Extraction of Polyphenols from Oregano and Thyme by Maceration using Glycerine

 

Wissam Zam¹*, Ali Ali², Farah Husein²

¹Department of Analytical and Food Chemistry, Faculty of Pharmacy,

Al-Andalus University for Medical Sciences, Tartous, Syria.

²Department of Food Technology, Faculty of Technical Engineering, Tartous University, Tartous, Syria.

 

ABSTRACT:

Oregano and thyme are plants belonging to Lamiaceae family and are considered a rich source of polyphenols. The aim of this study is to determine the amount of polyphenols and the antioxidant capacity of oregano, thyme and their 1:1 mixture. Extracts were prepared by maceration (from 1 to 15 days) using glycerin at a concentration of 95%. The phenolic content of these extracts was calculated by Folin-Ciocalteau assay, whereas DPPH (2,2-diphenyl-1-picrylhudrazyl) test was used to determine the antioxidant capacity. Oregano had the highest phenolic content and antioxidant capacity while thyme had the lowest values. The best time of maceration was at 10 days for all extracts. In conclusion, results showed that glycerin could be used as an important green solvent in the extraction of polyphenols from plant materials; also increasing the maceration period to 10 days is better for both phenolic content and antioxidant activity.

 

 

1. INTRODUCTION:

Oregano (Origanum vulgare) and Thyme (Thymus vulgaris) are flowering plants belonging to the Lamiaceae family^[1]. They have been used in cooking to improve the flavor of food and as preservative agent long time ago^[2]. Recent researches have proved that thyme and oregano could be an important source of bioactive polyphenols and have antioxidant properties. They also have great effects on the human health such as anti-inflammatory, antimicrobial and anticarcinogenic activities^[3-5]. Recently, there has been a rising awareness to the importance of high content of phenolic compounds due to their antioxidant properties which could prevent the oxidative degradation in food and protect oils and lipids^[6-10].

 

Oregano and thyme are widely used in Mediterranean Basin for different purposes. Thyme and oregano leaves are used either dried or fresh and the flowers could also for the extraction of essential oils that are used as aromatic additives in food^[11]. They also contain many important compounds such as carvacrol and thymol; the carvacrol content in oregano can be up to 95%, and it is considered the most powerful effective bioactive compound against followed by thymol^[12].

 

The most polyphenols reported to be present in thyme and oregano are hydroxycinnamic acids and flavons. Researchers have also found a high content of rosmarinic acid in plants belonging to Lamiaceae family. Results have shown that oregano contains three major phenolic compounds that have an effective antioxidation activity; these major compounds are caffeic acid, protocatechuic acid, and rosemarinic acid. Whereas salvianolic acid, rosemarinic acid, luteolin 7-O-B glucuronide and luteolin glucoside are considered the main phenolic compounds in thyme^[13].

 

Glycerin is an important solvent recently used due to its important properties in the extraction of polyphenols from plants. It is considered inexpensive non-toxic and non-flammable solvent with a high boiling point^[14]. Glycerin has also a dielectric constant that is similar to methanol and ethanol and lower than water, thus glycerin is less polar and more suitable for the extraction of polyphenols from plant materials^[15]. Recent studies have indicated the high capacity of aqueous glycerin solutions to be used as a solvent and have proven their efficiency in extracting polyphenols from green extracts^[16].

 

The aim of this study is to determine the antioxidant power and phenolic compounds found in thyme and oregano using glycerin as extraction solvent.

 

2. MATERIALS AND METHODS:

2.1 Chemicals:

Folin–Ciocalteu reagent, sodium carbonate, 2,2-diphenyl-1-picrylhydrazyl reagent, methanol, pure glycerin for medical use were used.

 

2.2 Sample preparation:

Thyme and oregano were obtained from the mountains of the Syrian coast and the plants were harvested in the flowering stage. The plants were left to dry under shade, then the leaves and the flowers were wrapped in dark black bags and placed in a dry and dark place.

