Antioxidant Activity and Phenolic Content of Sonication- and Maceration-Assisted Ethanol and Acetone Extracts of Cymbopogon citratus Leaves

Abstract

Cymbopogon citratus is a medicinal and well-known aromatic plant which is usually used as a substitute for green-tea with extraordinary phytomedicinal potential. It is of great importance because it offers several promising health effects. The objective of the study was to investigate the antioxidant activity and total phenolic content of lemongrass leaves extracted by maceration and ultrasound-assisted extraction techniques. Different concentrations of both acetone and ethanol (50% and 70%) as solvents were tested for their radical scavenging activity against the stable free radical DPPH in quantization using a spectrophotometric assay. The ultrasound-assisted extraction technique had advantageous results in the polyphenols extraction yield (26.68%), TPC (61 mg GAE/g of extract) and DPPH (73.85%) compared to the maceration technique. Overall, the results were significant from 50% ethanol and the DPPH results were more significant from 70% ethanol than other concentrations. It is concluded that the ultrasound-assisted extraction is a better extraction technique for polyphenols than other conventional techniques.

1. Introduction

Lemongrass (Cymbopogon citratus), also called ‘‘Squinant’’ or ‘‘Citronella’’ in English or other informal names [1,2,3], belongs to one of the largest flowering plants family, Poaceae, and genus, Cymbopogon [4]. C. citratus is a distinguished aromatic plant typically used as an alternative to green-tea. Due to the production of secondary metabolites, lemongrass is used to heal many medical conditions [5,6,7,8]. Studies showed that lemongrass possesses effectiveness against several bacteria such as Salmonella Enteritidis and E. coli [9,10] and fungi [11,12]. Lemongrass leaves are useful in both food and health field as these possesses several biological activities (tumor and hypotensive suppressive effect, antioxidant, hypolipidemic, immune boosting agent, anti-atherosclerotic) and are helpful in the treatment liver diseases, cardiovascular diseases and tissue inflammations. In addition, they regulate the blood cholesterol and sugar level [13,14,15,16]. A study showed that various health benefits could be offered by lemongrass extracts [17].

In the body, the uncontrolled production of reactive oxygen species (ROS) and free radicals as the aerobic metabolism’s secondary products leads to oxidative stress. In the oxygen-rich atmosphere, oxidative stress is an inevitable outcome of life and is contemplated as the major cause of cardiovascular disease, cancer, atherosclerosis, diabetes mellitus, rheumatoid arthritis, hypertension, aging, reduced immunity and others [18,19,20,21,22].

Antioxidants have a vital role in human health because they have antioxidant enzyme cofactors that scavenge free radicals. In the body, they provide protection to dietary antioxidants, i.e., vitamin E and C, and pro-oxidant metal ion chelation [23,24,25]. Commonly, antioxidants are used in the synthetic form, i.e., butyl hydroxy anisole, butyl hydroxy toluene and tert-butyl hydroxyquinone. Nowadays, synthetic antioxidants are not mostly used due to human health concerns [26].

Generally, the extraction of polyphenols is done by means of organic solvents such as acetone, ethanol, methanol and ethyl acetate [27,28]. Extraction using only the organic solvent may not be efficient. However, using different assistive novel extraction technologies (sonication, microwave, pulsed electric field, etc.) could be very effective and cost-saving [29,30]. Maceration is a solid–liquid conventional extraction technique involving extraction by keeping the sample soaked in the solvent at room temperature, with/without agitation, for a long period, so that the sample’s bioactive compounds become entirely soluble in the solvent [27,31]. Meanwhile, ultrasound-assisted extraction or sonication is an innovative extraction technique in which samples are mixed with organic solvents in flasks or beakers and then these flasks are placed at a pre-set time, which is generally l hour or less, and temperature in the ultrasonic water bath. During the process, the produced sound waves lead to the release of polyphenolic compounds by rupturing the cell walls of the samples. Ultrasonic extraction is a more effective technique as it yields 6–35% more extraction of polyphenols and is more time-saving than other traditional techniques [29,32,33,34].

We investigated the antioxidant potential of C. citratus by employing the maceration and ultrasound-assisted extraction (UAE) processes and explored the effects of different concentrations of acetone and ethanol on the yield, TPC and DPPH. This study may provide a knowledge for pilot-scale and large-scale industrial applications of antioxidants extracted from leaves of C. citratus.

2. Materials and Methods

2.1. Plant Material

Lemongrass (Cymbopogon citratus) was procured from Tramari, Islamabad-Pakistan, and taken to FSRI (Food Science Research Institute, Islamabad, Pakistan) labs at NARC (National Agricultural Research Center, Pakistan, Islamabad), Islamabad-Pakistan. Lemongrass leaves were weighed and put in a hot air oven at 50 °C for 72 h. until the moisture content was less than 10%. Firstly, dried leaves were crushed with mortar and pestle. Secondly, fine powder of crushed leaves was prepared by being ground in Cyclotech mill (Foss A/S, Hillerød, Denmark) with sieve (0.5 mm size). Then, lemongrass leaves powder was stored at 4 °C by packing in polyethylene bags that were air-tight zip bags.

