[ ARTICLE ]

Korean Journal of Medicinal Crop Science - Vol. 24, No. 5, pp.351-359

ISSN: 1225-9306 (Print) 2288-0186 (Online)

Print publication date Oct 2016

Received 12 Apr 2016 Revised 16 May 2016 Reviewed 6 Jul 2016 Reviewed 25 Jul 2016 Accepted 2 Aug 2016

DOI: https://doi.org/10.7783/KJMCS.2016.24.5.351

Effects of Roasting and Peeling Process and Extraction Temperature on the Antioxidant Activity of Burdock Tea

Mi Young Park^*^{, †} ; Chengguang Yu^** ; Young Hyun Park^**

*Department of Food and Nutrition Education, Graduate School of Education, Soonchunhyang University, Asan 31538, Korea.
**Department of Food Science and Nutrition, Soonchunhyang University, Asan 31538, Korea.

우엉차의 껍질 제거와 로스팅에 따른 추출온도별 항산화 효과

박미영^*^{, †} ; 우등광^** ; 박영현^**

*순천향대학교 교육대학원 영양교육전공
**순천향대학교 자연과학대학 식품영양학과

†Corresponding author: +82-41-530-1259 phy012@sch.ac.kr

© The Korean Society of Medicinal Crop Science. All rights reserved.
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/3.0 ) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background:

We investigated the optimal aqueous extraction conditions for recovery of high yields of total phenolic compounds from roots of Arctium lappa L. (burdock, Asteraceae), and we compared their antioxidant capacity.

Methods and Results:

The antioxidant activity of the extracts was tested using 2,2-diphenyl-1-picrylhydrazyl, 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)-diammonium salt, and oxygen radical absorbance capacity assays. In addition, the major phenolic compounds present in the extracts were determined by high performance liquid chromatography analysis. Our results suggest that the roasted burdock 100°C, 15 min extract exhibited the strongest radical scavenging activity and possessed the highest concentration of phenolic compounds. The polyphenol content of both dried burdock and roasted burdock significantly increased with increase in the extraction temperature and time.

Conclusions:

These results indicated a relationship between phenolic compound levels in burdock and their free radical scavenging activities. This suggests that phenolic compounds significantly increase the antioxidant potential of burdock extracts.

Keywords:

Arctium lappa L., 2,2’-Azino-Bis (3-Ethylbenzothiazoline-6 Sulfonic Acid)-Diammonium Salt, Antioxidant Activity, 2,2-Diphenyl-1-Picrylhydrazyl, Oxygen Radical Absorbance Capacity

INTRODUCTION

Considerable epidemiological evidence has revealed an association between diets rich in vegetables and fruits and a decreased risk of cardiovascular diseases and certain forms of cancer (Gaté et al., 1999; Yeum et al., 2003). Research has shown that, these diseases occur result from a variety of exogenous and endogenous free radicals in the external environment.

The most widely used natural antioxidants are carotenoids, vitamin C, and vitamin E. These antioxidants play a significant role in reducing excessive oxidative stresses, which leads to loss of cell function and regulation (Nordberg and Arnr, 2001). Excessive levels of reactive oxygen species (ROS) or loss of antioxidant defense systems can injure cells through lipid peroxidation, and cause DNA and protein damage (Halliwell, 1996; Shahidi et al., 1992). Therefore, improving antioxidant potential is an important method to prevent the development of some chronic diseases.

Herb- and plant-based medicines contain phenolic compounds and have played an important role in human health. Accumulating evidence suggests that phenolic compounds function as reducing agents, hydrogen donors, and free- radical eliminators (Lee et al., 2011; Seo et al., 2009; Shahidi et al., 1992). However, a lot of research is still needed to evaluate their properties and mechanisms of action.

Arctium lappa L. commonly known as burdock, is a perennial plant of the Asteraceae (Compositae) family. Burdock root is usually consumed as a tea and a side dish in Korea. It has been reported to have a variety of biological activities including anti-inflammatory, antiproliferative, antiviral, and antioxidant effects (da Silva et al., 2013; dos Santos et al., 2008; Huang et al., 2010; JianFeng et al., 2012; Pereira et al., 2005; Wang et al., 2014). Previous studies have focused on the antioxidant activity of organic solvent extracts of burdock root; however, it is unknown whether water extracts of burdock root possess antioxidant properties (da Silva et al., 2013; dos Santos et al., 2008; Tian et al., 2014). Because burdock tea is prepared from water, it is necessary to assay the antioxidant activity of its water extract. During the preparation of burdock tea, it is dried and roasted without being peeled in the food industry; however, the skin is often peeled following a preliminary wash in the home. Therefore, the antioxidant activity of non-peeled and peeled burdock root should be studied.

