ANTIOXIDATIVE CHARACTERISTICS OF BEVERAGES MADE FROM A MIXTURE OF LEMONGRASS EXTRACT AND GREEN TEA

Combination of lemongrass extract and green tea infusion is expected to produce functional beverage that has potential antioxidant activity as compared to individual materials. This research was aimed to investigate the appropriate solvent type and extraction methods to produce lemongrass extract with highest antioxidant activity and also to investigate the antioxidant characteristic of beverages prepared from a mixture of lemongrass extract and green tea. Lemongrass were subjected to extraction under reflux and maceration using three type of solvents (distilled water, ethanol, and ethyl acetate). The resulting extracts were assessed for their total phenolic content, total flavonoid content, and antioxidant activity. Extraction under reflux and using ethanol as solvent resulted in extract with best antioxidant activity (IC50=158.70±47.88 mg/L for reflux method and 103.73±8.03 mg/L for ethanol). This extract was combined with green tea with several ratios (lemongrass extract to green tea [w/w] = 1:3; 1:2; 1:1; 2:1; 3:1). Four level of stevia were used in the beverages (0, 200, 400, 600 ppm). Beverages made with lomogass: tea ratio of 1:3 and 1:1 (w/w) and 600 ppm stevia turned out to be beverages with the highest antioxidant activity (IC50=271.83±44.48 mg/L and 287.15±36.25 mg/L for ratio 1:3 and 1:1 [w/w] consecutively, 301.59±80.89 mg/L for stevia level 600 ppm). Hedonic testing on beverages revealed that beverage made from lemongrass: tea ratio 1:1 [w/w] ratio with addition of stevia at 600 ppm is the most preferred based on colour, aroma, and overall acceptance.


INTRODUCTION1
Antioxidant acts as radical scavenger which donates hydrogen ion to neutralize free radical in human cells and inhibits occurence of cancer.Incorporation of antioxidant in the diet might help in reducing oxidative stress and occurrence of degenerative disease such as cancer (Cheel et al. 2005).Lemongrass (Cymbopogon spp.) plant grows in tropical and subtropical region and is commonly cultivated as spices or essential oil.Lemongrass extract was reported to inhibit colon cancer in animal models (Puatanachokchai et al. 2002).Juntachote et al. (2006) reported that ethanolic extract of lemongrass contains 535.44 to 1007.35 mg/100 g total phenolic and have 80.38 to 93.31% antioxidant activity.
Other than lemongrass extract, green tea infusion also has high antioxidant activity.As mentioned by Kirakosyan et al. (2010), pairs of compounds with high antioxidant capacity showed strong synergistic interactions.This finding leads to possibility of synergistic interactions between lemongrass extract and green tea in terms of antioxidant activity.Addition of stevia as sweetener was expected to mask undesirable taste that might be contributed by mixture of lemongrass extract and green tea.Lemongrass extract is a potential natural antioxidant however a proper solvent and method of extraction are yet to be determined.Madan et al. (2010) mentioned that steviol glycoside as main component of stevia has antioxidant activity.

Materials
Materials used in this research were lemongrass leaves from a plantation in Garut-East Java, commercial stevia powder 90% Aoxing, green tea from Gunung Mas tea plantation, and drinking water.The lemongrass was dried using cabinet dryer (40°C, 24 h), grinded, and sieved.Particle size of 30 mesh was considered as sufficient for extraction of lemongrass antioxidant.It referred to particle size of wheat bran in extraction of wheat bran antioxidant (Yu, 2008).The solvents used were ethanol 80%, ethyl acetate 80%, and distilled water.Concentration of solvent for optimum concentration of ethanol extraction of lemongrass as mentioned by Jutanchote et al. (2006), is 1:10 [w/v] (powder : solvent).Two types of extraction methods were applied in this study were maceration (25°C, 48 h) (modified from Tachakittirungrod et al. 2007 in terms of sample amount) and reflux extraction (boiled, 30 min) (modified from Cheel et al. 2005 in terms of solvent type).