2.2. Physicochemical Analysis of Lemongrass Leaves

Lemongrass leaves powder was prepared and analyzed for ash, moisture, crude fat, crude fiber and crude protein in accordance to [35] standards methods. Carbohydrates in lemongrass leaves powder were calculated by subtracting sum of % moisture, % ash, % crude protein, % crude fat and % crude fiber from 100.

2.3. Methods of Polyphenols Extraction

Polyphenols were extracted from lemongrass leaves powder by employing maceration and sonication techniques.

2.4. Maceration Technique

Lemongrass leaves powder was subjected to maceration technique in accordance to [36] method with some modifications. Different solvents, i.e., ethanol and acetone, at two solvent concentrations (50% and 70%) with sample-to-solvent ratio of 1:15 at 40 °C, were used to carry out extraction. Concisely, 3 g lemongrass powder sample was taken into conical flasks. Then, 50% and 70% ethanol and 50% and 70% acetone was added to the flasks containing sample. The flasks were covered with aluminum foil to prevent the evaporation of ethanol and acetone and the mixture pouring out of flasks during operation. The conical flasks were placed in shaking water bath to shake the raw material. Rotation was at shaking speed of 9 and at temperature of 40 °C for 24 h. After removing the extracted sample from Maceration water bath, Whatmann filter paper 41 was used to filter the samples. Then, the samples were centrifuged for 10 min at 5000 rpm. Polyphenols extracted were then separated from ethanol and acetone by evaporating solvent through rotary evaporator under vacuum at 45 °C. The sample extracts were placed in the oven overnight at 40 °C for complete drying of extract. After drying, extracts were stored at 4–6 °C.

2.5. Sonication Technique

Lemongrass leaves powder was subjected to sonication technique in accordance to the method defined by [37], with some amendments. Different solvents, i.e., ethanol and acetone, at two solvent concentrations (50% and 70%), with sample-to-solvent ratio of 1:20 at 45 °C for 60 min, were used to carry out extraction. Concisely, 3 g lemongrass powder sample was taken into conical flasks. Then, 50% and 70% ethanol and 50% and 70% acetone were added into the 125 mL reagent bottles containing sample. The reagent bottles were covered with aluminum foil to prevent the evaporation of ethanol and acetone and the mixture pouring out of reagent bottles during operation. The reagent bottles were placed in sonication water bath (Tecator 1024, Pal Anders vag, Hoganas, Sweden) at 35 kHz frequency. As in maceration, the filtration, centrifugation, solvent vacuum evaporation, drying, collection in reagent bottles and storing at 4–6 °C of sample extracts was done.

2.6. Yield of Lemongrass Leaves

The yield of lemongrass leaves extracts by maceration and sonication techniques was calculated as followed:

% extract yield = (weight of the extract ÷ sample weight) × 100

2.7. Total Phenolic Content

Total polyphenolic content of lemongrass leaves determined in accordance to Folin-Ciocalteu Method, as defined by [38]. For the analysis, methanolic lemongrass leaves extracts of 10 mg/mL were prepared. Concisely, methanolic extract solution (0.5 mL) was mixed with 10% Folin-Ciocalteu reagent dissolved in water (2.5 mL) and 7.5% sodium carbonate (2.5 mL). Blank was concomitantly prepared, containing 0.5 mL methanol, 2.5 mL diluted Folin–Ciocalteu reagent dissolved in water and 2.5 mL 7.5% sodium carbonate. Then, the incubation of samples was carried out for 30 min at 25 °C until blue-color development. The UV-visible spectrophotometer was used to determine the absorbance at 765 nm. Therefore, spectrophotometer was first calibrated by using pure methanol as a blank. For Gallic acid standard solution, same procedure was carried out. Total polyphenolic content was expressed as (mg GAE)/g of extract.

2.8. Antioxidant Activity of Lemongrass Leaves

In vitro antioxidant activity evaluation of polyphenols implicates the free radicals’ production and to-be-tested antioxidants’ radical scavenging ability by using reagents and chemicals which may be measured. Several assays are used for determining the antioxidant activity, but DPPH is broadly used to calculate antioxidant capacity in fruits, plants, vegetables, etc., [39,40].

1,1-diphenyl-2-picryl-hydrazyl of lemongrass was determined in accordance to method of [41], with some amendments. Concisely, by dissolving 24 mg DPPH with 100 mL methanol, stock solution was prepared and stored at 20 °C. The spectrophotometer was used to obtain an absorbance of about 0.980 at 517 nm by preparing working solution obtained by mixing DPPH solution with methanol—100 μL of the samples (400, 200, 100, 50, 25) in test tube along with 3 mL DPPH solution. The solutions in the test tubes were shaken well and were incubated for 30 min in dark at room temperature. After that, the samples were run at 517 nm. The scavenging activity was estimated based on the percentage of DPPH radical.

DPPH radical% Inhibition = Control absorbance (A₀) − Lemongrass leaves extract absorbance (A₁) × 100

2.9. Statistical Analysis

Resulted data from research was employed to Minitab software (Version 18.1, Minitab, Borough, PA, USA) to carry out statistical analysis. Analysis of variance (ANOVA) technique was applied to data for statistical analysis to find out level of significance. Least significant differences (LSD) amid means were calculated for better interpretation of results.