The purpose of this study was to optimize water extraction conditions for high yield total phenolic content recovery. The antioxidant capacity of water extracts of burdock root was assessed and compared using three different antioxidant assays. Additionally, the contents of major phenolic compounds in burdock root extracts were determined by high performance liquid chromatography (HPLC) analysis. These findings can be considered for the further isolation and purification of phenolic compounds from A. lappa root for potential use in the nutraceutical or pharmaceutical industries.

MATERIALS AND METHODS

1. Sample preparation

The roots of dried- and roasted-burdock were obtained from Jinjusi, Gyeongsangnam-do, South Korea, and fresh roots were obtained from Asan-si, Chungcheongnam-do, South Korea. The fresh burdock roots were divided into two groups. One group contained peeled burdock roots and the other contained non-peeled burdock roots. The burdock roots were cut into thin slices (0.5㎝), and then dried for 6 h at 60℃ using a drying oven (J-300M, JISICO Co., Ltd., Seoul, Korea). After drying, the two groups of burdock were divided into further two subgroups, one of which was roasted separately in a fryingpan for 3 min at 150℃ (Fig. 1). Water was selected for this study as it is safe, environmentally friendly, accessible, and cheap in comparison to the organic solvents utilized in previous studies (Vuong et al., 2014).

Fig. 1

The method for fresh burdock root preparation.

The roots of dried burdock (DB) and roasted burdock (RB) (1 g) were extracted with water (20, 80, 100℃, 100㎖) for 5, 15, and 30 min. Furthermore, the nonpeeled and peeled burdock roots (1 g) were extracted with 80℃ water (100㎖) for 30 min, next, all the burdock extracts were filtered through 0.45㎛ FP-Vericel™ membrane filter (VWR International, LLC., Radnor, PA, USA). All other chemicals and reagents used were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).

2. Total phenol determination

Total phenol contents were determined using the Folin- Ciocalteu reagent according to the method described by Ainsworth and Gillespie (2007). Briefly, 50㎕ samples of various fractions were assayed with 250㎕ Folin reagent and 500㎕ sodium carbonate (20%, w/v). The mixture was vortexed and diluted with water to a final volume of 5㎖. After incubation for 30 min at room temperature, the absorbance was read at 734㎚ using UV-VIS spectrophotometer (V-530, Jasco Co., Tokyo, Japan). Total polyphenol contents of burdock extracts were reported as ㎎• gallic acid equivalent/g (㎎• GAE/g).

3. Total flavonoid determination

Total flavonoid contents were determined using the method described by Ahmed et al. (2014). Burdock extracts (10㎎/㎖) were added to the test tube, then 2.5㎖ distilled water and 75㎕ 5% NaNO2 solution were added, and the samples were shaken. After 5min, 150㎕ 10% AlCl₃ solution was added, and after 6min, 500㎕ 1N NaOH solution was added and the mixture was shaken. After incubation for 10min at 37℃, the absorbance was determined at 415㎚ using UV-VIS spectrophotometer (V- 530, Jasco Co., Tokyo, Japan). Total flavonoid contents of burdock extracts were expressed as ㎎• quercetin equivalent/g (㎎• QE/g).

4. DPPH radical scavenging activity

The antioxidant activity of the extract and fractions was determined on the basis of their ability to scavenge the stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical, using the method described by Braca et al. (2001). The absorbance at 517㎚ was determined by using UV-VIS spectrophotometer (V-530, Jasco Co., Tokyo, Japan). The DPPH radical scavenging activity was calculated by the following formula:

$DPPH radical scavenging activity (%)= A 0 − A A 0 × 100$

where A₀ is the absorbance of the control and A is the absorbance of the burdock extracts or the standard.