Extract preparation (modified from
The resulted extracts were filtered using Whatmann no.1 and were concentrated using rotary evaporator at 40 o C. The concentrated extracts were analyzed for their moisture content, yield (%), total phenolic content, total flavonoid content, antioxidant activity, phytochemical test, and toxicity test.

Beverages preparation (Tagami et al. 2012)
The beverage was prepared from combination of lemongrass extract and green tea with addition of stevia powder as sweetener to mask undesirable taste.The amount of stevia powder added was based on regulation by World Health Organization (WHO, 2009).The ratios of lemongrass extract to green tea were 1:1; 1:2, 1:3, 2:1, 3:1 [w/w] ( Kirakosyan et al. 2010).The mixture of lemongrass extract and green tea leaves was brewed with water at 90°C for 1 minutes.The ratio of green tea to water is 1 to 43 (w/v).The brewing method was adopted from Standard Table of Food Composition in Japan (Tagami et al. 2012).The beverages were analyzed for total phenolic content, flavonoid, and antioxidant activity.Hedonic test was done for sample with the best characteristics.

Antioxidant activity using DPPH method (Shim and Lim, 2009)
Sample was measured at 0.1 mL and was mixed with 2.9 mL of 0.1 mM of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH).The mixture was homogenized by using vortex.Afterwards the mixture was put aside in dark place for 30 minutes.The absorbance of the mixture was measured by using visible spectro-photometer at wavelength 517 nm.The control was made by mixing 0.1 mL of ethanol and 2.9 mL of 0.1 mM DPPH.
Total phenolic content using Folin-Ciocalteu method (Makkar et al. 2007) (modified in terms of sample amount) The sample was put in reaction tube (1 ml) and was added with 0.5 mL of 1 N Folin-Ciocalteu reagent and 2.5 mL of 20% sodium carbonate solution.The solution was mixed well using vortex and incubated under dark conditions for 40 minutes.The absorbance of the solution was measured using visible spectrophotometer with wavelength 725 nm.The total phenolics content was determined from calibration curve with tannic acid as a standard.

Total flavonoid content (Meda et al. 2005)
For total flavonoid content analysis, 5 mL of sample was mixed with 5 mL of 2% aluminum chloride.This mixture was mixed well by using vortex and incubated for 10 minutes at room temperature.Absorbance of mixture was measured by using visible spetrophotometer at 415 nm.Blank was prepared from 5 mL sample and 5 mL ethanol.Calibration curve was made by using quercetin as standard.

Toxicity test (Juniarti et al. 2009)
The toxicity test of lemongrass extract was done at LPSB (Laboratorium Pusat Studi Biofarmaka) IPB, Bogor.Brine Shrimp Lethality Test (BSLT) method was applied to measure lethality level of the brine shrimp larvae treated with lemongrass extract.Toxicity of extract was stated as on LC50 value (concentration where sample causes 50% death of shrimp larvae).

Consumer acceptance test by 7 hedonic scale (Moskowitz et al. 2006)
The consumer acceptance was evaluated regarding colour, aroma, taste, and overall acceptance of beverage.The test required 50 panelists and counterbalancing was also done to the samples.The test was done by using seven scales from 1 (extremely dislike) until 7 (extremely like).

Statistical analysis
All statistical analysis were done using SPSS version 17. Complete factorial randomized designs with two repetitions were applied to all experiments.For the extract preparation step, two factors were analyzed (solvent types and methods of extraction), for beverage preparation step, another two factors were analyzed (ratio of lemongrass extract to green tea and stevia level).Significant effects of factors are analyzed further using Duncan test.

