ANTIOXIDANT ACTIVITY AND ISOLATION BIOACTIVE COMPOUNDS FROM ROSELLE ( HIBISCUS SABDARIFFA ) FROM VARIOUS METHODS AND DRYING TIME

The focus of this study was to evaluate the effect of different drying methods and drying level on the bioactive compounds of roselle (Hibiscus sabdariffa L.)as antioxidant agent. The experiment has been conducted in cabinet dryer and by a direct exposure on sun light as weel as by using a green house effect panel with duration of driying: 1, 3, 5, 7 hours. The observed parameters were Colour, levels of anthocyanins,vitamin C, and free radical trapping capacity (DPPH). The data obtained were analyzed using analysis of variance (ANOVA) and further test DMRT (Duncant Multiple Range Test). The best results from these study was the cabinet drying with 7 h of drying time which have value for color (L, a*, b*), total anthocyanins, vitamin C, and antioxidant activity in respective order were 21.633; 6.80; 4.53; 3.22mg/100g; 1238.29mg/100g; 74,63%. Keyword antioxidant, roselle, anthocyanins, vitamin C

Roselle (Hibiscus sabdariffa L.) is one of the Indonesian indigenous plants including herbaceous plants.Petals are bright red can be dried into dried rosella known by the public as red tea.The red color of roselle caused by it contains anthocyanin pigments that can act as antioxidants.Roselle calyx is containing of: flavonoid, gossypetin, sabdaretin, hisbicetin and anthocyanin (Azza et.al, 2011;Chumsri et.al, 2008).Purbowati et al. (2015) stated that roselle calyxes are containing a lot of phenolic compounds, anthocyanin and vitamin C. Dried roselle calyx contains, in respective order : 23.77 ± 0.25; 14.80 ± 0.08;10.74± 0.14 mg/100g.
Wet roselle is a highly perishable material subject because it contains 90.42% of water (Chumsri et al., 2008).Therefore, dehydration process is the important step to extend shellife of raw material and keeping its native properties before utilization for different purposes.The primary objective of drying is the removal of water from foods, which microorganisms require for growth, resulting in a more shelf stable, smaller and lighter food.Reduction in moisture discourages growth of spoilage or pathogenic microorganisms (Boyer and Huff, 2008).
One of the major set back for roselle was in dried roselle form, the antioxidant activity lower than the fresh one in the same dried weight most probably due to drying method employed.Chumsri et al. (2008) stated that EC50 from the fresh roselle was 39.37mg/ml lower than the dried ones 47.53 mg/mL.This condition caused by anthocyanin as a bioactive compound from roselle will detariote under exposed by heat and light (Laleh et al.,2006;Rahmawati (2011):) There are several methods of drying including sun or solar, conventional, and cabinet drying.There are different forms of solar drying, namely: traditional, solar box and sun oven.Traditional sun drying is low/no cost and crops are exposed to the environment.It is the preferred choice of drying for farmers in developing countries because of finance.This method of drying however is worth with many challenges including unpredictable weather, infestation of insects, pests and microbial contamination (Ekechucwu, 2010), and it poses a food safety problem for consumers.Solar box is a box fabricated with wood and glass cover of varying sizes while sun oven is a box with reflective panels.Food is placed in a pan painted black in the box.Both solar box and sun oven are left outside to absorb energy from the sun and are safer alternatives to the traditional concrete floor, open air drying and more beneficial.Although solar box and sun oven drying are equally affected by weather as the traditional drying, they are not plagued by pests' problems.Conventional drying uses electricity and is the most practical and reliable.However, due to high cost and epileptic supply of electricity low scale farmers in developing countries cannot afford this method of food drying.
The selection of the dehydration method depends on raw material properties, the desired characteristics of the dried product, the restriction on the operating conditions and costs.These research objective were to determine the effect of various drying method and time on anthocyanin and vitamin C isolation and antioxidant activity of dried roselle..

Tools
The equipment used in this study includes cabinet dryer, greenhouse chamber effect, microwave electrolux, vacuum evaporator, burette, shaker, micro pipette, multiwell plates, spectrophotometer, analytical scales.Also needed glassware for analysis such as test tube, erlenmeyer, measuring cups and measuring flask.