5. ABTS radical scavenging activity

The spectrophotomeric analysis of 2,2'-azino-bis (3- ethylbenzothiazoline-6-sulfonic acid)-diammonium salt (ABTS) radical scavenging activity of burdock extracts was performed according to the method described by Re et al. (1999). The absorbance at 734㎚ was determined by using UV-VIS spectrophotometer (V-530, Jasco Co., Tokyo, Japan). The ABTS^•+ radical scavenging activity was calculated using the following equation:

$ABTS · + scavenging effect (%) = [1 − (A s a m p l e − A c o n t r o l)] × 100$

where A_sample is the absorbance of the burdock extracts or the standard and A_control is the absorbance of the control.

6. ORAC value assay

The oxygen radical absorbance capacity (ORAC) value was determined using the modified method described by Ou et al. (2001). The reaction was carried out in 75mM sodium phosphate buffer (KH₂PO₄-K₂HPO₄ buffer, pH 7.4, 25㎕) and fluorescein (60㎚, 150㎕) with burdock extracts (100㎍/㎖, 25㎕), 2,2’-azobis (2-methylpropionamidine) dihydrochloride (AAPH, 25㎕) and trolox (25㎕), respectively. Samples were mixed was done in black-walled 96-well plates at 37℃, and testing started immediately after mixing. Fluorescence was measured at an excitation wavelength of 485㎚ and emission wavelength 535㎚ every 1min for 120min using a microplate reader Fluostar Omega spectrophotometer (BMG Labtech, Ortenberg, Germany).

The trolox calibration curve was calculated based on the trolox concentration range of 0 - 50㎍/㎖ (1, 5, 10, 30, and 50㎍/㎖ final concentrations). The area under the curve (AUC) was calculated for each sample by integrating the relative fluorescence curve. The net AUC of the sample was calculated by subtracting the AUC of the blank. The regression equation between the AUC and the trolox concentration was determined, and ORAC values are expressed as ㎎• trolox equivalent/100 g (㎎• TE/100 g) of samples.

7. Quantification of phenolic compounds by HPLC analysis

Phenolic compounds were identified and quantified by using a HPLC Spectra System SCM 1000 (Thermo Fisher Scientific Inc., Waltham, MA, USA) equipped with UVvisible detector (Shimadzu, Kyoto, Japan). Reverse phase chromatographic analyses were carried out under gradient conditions using a packed C₁₈ column (4.6㎜ × 120㎜); the mobile phase was water containing 0.5% phosphoric acid and 20% ACN. The burdock extracts as well as the mobile phase were filtered through a 0.45㎛ membrane filter (Merck Millipore, Billerica, MA, USA) and then degassed in an ultrasonic bath prior to use. The extracts were analyzed for the presence or absence of gallic acid (GA), chlorogenic acid (CGA), epigallocatechin gallate (EGCG), and caffeic acid (CA). Those compounds were identified by comparing their retention time and UV absorption spectra with those of the commercial standards. The flow rate was 0.5㎖/min, injection volume was 10㎕ and the wavelength was 280㎚.

8. Statistical analysis

All tests were carried out in triplicate and the data are presented as means ± standard deviation (SD). Data were analyzed by One-way analysis of variance (ANOVA) followed by Duncan’s Multiple Range Tests; p < 0.05 was considered as statistically significant.

RESULTS

1. Total phenolic and flavonoid contents of DB and RB extracts

Plant phenolics and flovonoids are generally highly effective free radical scavengers and antioxidants (Mustafa et al., 2010). The content of total phenolic compounds in the extracts from DB and RB are shown in Table 1. The total polyphenol contents of DB and RB ranged from 10.86 ± 0.33 to 38.75 ± 0.48, and 27.20 ± 0.42 to 83.97 ± 2.02㎎• GAE/g, respectively. The total flavonoid contents of DB and RB ranged from 2.17 ± 0.09 to 25.33 ± 0.41 and from 4.68 ± 0.41 to 33.32 ± 0.25, ㎎• QE/g, respectively (Table 1). Longer extraction time and higher extract temperature resulted in the extraction of higher phenolic and flavonoid compounds. In the comparison between the DB and RB extracts, using the same extraction method, the RB extracts showed higher total polyphenol contents and total flavonoid contents than the DB extracts.

Table 1

Total polyphenol and total flavonoid contents of DB and RB extracts by different extraction method.

In Table 2, the total phenolic and flavonoid contents of the non-peeled RB extracts were higher than those of the other extracts (p < 0.05).

