and flavonoid from olive fruit extracts. “Regarding a significant increase in the extraction yield for MAE with polar solvents, Hence, the microwave-assisted method will have many advantages due to its less extraction time, higher extraction efficiency, high extraction selectivity, and less labor, which makes it a satisfactory method in the extraction of phenolics and flavonoid compounds from olive fruits”.

References:

Akhtar, I., Javad, S., Ansari, M., Ghaffar, N., & Tariq, A. (2020). Process optimization for microwave-assisted extraction of Foeniculum vulgare Mill using response surface methodology. Journal of King Saud University-Science, 32(2), 1451-1458.

Brahmi, F., Mechri, B., Dhibi, M., & Hammami, M. (2013). Variations in phenolic compounds and antiradical scavenging activity of Olea europaea leaves and fruit extracts were collected in two different seasons. Industrial Crops and Products, 49, 256- 264.

Chan, Y. H., Quitain, A. T., Yusup, S., Uemura, Y., Sasaki, M., & Kida, T. (2018). Optimization of hydrothermal liquefaction of palm kernel shell and consideration of supercritical carbon dioxide mediation effect. The Journal of Supercritical Fluids, 133, 640-646.

Da Rosa, G. S., Vanga, S. K., Gariepy, Y., & Raghavan, V. (2019). Comparison of microwave, ultrasonic and conventional techniques for extraction of bioactive compounds from olive leaves (Olea europaea L.). Innovative Food Science and Emerging Technologies, 58, 102234.

Farhangi, H., Ajilian, M., Saeidi, M., & Khodaei, G. H. (2014). Medicinal fruits in the holy Quran. International Journal of Pediatrics, 2(3.2), 89-102.

Kadi, H., Moussaoui, R., Djadoun, S., & Sharrock, P. (2016). Microwave-Assisted Extraction of olive oil pomace by acidic hexane.

Khayyal, M. T., El-Ghazaly, M. A., Abdallah, D. M., Nassar, N. N., Okpanyi, S. N., & Kreuter, M. H. (2002). Blood pressure-lowering effect of an olive leaf extract (Olea europaea) in L-NAME induced hypertension in rats. Arzneimittel forschung, 52(11), 797-802.

Kırbaşlar, Ş. İ., & Şahin, S. (2021).Recovery of bioactive ingredients from biowaste of the olive tree (Olea europaea) using microwave-assisted extraction: a comparative study. Biomass Conv. Bioref. https://doi.org/10.1007/s13399-020-01194-y.

Markin, D., Duek, L., & Berdicevsky, I. (2003). In vitro antimicrobial activity of olive leaves. Antimikrobielle Wirksamkeit von Olivenblättern in vitro. Mycoses, 46(3‐ 4), 132-136.

Nicolì, F., Negro, C., Vergine, M., Aprile, A., Nutricati, E., Sabella, E., & De Bellis, L. (2019). Evaluation of phytochemical and antioxidant properties of 15 Italian Olea europaea L. cultivar leaves. Molecules, 24(10), 1998.

Nile, S. H., Nile, A. S., & Keum, Y. S. (2017). Total phenolics, antioxidant, antitumor, and enzyme inhibitory activity of Indian medicinal and aromatic plants extracted with different extraction methods. Biotechnology, 7(1), 1-10.

Rahmani, A. H, Albutti, A. S., & Aly, S. M. (2014). Therapeutics role of olive fruits/oil in the prevention of diseases via modulation of antioxidant, antitumor, and genetic activity. Int J Clin Exp Med, 7(4), 799–808.

Rebey, I. B., Jabri-Karoui, I., Hamrouni-Sellami, I., Bourgou, S., Limam, F., & Marzouk, B. (2012). Effect of drought on the biochemical composition and antioxidant activities of cumin (Cuminum cyminum L.) seeds. Industrial Crops and Products, 36(1), 238-245.

Rivas, C., Espín, J. C., & Wichers, H. J. (2000). Oleuropein and related compounds. Journal of the Science of Food and Agriculture, 80(7), 1013-1023.

Schwingshackl, L., Lampousi, A. M., Portillo, M. P., Romaguera, D., Hoffmann, G., & Boeing, H. (2017). Olive oil in the prevention and management of type 2 diabetes mellitus: a systematic review and meta-analysis of cohort studies and intervention trials. Nutrition and Diabetes, 7(4), e262-e262.

Yigit, A., Sahan, Y., & Korukluoglu, M. (2001). Antimicrobial substances are found in olive leaves and olive. 2nd International AltInoluk “Antandros” Olive Busines Symposium.

Zhao, S., & Zhang, D. (2014). Supercritical CO₂ extraction of Eucalyptus leaves oil and comparison with Soxhlet extraction and hydro-distillation methods. Separation and Purification Technology, 133, 443-451.

Zin, M. M., Anucha, C. B., & Bánvölgyi, S. (2020). Recovery of phytochemicals via electromagnetic irradiation (microwave-assisted extraction): Betalain and phenolic compounds in perspective. Foods, 9(7), 918.