Decreased moisture of roselle petals
The roselle calyxes were old, whole and clean separated from damaged or rotten calyxes and dispose of any defects in the calyxes.The calyxes are separated from the seeds and then wash with clean water and drain the water.After that, drying is done using the cabinet dryer method, Greenhouse Effect and solar dryer each with drying time 1,3,5,7 hours.Then the resulting roselle petals wrapped in a tight plastic to avoid the effects of moisture.

Extraction
Ten gram of the dried roselle calyxes with various drying method and time were ground for 1 minute using grinder and sieved in 60 mesh.The extraction were done in 100 ml aquadest, microwave power extraction 250 W and 5 minutes time of extraction for each unit combinations.
Microwave used for extraction was Electrolux EMM 2007X.The slurry was radiated in microwave oven at regular intervals (one minute radiation and two minute off) to keep the temperature not rise above the boiling point (Purbowati et al., 2016).Roselle extract was filtered and concentrated with vacuum evaporator at 70oC, 44 cmHg and blowing with N2 to ensure the solvent totally evaporate.

Color measurement (Tanska et al., 2005)
The result were expressed in CIEL*a*b*L color space, where L* is the lightness, a* is the greenness-redness and b* is the blueness-yellowness.The L* component was in range of 0-100% while a* and b* component were in the range from -120 to +120.Before analysis, the calibration to standard white respective plate was done.

Antioxidant capacity
Capacity antioxidant test using DPPH method (Blois, 1985).100 mL of sample add with 3 mL of methanol.This solution was adding with 1 mL of DPPH and keep in the dark for 15 minutes in room temperature.The decreasing of DPPH absorbance measure with spectrophotometer using λ 517 nm.As standard was used gallic acid.

Data Analysis
The determination of chosen drying method and time statiscally using Statistical Product and Service Solutions (SPSS) software through Independent T-Test.

RESULT AND DISCUSSION Color Measurement
The color of roselle extract from the various drying methods and times were measured using digital image anlysis according to the methods describe by Tanska et al. (2005).Result of color measurement of the extract expressed by L*, a* and b*.All extract were not light (L* value average 26.67 -21.33) and had a red-yellow ( positive a* and positive b*).The statistic showed there were significant differences in extract color obtained by different both drying method and drying time.(2017), the longer drying time was caused higher temperature leads to lightening.But, it showed in Figure 1, that the increasing of drying time until 7 h, make the extract darker.This occur as a consequence of the presence of phenolic substance esspesialy anthocyanin which responsible for red color in roselle extract.According to Kharisma et al (2015), the increasing of anthocyanin content increased the redness level of the extract.Hosain et al.( 2010) showed significant higher content of phenolic substance and antioxidant capacity of the air dried lamiacea herbs versus the fresh ones.It mean that during drying process, bioactive compounds were concentrated in plant cell.
The same reasons used for a* and b*. Figure 2 showed that the value of a* is increasing with the increasing of drying time.In the other side, higher value of anthocyanin has a negative correlation with b* Furthermore, it was noted that the higher temperature tends to lighter (Figure 3).It is as a result of the absence of anthocyanin caused the b* value higher .Drying method made siqnificantly difference to the a* and b* value (Figure 4).Cabinet drying had no difference effect with sun drying method on b* value, although the value of that resulted from cabinet drying (8.55) was higher than that of sun drying method (6.99).In the other side, value of a, for cabinet drying, sun drying and ERK method were, in respectively order, 5.75; 5.26; 4.69.