Table 2

Total polyphenol and total flavonoid contents of nonpeeled and peeled burdock extracts.

2. DPPH radical scavenging activity of burdock extracts

The free-radical scavenging activity of burdock extracts was assessed by DPPH assay. As shown in Fig. 2A, the RB 100℃, 30 min extract showed the highest scavenging activity. The RB extracts showed higher DPPH radical scavenging activity than DB extracts with the same extraction method.

Fig. 2

DPPH radical scavenging activity of DB, RB, NP-B and P-B extract.A; black or white bar represents roasted-burdock (RB) or dried-burdock (DB), respectively. B; non peeled-dried burdock (NP-DB), non peeled-roasted burdock (NP-RB), peeled-dried burdock (P-DB), peeled-roasted burdock (P-RB). Each bar represents the mean ± SD. The bars with different letters are significantly different (p < 0.05) from each other.

The DPPH radical scavenging activity of non-peeled and peeled burdock extracts decreased in the order non-peeled RB (88.98 ± 0.65%) > peeled RB (57.53 ± 2.74%) > non-peeled DB (47.82 ± 1.50%) > peeled DB (22.89 ± 0.81%). The RB extracts showed higher scavenging activity than DB extracts, and non-peeled burdock extracts showed higher scavenging activity than peeled burdock extracts (Fig. 2B).

3. ABTS radical scavenging activity of burdock extracts

The ABTS assay is based on the reaction between ABTS and potassium persulfate, which generates a blue/ green ABTS radical (ABTS^•+). The ABTS radical scavenging activities of various extracts increased with extract time and temperature (Fig. 3A). The ABTS radical scavenging activity of non-peeled and peeled burdock extracts decreased in the order non-peeled RB (30.43 ± 0.48%) > peeled RB (17.45 ± 0.41%) > non-peeled DB (12.99 ± 0.34%) > peeled DB (10.27 ± 0.44%). Similar to the results obtained by the DPPH radical scavenging assay, the RB extracts showed higher scavenging activity than DB extracts, and nonpeeled burdock extracts showed higher ABTS radical scavenging activity than peeled burdock extracts (Fig. 3B).

Fig. 3

ABTS radical scavenging activity of DB, RB, NP-B and P-B extract.A; black or white bar represents roasted-burdock (RB) or dried-burdock (DB), respectively. B; non peeled-dried burdock (NP-DB), non peeled-roasted burdock (NP-RB), peeled-dried burdock (P-DB), peeled-roasted burdock (P-RB). Each bar represents the mean ± SD. The bars with different letters are significantly different (p < 0.05) from each other.

4. ORAC value of burdock extracts

The ORAC assay is based on radical scavenging by the AAPH radical, and the ORAC has been widely employed to evaluate the antioxidant capacity of beverages, food, and plant extracts (Prior et al., 2005). Similar to the DPPH and ABTS scavenging activity, obtained in the same extract time, the ORAC value increased as the temperature increased, and the RB extracts showed higher values than the DB extracts (Fig. 4A). Non-peeled burdock extracts showed higher ORAC values than peeled burdock extracts (Fig. 4B). The ORAC values of nonpeeled and peeled burdock extracts decreased in the order non-peeled RB (7993.29 ± 74.90㎎• TE/100 g) > non-peeled DB (3306.03 ± 41.72㎎• TE/100 g) > peeled RB (2322.49 ± 76.94㎎• TE/100 g) > peeled DB (1,081.85 ± 40.90㎎• TE/ 100 g). These data indicate that the presence of phenolic and flavonoid compounds in extracts contributes significantly to their antioxidative potential.

Fig. 4

ORAC value of DB, RB, NP-B and P-B extract.A; black or white bar represents roastedburdock (RB) or dried-burdock (DB), respectively. B; non peeled-dried burdock (NP-DB), non peeled-roasted burdock (NP-RB), peeled-dried burdock (P-DB), peeled-roasted burdock (P-RB). Each bar represents the mean ± SD. The bars with different letters are significantly different (p < 0.05) from each other.

5. Quantification of phenolic compounds by HPLC analysis

Previous studies have identified GA, CGA, and CA in burdock roots (Tezuka et al., 2013; Saleem et al., 2009). As the extract temperature and time were increased, the polyphenol contents of both DB and RB significantly increased (Table 3).