3. Results and Discussion

3.1. Physicochemical Analysis

The raw material quality’s significance can be assessed by the estimation of a proximate composition. The data of the proximate composition of lemongrass are presented in

Table 1. Composition of Lemongrass Leaves Powder.

Moisture (%)	Ash (%)	Crude Fat (%)	Crude Protein (%)	Crude Fiber (%)	Carbohydrates (%)
9.93 ± 0.23	4.83 ± 0.07	2.23 ± 0.03	3.82 ± 0.04	20.61 ± 1.13	58.58 ± 2.15

. The ash (4.83%), fat (2.33%) and fiber (20.61%) contents were found in smaller amounts, while the protein (3.25%), carbohydrate (59.15%) and moisture (9.93%) contents were found in appreciable amounts in lemongrass.

Earlier, [42] found 2.83% fibers, 11.67% ash, 11.14% protein and 87.63% carbohydrates in lemongrass. Similarly, [43] observed 7.01% moisture, 11.15% protein, 65.78% carbohydrates, 1.45% fat and 11.28% ash in lemongrass powder.

3.2. Extraction Yield

Overall, the yield of the polyphenols in the lemongrass extract was significantly higher with the sonication than the maceration technique. The highest yield (%) was achieved with ethanol at two different concentration levels, either through the ultrasound-assisted extraction technique or the maceration extraction technique (Figure 1). In the maceration technique, the extraction of the polyphenols with 70% ethanol gave the maximum yield (20.3%), followed by the 50%-ethanol extraction yield (19.4%), while the low extraction yield (17.4%) was observed in samples extracted with 50% acetone. With regards to sonication, 50% ethanol had a comparatively higher yield (26.7%) than the lemongrass samples extracted with solvent acetone.

The sonication technique showed the highest yield% with the 50% ethanol concentration. Meanwhile, the maceration technique showed the highest yield% with the 70% ethanol concentration. Ref. [26] reported that the maximum 7.7% yield was obtained from 70% ethanol extract, rather than the other 30% and 96% ethanol extracts. Refs. [27,43] also reported a better yield with sonication.

3.3. Total Phenolic Contents

It is observed from the data that sonication resulted in a comparatively higher phenolic content and was a more efficient extraction technique than maceration (Figure 2). The maceration extraction technique results showed that acetone was the most effectual solvent, while ethanol showed the lowest phenolic content. The highest total phenolic content (55.2 mg GAE/g of extract) was extracted with solvent acetone at a 50% concentration, whereas with 70% ethanol, the least polyphenols (32.9 mg GAE/g of extract) were obtained. Meanwhile, the sonication technique results showed that with 50% ethanol (61.2 mg GAE/g of extract), the maximum polyphenols were extracted, while 70% acetone (50.9 mg GAE/g of extract) extracted the minimum phenolics.

The sonication technique showed the highest total phenolic content with the 50% ethanol concentration. Meanwhile, maceration showed the highest total phenolic content with the 50% acetone concentration. Ref. [26] reported that the 30% ethanol extract showed the maximum total phenolic content of lemongrass leaves (50.0 mg GAE/g). Similarly, ref. [44] reported the maximum total phenolic content (67.3 mg GAE/g) with the 40% ethanol extract in lemongrass leaves. Refs. [27,43] also reported a better total phenolic content with sonication. Ref. [45] reported the better recovery of flavonoids and phenolics with ethanol as the co-solvent.

3.4. DPPH Radical Scavenging Activity

The DPPH radical scavenging activity of lemongrass leaves extracts at different solvents and their concentration levels (Figure 3 and Figure 4) discloses the high antioxidant activity of all sample extracts. With the 70% ethanol concentration level, scavenging activity (73.8 ± 0.9) was exhibited by the samples extracted, while the samples extracted with 70% acetone showed the lowest scavenging activity (67.9 ± 0.7). In the case of sonication, the 50% acetone concentration showed the highest radical scavenging activity, while maceration showed radical scavenging activity with the 70% ethanol concentration.

A study reported that the 70% ethanol extract (79.4 mg/L) had the highest DPPH inhibition which resulted in the lowest IC₅₀ value of lemongrass leaves extracts [26]. Similarly, ref. [46] reported 1140 mg/L with 80% methanol using fresh lemongrass extract. Refs. [27,43] also reported a better DPPH radical scavenging activity content with sonication.

4. Conclusions

Lemongrass leaves are potential sources of polyphenols with high antioxidant properties. The sonication technique resulted in a comparatively high yield (26.7%) and a higher phenolic content (61.2 mg GAE/g of extract) than the maceration technique. Furthermore, it was found that ethanol is an efficient solvent in extracting polyphenols, specifically, 50% ethanol yielded the maximum extract. Ethanol is also classified under the GRAS status and favored due to its utilization in food industries to prepare functional foods. Further studies may be carried out to investigate the individual phenolic compounds of lemongrass by HPLC and other advance techniques.