RESULTS AND DISCUSSION
Prepared extract yield (%) Figure 1 shows the result of yield (%) for extracts obtained using solvents (distilled water, ethanol 80%, and ethyl acetate 80%) and two types of method (maceration and reflux).Based on statistical analysis result, there is a significant effect of interaction of solvent types and methods of extraction on yield (%) (α=0.05).
Note: Different superscript in the graph means there is significant difference at α=0.05It can be noted that reflux and maceration with ethanol as solvent resulted in highest yield (%). Previous study showed that extraction using reflux results in higher amount of yield compared to maceration (Sultana et al. 2009).It is possible that heat generated during reflux destructs cell wall of lemongrass leaves so the content of cell leaks and dissolves in solvent extraction.It is important to note that yield (%) does not represent antioxidant activity since the extract obtained is crude extract that may contains compounds other than antioxidant compounds.It was reported that organic solvent has ability to cleave hydrogen bond or hydrophobic bond between polyphenols and protein or polysaccharide in plant tissue (McManus et al. 1985) herefore organic solvent is suitable to extract polyphenols.On the other hand, polarity of solvent is responsible to determine type of extracted components (Islam, 2008).Ethanol is suitable to extract compounds with polar characteristic (Sultana et al. 2009).It can be inferred that polar compounds dominate lemongrass because the yield (%) is the highest in extraction using ethanol.

Total phenolic content of extracts
The results of total phenolic content of extracts can be seen in Figure 2. Based on statistical analysis on total phenolic content of extracts, there is a significant effect of interaction of solvent types and methods of extraction (α=0.05) on total phenolic content.Reflux extraction using ethanol as a solvent resulted in significantly higher total phenolics than other treatments (126.66±4.59mg TAE/g extract).It can be inferred that phenolics present in lemongrass dissolve better in organic polar solvent.It was reported by Sultana et al. (2009) that regardless the extracting solvent used, total phenolic content of plant materials extracted using reflux technique is decreased.This might be caused by thermal decomposition of some phenolics.However different result was found in extraction of Vogel seeds under reflux using aqueous ethanol.It can be inferred that effective extraction condition varied among different plant phenolics.Antolovich et al. (2000) also studied that extraction under reflux condition can lead to the release of some bound phenolics, thus increase the amount of phenolics.Beside that, Iso-orientin and orientin, the two main water-soluble antioxidant compounds in lemongrass (Cheel et al. 2005) are able to withstand high temperature as high as spray drying (Viljouen, 2008).These explain the high total phenolic contents of extract under reflux treatments.

Total flavonoid content of extracts
The result of total flavonoid analysis of all extracts can be seen in Figure 3. Based on statistical analysis of total flavonoid content, there is a significant effect of interaction (α=0.05) of solvent types and methods of extraction on total flavonoid content.This result is in agreement with total flavonoids of Aloe barbadensis leaves and Ficus religiosa fruits that increased under reflux extraction with aqueous methanol and aqueous ethanol consecutively (Sultana et al. 2009).

Antioxidant activity of extracts
Solvent types and method of extraction gave significant effect towards antioxidant activity (α=0.05).However, there is no significant effect of interaction of solvent types and methods of extraction (α=0.05) on antioxidant activity of extracts.Effect of method on antioxidant activity is as shown in Table 1.Reflux method resulted in extract with statistically highest antioxidant activity (IC50 158.70±47.88mg/L).Extract obtained using ethanol 80% as solvent also resulted in statistically highest antioxidant activity (IC50 103.73±8.03mg/L).Usually extraction under reflux results in extract with lower antioxidant activity as compared to maceration.However this is not solely the case.Previous study showed that ethanolic extracts of roots of Moringa oleifera, leaves of Aloe barbadensis, and fruit of Ficus religiosa obtained from reflux method has higher scavenging activity as compared with those of obtained from maceration method (Sultana et al. 2009).Reflux is suitable to extract sample that has resistance to high temperature.
It was reported that antioxidant activities of the plant materials are strongly correlated with nature of extracting solvents.Ethanol as a polar solvent is suitable to extract antioxidant compounds from various plants and plant-based foods such as plum, pomegranate, broccoli, rosemary, sage, rice bran, and citrus peel (Sultana et al. 2009).In this case ethanol is also suitable to extract antioxidant constituent of lemongrass since it resulted in extract with highest antioxidant activity.