 

The following amounts were taken from both thyme and oregano:

5g oregano, 5g thyme, oregano:thyme 1:1 (2.5 g+2.5 g); these samples were crushed by the coffee melt and the powders were then placed in 100ml flasks. Each 95 g of pure glycerin was packed to the flasks with well-packed shakers. The flasks were wrapped by aluminum foil and incubated at 55°C in oven at different intervals (1-2-4-6-8-10-12-15) days. 

 

2.3 Antioxidant capacity:

The antioxidant capacity of the phenolic compounds was studied by DPPH assay as indicated by Brand-williams ^[17,18]. The samples were diluted by distilled water to a ratio of 1/50, then 200 μl of the diluted samples was added to 2ml of DPPH solution. Samples were well shaken and then incubated for an hour in the dark at room temperature. The absorption was measured by a spectrophotometer at 520nm^[19].

 

The percentage of DPPH scavenging activity was calculated using the following equation:

 

Scavenging of DPPH % = [(A0-A1)/A0}] x 100

 

Where A0 is the absorbance of the control and A1 is the absorbance of the sample.

 

2.4 Measuring the phenolic content:

The total phenolic content of the samples was measured by Folin-Ciocalteu test^[20,21]. The samples were diluted by 1/50 with distilled water; then 1ml of the extract was taken and placed in a test tube with Fuline-Ciocalteu reagent and 4ml of sodium carbonate (7.5%). Samples were incubated at 40°C within a water bath for 30 minutes. The absorbance was measured by a spectrophotometer at a wavelength of 734nm. Measurement and quantification were based on standard calibration curve of gallic acid.

 

2.5 Statistical analysis:

The coefficients were distributed according to two ways completely randomized design by using the ANOVA test and LSD was calculated to determine the significant differences of the coefficients, at a significant level of 0.05. Costat software was used to conduct the test.

 

3. RESULTS AND DISCUSSION:

3.1 Total phenolic content:

The total phenolic content of thyme and oregano samples was determined by Folin–Ciocalteu test. The absorbance was measured by spectrophotometer at 734 nm. The phenolic content was estimated at g/100g dry matter (DW).

 

Table 1: The total phenolic content values for all samples in gram per 100 gram (DW).

 

As shown in table 1, the best results were obtained at maceration time of 10 days. The higher amount of polyphenols was in oregano (4.67g/100g) and the lowest content was in thyme (3.58g/100g) at 10 days.

 

It is clear from the results that oregano exceeded both thyme and the mixture as it had the best polyphenols content during all maceration times, with no significant differences in the amount of oregano polyphenols during the period from 6-15 days. In accordance with our results, Adamkova et al. (2015) pointed out that oregano had a higher amount of polyphenols (4.66g/100g) than thyme (2.21g/100g)^[22].

 

3.2 Antioxidant activity:

The antioxidant capacity was estimated by DPPH test. Table 2 shows the values of the results obtained for oregano, thyme and the mixture.

 

The table shows that the best maceration time for each of the extracts was at 10 days as the antioxidant capacity began to decrease after 12 days. It has been shown from table 2 that oregano has an antioxidant capacity greater than the mixture and thyme at all times of maceration.

 

Table 2: The antioxidant activity for all samples estimated as a percentage (%).

 

Spiridon et al. (2011) in their study on a number of plants of the Lamiaceae family found that the methanolic extracts of oregano have the highest antioxidant capacity and phenolic content compared to other plants, including thyme^[23]. In this study, oregano had the highest values of antioxidant activity followed by the mixture then thyme^[23].

 

In a study conducted by Magulska et al. (2018) on a number of medicinal plants, including oregano and thyme, it was found that oregano and thyme have significantly different phenolic content and antioxidant activity. Phenolic content values of thyme ranged from 42.4-240.8mg GA/g while the values for oregano ranged from 104.3-325.1 mgGA/g^[24]. The estimated antioxidant capacity in thyme ranged from 32.5–79.3% and for oregano from 46.8-87.2%^[24]. These values are very close to the results obtained in our study. Note that the solvent used in the previous study is methanol while glycerin is the solvent used in our study. This confirms the importance and efficiency of glycerin as an important solvent in the process of plant extraction.