Anthocyanin
This research showed that the increasing of drying time untill 7 hours will cause the increasing of anthocyanin content in the extract.Figure 5 showed that for 7 hours drying time, anthocyanin content in roselle extract was 2.86 mg/100 g.The increasing for time drying made the increasing of anthocyanin content.
Figure 5 the influence of drying time on the antocyanin content of roselle extract Figure 6 shows that, although the position of the cabinet drier line above sun drier, the slope or rate of increase of anthocyanin levels in the extract using cabinet drier is 0.46 lower compared with the sun drier 0.48.However, up to 5 hours of drying, anthocyanin levels in the extracts using the cabinet drier higher than the sun drying.This condition caused by the cabinet drying used a temperature of 50°C and is relatively constant.As for sun drying method, the average temperature measured was 40°C and fluctuated depended the climate and season at the time of drying.According to Husni et al. (2014) water content of padina sp which treated by sun light exposure can not evaporate constanly caused by the drying temperature using this method was fluctuated.As a result the water content in the material was decreased faster using the cabinet until the 5th hour of drying time.But finally at 7 hours of drying, the amount of anthocyanin in the extract using cabinet dryer (3.42 mg/100g) was lower than the extract produced from dried roselle using a sun drying method (3.74 mg/100g) According to Santosa and Dewi (2009) drying process could release fenolic acids bonds from cell tissue and then bioactive compounds was extracted out.Sari et al. ( 2012) stated that the optimum dirying temperature to get maximum fenolic substances was 60°C.Drying method with used temperature more than 60°C, after 4 minutes will degrade the amount of fenolic substances.
According to Purbowati et al. (2016), that the stability of anthocyanin is affected by temperature.The higher the temperature the greater the likelihood of anthocyanin degradation.The damage will increase with the heating temperature.
The amount of anthocyanin of roselle extract produced from the cabinet dying method (2.08 mg/100 g) was the highest one compare with sun drying method (1.57mg/100 g) and ERK (0.72 mg/100 g).

Vitamin C
Vitamin C is a type of water soluble vitamin.Reduction in the amount of water in the cells significantly affect the vitamin C content produced.Figure 8 showed Vitamin C content on different drying method.Vitamin C content in dried roselle calyxes, which is drying using the cabinet method (47.721mg/100g) was higher than the drying method using sunlight (39.27 mg/100g) and greenhouse effect (3.061 mg/100g).This is because in the drying using the cabinet, the resulting water content of roselle is lower than the water content of dried roselle water using greenhouse effect and sunlight.So for the same material weight, the water content for the dried roselle with greenhouse and sun effect is more than the drying of the cabinet.
Figure 6 the influence of drying time on the anthocyianin roselle extract Figure 7 The influenced of drying method on the anthocyanin content of roselle extract The drying time has a significant effect on vitamin C (Figure 9).The longer the drying time of the water content in the ingredients is less.So with the same weight of water content in the roselle with a longer drying time will have less water content.This results in more vitamin C content.For drying time 1 and 3 hours of vitamin C content is relatively the same, because it is still estimated adaptation conditions.

Antioxidant activity
Antioxidant activity is influenced by the amount and type of antioxidant compounds.In general, the more amount of antioxidant compounds, the activity of antioxidant compounds in neutralizing free radicals will be higher (Karisma et al., 2015).At each treatment, the higher the concentration of extract used, the radical inhibitory activity is also increasing (Figure 10).The highest inhibition (74.676%) was obtained from cabinet drier for 7 hours of drying.Anova analysis showed that drying method and drying time had significant effect on antioxidant activity.The activity determined largely by its reactivity as a hydrogen donor agent, its reactivity with other antioxidants, the metal bonding transition potential and its ability to stabilize and delocalize unpaired electrons.Roselle extract has anthocyanin content as part of phenolic compound.In addition, the content of vitamin C in the extract was quite high 20.47 mg/100g.According to Tsai et al (2002) antioxidant activity of roselle extract has a strong correlation with anthocyanin content.Falade et al. (2005) states that roselle extract has a high vitamin content.Vitamin C is known as an antioxidant compound.This result is possible because the antioxidant activity is not only caused by phenolic content, but it can be caused by some other phytochemicals such as ascorbic acid, tocopherol and other pigments with synergistic mechanisms which also determine the activity of antioxidants.
A decrease in phenolic content does not lead systematicaly to a decrease of antioxidant activity.Indeed, with lower phenolic content, one also can have a higher antioxidant activity (Buchner et al., 2006).

Figure 1
Figure 1 The influenced of drying time to the lightness (L*) of roselle extract According to Ogrodowska et al.(2017), the longer drying time was caused higher temperature leads to lightening.But, it showed in Figure1, that the increasing of drying time until 7 h, make the extract darker.This occur as a consequence of the presence of phenolic substance esspesialy anthocyanin which responsible for red color in roselle extract.According toKharisma et al (2015), the increasing of anthocyanin content

Figure 2 Figure 3
Figure 2 the influenced of drying time to the a* and b* color