Phytochemicals of selected extract detected qualitatively
Ethanolic extract obtained under reflux was chosen as extract with highest antioxidant activity and also chosen to be applied in beverages.Therefore, this extract was subjected to qualitative phytochemical test.The result of qualitative analysis on phytochemicals present in ethanolic extract obtained under reflux is as shown in Table 2. Result of qualitative analysis on lemongrass extract suggested that flavonoids present in more abundant level as compared to phenolics.In fact flavonoids is a part of phenolics so the amount of phenolics should be higher than flavonoids.It is important to be noted that one can not determine amount of compound through qualitative analysis.Since qualitative analysis merely serve as rough estimation of phytochemicals present in the extract.However qualitative analysis on phytochemical is also important to give information of compounds that might present in extract.

Toxicity analysis on selected extract
Toxicity analysis on ethanolic extract obtained under reflux was conducted to confirm the safety of extract considering the extract was applied to food product.Result of toxicity analysis was expressed as LC50 with value of 289.23 ppm.According to Moshi et al. (2010), the brine shimp lethality test results are interpreted as non toxic when the value of LC50 is higher than 100 ppm.The result of interpretation confirms the safety of extract to be consumed and therefore the extract can be applied to food product.

Prepared beverage total phenolic content
The result of total phenolic analysis of the beverages is shown in Figure 4.There is a significant effect of interaction (α = 0.05) of lemongrass extract to green tea ratio and level of stevia in affecting total phenolic content of beverages.The ratio of lemongrass extract to green tea at 1:3 [w/w] and level of stevia at 600 ppm resulted in statistically highest total phenolic content in the tested beverages (446.93±12.61mg TAE/g beverages).
Interaction between ratio of lemongrass extract to green tea and level of stevia gave various effects regarding total phenolic content.It may be contributed by complex interaction among phenolics from lemongrass extract and green tea and glycosides from stevia present in mixture.Interestingly, stevia level at 600 ppm resulted in highest phenolic in each lemongrass extract to green tea ratio.Jahan et al. (2010) found that total phenolics content from stevia leaves (Stevia rebaudiana) extracted with hot water is as much as 31.25±0.25 mg GAE/g.Therefore, increasing amount of stevia might affect amount of total phenolic in beverages.

Total flavonoid content of beverages
The result of total flavonoid analysis can be seen in Figure 5. Based on statistical analysis, there is a significant effect of interaction (α=0.05) of lemongrass extract to green tea ratio and stevia level on total flavonoid content.Ratio of lemongrass extract to green tea at 1:1 [w/w]) and level of stevia at 600 ppm resulted in statistically highest total flavonoid content beverage (144.25±7.74mg QE/g beverage).Again, interaction between each constituent, in this case flavonoid constituent may occur and affect total flavonoid content in some ways either decrease or increase total flavonoid content.According to Hidalgo et al. (2010), it is well-known that flavonoids have tendency to bind each other when they are mixed together.It is possible that these bonds hinder the availability of flavonoid constituent to react with reagent during analysis.Hence it may decrease total flavonoid content analyzed.

Antioxidant activity of beverages
Based on statistical analysis, there is no significant effect of interaction (α=0.05) of lemongrass extract to green tea ratio and level of stevia on antioxidant activity of beverages.Effect of lemongrass extract to green tea ratio and level of stevia to antioxidant activity of beverage can be seen in Table 3.
Ratio of lemongrass extract to green tea at 1:3 and 1:1 [w/w] resulted in statistically highest antioxidant activity (IC50 271.84±44.48mg/L and 287.15±36.25 mg/L respectively) among treatments.On the other hand, stevia level at 600 ppm resulted in statistically highest antioxidant activity (301.60±80.89mg/L) among treatments.Lemongrass extract to green tea ratio affected value of IC50 in various ways.It can be inferred from the result that chemical interactions or binding may be occurring among antioxidant components (Hidalgo et al. 2010) and leads to unpredictable result of antioxidant activity.
Study regarding antioxidant activity of combined flavonoid constituents revealed that combination of certain constituents may result in either synergistic or antagonistic interaction.Again, the result is unpredictable since there is no certain rule regarding either synergistically or antagonistically interacted combination (Hidalgo et al. 2010).For example, Kadoma et al. (2006) studied the synergistic interaction between epicathecin and epigallocatechin gallate (bioactive compounds in green tea) with tocopherols in an in vitro model.Meanwhile another study (Hidalgo et al. 2010) showed antagonistic interaction of antioxidant activity when epicathecin and epigallocatechin gallate were combined with myricetin and quercetin.
In comparison with vitamin C, this beverage was less effective as antioxidant, which showed the IC50 of 4.14 ppm (Witoko, 2012).On the other hand it is comparable with the IC50 of black tea, which is 288.60 ppm (Moraes- de-Souza et al. 2008).