 

Numerous studies have addressed the importance of glycerin as an important solvent in the extraction of polyphenols. In a study of olive leaves extracts conducted by Mourtzinos et al. (2016) using glycerin as a solvent; there was clearly a positive effect of glycerin at a concentration 60% (w/v) at a temperature 60°C^[25]. It was noted that increasing the amount of glycerin up to 90% (w/v) in the extraction process led to an increase in the amount of phenolic content and therefore glycerin can be used as a green alternative solvent, which is better than other solvents such as methanol and ethanol^[25].

 

In another study, the mass of yield of rosemary plant extracts was measured using 70% ethanol as a solvent and maceration was carried out for 7 and 15 days to extract phytochemicals including phenols where the yield was weighed and the results were estimated as a percentage^[26]. The results showed that there was no significant difference between maceration for 7 and 15 days, the yield of extraction at 7 days was 5.3% while at 15 days was 5%^[26]. This is in accordance with our results as there was no significant difference between the amount of polyphenols between 6 and 15 days while there was a slight decrease in antioxidant capacity from the tenth day to 15 day of maceration.

 

4. CONCLUSION:

In this article, the importance of glycerin as a green solvent like other solvents in the extraction of polyphenols from oregano and thyme was emphasized. It was proved that maceration at large intervals up to 10 days could increase both the antioxidant capacity and the phenolic content in both thyme and oregano.

 

5. REFERENCES:

1.      R. Amarowicz, Z.Z. Egarska, R. Rafałowski, R.B. Pegg, M. Karamac and A. Kosinska. Antioxidant activity and free radical-scavenging capacity of ethanolic extracts of thyme, oregano, and marjoram. Eur. J. Lipid Sci. Technol. 2009; 111: 1111-1117.

2.      H.F. Hasan, S.L. Hassan and O.K. Luaibi. Effect of Thymus vulgaris leaves Extract and Ceftriaxone on induced Pyometra in mice. Research J. Pharm. and Tech. 2019; 12(10):4683-4688.

3.      A. Vallverdú-Queralt, J. Regueiro, M. Martínez-Huélamo, J. Alvarenga, L. Leal and R. Lamuela-Raventos. A comprehensive study on the phenolic profile of widely used culinary herbs and spices: rosemary, thyme, oregano, cinnamon, cumin and bay. Food Chemistry. 2014.

4.      A. Thennarasu. Quercetin in Health and Disease. Research J. Pharm. and Tech. 2013; 6(12): 1397-1399.

5.      U.S. Mahadeva Rao, K.S. Mohd, S.Z. Bt Abd Halim and M. Bt Khamis. Screening of Phytochemicals and Comparative Antioxidant activity of Leaf and Fruit of Malaysian Mengkudu Using Aqueous and Organic Solvent Extracts. Research J. Pharm. and Tech. 2013; 6(9): 1064-1072.

6.      A. Yashin, Y. Yashin, X. Xia and B. Nemzer. Antioxidant Activity of Spices and Their Impact on Human Health: A Review. Antioxidants. 2017.

7.      Mahadeva Rao US, Mohd KS, Bt Abd Halim SZ and Bt Khamis M. Screening of Phytochemicals and Comparative Antioxidant activity of Leaf and Fruit of Malaysian Mengkudu Using Aqueous and Organic Solvent Extracts. Research J. Pharm. and Tech. 2013; 6(9): 1064-1072.

8.      T. Das. Role of Antioxidants in Health and Diseases-A Review. Research J. Pharm. and Tech. 2015; 8(8): 1033-1037.

9.      P. Vishnu Mohan Reddy, K. Venkata Ratnam, R.R. Venkata Raju. In vitro Antioxidant Properties of Alangium salvifolium Seed Extracts. Research J. Pharm. and Tech. 2019; 12(10):4714-4718.

10.   R. Selvam, D. Anandhi, D. Saravanan, K. Revathi. Antioxidant Properties of Punica grantum Fruit rind extract against liver Hepatocellular Carcinoma studied in HEPG2 Cell Line. Research J. Pharm. and Tech. 2019; 12(10): 4719-4723.

11.   T. Rababah, F. Banat, A. Rababah, K. Ereifej and W. Yang. Optimization of Extraction Conditions of Total Phenolics, Antioxidant Activities, and Anthocyanin of Oregano, Thyme, Terebinth, and Pomegranate. Journal of Food Science. 2010; 75.