Hedonic test on beverages with best antioxidant activity
Beverages selection for hedonic test was emphasized on antioxidant activity (as IC50) of beverages.Beverages with best antioxidant activity were made with lemongrass extract to green tea ratio [w/w] at 1:3, 1:1, 3:1, with level of stevia at 600 ppm.
Based on statistical analysis (α=0.05), it can be seen that there is a significant effect of ratio of lemongrass extract to green tea on all parameters (colour, aroma, taste, and overall acceptance).Table 4 shows the effect of lemongrass extract to green tea ratio on colour, aroma, taste, and overall acceptance of beverages.It can be seen that although beverage made with ratio of lemongrass extract to green tea [w/w] at 1:1 was not significantly preferred as compared to beverage made with ratio (lemongrass extract to green tea [w/w]) 1:3, all attributes of this beverage were preferred by panel.It can be concluded that based on colour, aroma, taste, and overall acceptance this beverage was the most preferred by panel.The results also showed that the final product overall hedonic score is 4.4 (neutral to slightly liked), meaning there is an opportunity to improve its hedonic characteristic.

CONCLUSION
Solvent types and method of extraction interacts in affecting the yield (%), total phenolic content, and total flavonoid content of extract.Reflux method and ethanol resulted in statistically (α=0.05)highest antioxidant activity among other treatments (IC50 = 158.70±47.88mg/L for reflux method and 103.73±8.03mg/L for ethanol).
Ratio of lemongrass extract to green tea and level of stevia have significant effect of interaction on total phenolic content and total flavonoid content, but not to antioxidant activity of beverages.Beverages with best formulation are determined based on highest antioxidant activity.Beverages made with ratio of lemongrass extract to green tea [w/w] at 1:3 and 1:1 and level of stevia 600 ppm were beverages with the highest antioxidant activity.Hedonic testing on beverages revealed that beverage made from ratio of lemongrass extract to green tea [w/w] at 1:1 with addition of stevia at 600 ppm was the most preferred regarding colour, aroma, and overall acceptance (neutral to slightly liked).Therefore, beverage made from ratio of lemongrass extract to green tea [w/w] at 1:1 can be produced commercially as it exhibited beneficial characteristics.

Figure 1 .
Figure 1.Effect of solvent types and methods of extraction on yield (%)

05 Figure 2 .
Figure 2. Effect of solvent types and methods of extraction on the total phenolic content of extracts

05 Figure 3 .
Figure 3.Effect of solvent types and methods of extraction on total flavonoid content of extract

Figure 5 .
Figure 4. Effect of ratio of lemongrass extract to green tea and level of stevia on total phenolic content of beverages

Table 1 .
Effect of solvent types and methods of extraction on antioxidant activity of extracts (a)

Table 2 .
Result of qualitative analysis of phytochemicals on lemongrass ethanolic extracts extracted using reflux method

Table 3 .
Effect of lemongrass extract to green tea ratio and level of stevia on antioxidant activity of beverages (a)

Table 4 .
Effect of lemongrass extract to green tea ratio on colour, aroma, taste, and overall acceptance of beverages Note: Superscripts are to be read horizontally.Different superscripts among the same parameters indicate significant difference at α=0.05