12.   H. Sakkas and C. Papadopoulou. Antimicrobial Activity of Basil, Oregano, and Thyme Essential Oils. J. Microbiol. Biotechnol. 2017; 27: 429-438.

13.   T. Kulisic, V. Dragovi-Uzelac and M. Milos. Antioxidant Activity of Aqueous Tea Infusions Prepared from Oregano, Thyme and Wild Thyme. Antioxidant Activity of Herbal Tea Infusions, Food Technol. Biotechnol. 2006; 44: 485-492.

14.   I. Mourtzinos, E. Anastasopoulou, A. Petrou, S. Grigorakis, D. Makris and C.  Biliaderis. Optimization of a green extraction method for the recovery of polyphenols from olive leaf using cyclodextrins and glycerin as co-solvents. J Food Sci Technol. 2016; 53: 3939-3947.

15.   B. Karakashov, S. Grigorakis, S. Loupassaki, I. Mourtzinos and D. Makris. Optimisation of organic solvent-free polyphenol extraction from Hypericum triquetrifolium Turra using Box–Behnken experimental design and kinetics. Int J Ind Chem .2015; 6: 85-92.

16.   E. Shehata, S. Grigorakis, S. Loupassaki and D. Makris. Extraction optimization using water/glycerol for the efficient recovery of polyphenolic antioxidants from tow Artemisia species. Separation and Purification Technology. 2015; 149: 462-469.

17.   W. Brand-Williams, M.E. Cuvelier and C. Berset. Use of a Free Radical Method to Evaluate Antioxidant Activity. Lebensm.-Wiss. u.-Technol. 1995; 28: 25-30.

18.   W. Zam, G. Bashour, W. Abdelwahed and W. Khayata. Separation and Purification of Proanthocyanidins Extracted from Pomegranate’s Peels (Punica granatum). International Journal of Pharmaceutical Sciences and Nanotechnology. 2012; 5(3): 1808-1814.

19.   K.V.V.S. Krishna, B. Venkatesh, A. Madhavi, B. V. Padmavathi Devi, Christina Emette Jaldi, D. Sai Bhargavi. Evaluation of Antioxidant Activity of Panchachurnam: An Ayurvedic Polyherbomineral Formulation. Research J. Pharm. and Tech. 2019; 12(10): 4841-4847.

20.   M.A. Melakhessou, N. Benkiki and S.E. Marref. Determination of Antioxidant Capacity, Flavonoids and Total Phenolic Content of Extracts from Atractylis flava Desf. Research J. Pharm. and Tech 2018; 11(12): 5221-5226.

21.   W. Zam, G. Bashour, W. Abdelwahed and W. Khayata. Effective extraction of polyphenols and proanthocyanidins from pomegranate’s peel. International Journal of Pharmacy and Pharmaceutical Sciences. 2012; 4(3): 675-682.

22.   A. Adámková, L. Kouřimská and B. Kadlecová. The effect of drying on antioxidant activity of selected lamiaceae herbs. Potravinarstvo® Scientific Journal for Food Industry. 2015; 9: 252-257.

23.   I. Spiridon, R. Bodirlau and C. Teaca. Total phenolic content and antioxidant activity of plants used in traditional Romanian herbal medicine. Central European Journal of Biology. 2011; 6: 388-396.

24.   B. Magulska and M. Wesolowski. Total Phenolic Contents and Antioxidant Potential of Herbs Used for Medical and Culinary Purposes. Plant Foods for Human Nutrition .2019; 74: 61-67.

25.   I. Mourtzinos, E. Anastasopoulou, A. Petrou, S. Grigorakis, D. Makris and C.G. Biliaderis. Optimization of a green extraction method for the recovery of polyphenols from olive leaf using cyclodextrins and glycerin as co-solvents. J Food Sci Technol. 2016; 53: 3939-3947.

26.   L. Mutalib. Comparison between conventional and modern methods for extraction of Rosmarinus officinalis leaves. Zanco J. Med. Sci.2015; 19.

 

 

Accepted on 21.02.2020           © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(6): 2699-2702.