Table 3

Phenolic compounds contents of DB and RB extracts by different extraction method.

DISCUSSION

Burdock (Arctium lappa L.) root has been widely used as a folk medicine and vegetable in East Asia. In the present study, we investigated the optimal water extraction conditions for high-yield total phenolic content recovery, and the antioxidant capacity of the water extracts of A. lappa root.

In the DPPH radical scavenging assay, ABTS radical scavenging assay, and ORAC assay, the RB 100℃, 30 min extract showed the highest antioxidant activity, and non-peeled burdock extracts showed higher scavenging activity than peeled burdock extracts (Fig. 2, 3, 4). The ABTS radical scavenging activity of RB (80 - 100℃) is almost two-fold higher than that of DB (Fig. 3). As expected, the total phenolic acid and flavonoid contents of the RB 100℃, 30 min sample were higher than those of the other extracts (Table 1). These results indicate that there is a relationship between the phenolic compound concentrations in burdock and their free radical scavenging activities. Therefore, the presence of phenolic compounds significantly contributes to their antioxidant potential. These data are consistent with those from previous studies showing that a highly positive relationship exists between total phenolic contents and antioxidant activity in many plants (Chukwumah et al., 2009; Gursoy et al., 2009).

The phenolic and flavonoids compound contents of nonpeeled burdock extracts are greater than those of peeled burdock extracts (Table 2). These results support the conclusions of others studies showing that fruits and vegetable peels possess a higher content of these compounds than pulps (Asikin et al., 2012; Gorinstein et al., 2001).

Although caffeoylquinic acids, dicaffeoylquinic acids, CA derivatives, hydroxycinnamoylquinic acids, and CGA have been identified in burdock roots (Jaiswal and Kuhnert, 2011; Lin and Harnly, 2008; Liu et al., 2012; Saleem et al., 2009), most studies analyzed the organic solvent extract of burdock roots. Since burdock root has been consumed as a tea, we analyzed phenolic compounds in the water extract of burdock roots. As the extract temperature and time increased, the contents of phenolic compounds also increased (Table 3).

The CGA content was highest among the three phenolic compounds in DB (Table 3). The GA content increased as much as the CGA content in RB. The RB 100℃, 30 min extract contained 10-fold more CGA than the DB 100℃, 30 min extract (Table 3). Roasting treatment led to a significant increase in the level of GA, CGA, and CA, which correlates well with the observed antioxidant activity of burdock root extract. GA is a strong antioxidant compound and is used as a reference in the assessment of antioxidant activity. Its oxidation involves 4.6 electrons, whereas ascorbic acid involves two electrons (Hotta et al., 2002). CGA is the ester of CA and quinic acid and also possesses antioxidant properties (Sato et al., 2011; Xu et al., 2012). During the roasting process, GA may be released from tannin and CA may be generated from CGA degradation.

Thus, the antioxidant activity of the RB 100℃, 30 min extract is partly due to the presence of those phenolic compounds. The results indicate that the roasting process and conditions play an important role in the phenolic compound content of burdock tea. In addition to burdock root phenolic compounds, water-soluble polysaccharide, fructo-oligosaccharide, arctigenin and inulin are also known to have antioxidant activity (Li et al., 2008; Liu et al., 2014; Zhao et al., 2009). The antioxidant activity of burdock root may involve a synergistic effect among phytochemicals.

In this study, an assay of antioxidant activity was tested for the first time and the total phenolic and flavonoid contents of water extracts from burdock root were determined. Antioxidant and radical scavenging ability of the extracts were found to be extract temperature and time dependent.

A key finding of this study was that the RB 100℃, 30 min extract exhibited the strongest radical scavenging activity and possessed the highest concentration of phenolic compounds. Unfortunately, we did not analyze the contents of various phenolic compounds or those in non-peeled and peeled burdock extracts. However, the non-peeled RB extract was found to have a higher ORAC value than peeled RB extract, suggesting that non-peeled burdock extract has more antioxidant benefits than peeled burdock extract. Roasting treatment generated a high amount of phenolic compounds, in particular, GA.

Thus, non-peeled RB can be very beneficial for enhanced phenolic compound extraction with desirable benefits. Future studies should focus on the roasting condition, the identification of these antioxidants, and the purification of this plant ingredient to obtain agents with high efficacy and activity.

ACKNOWLEDGEMENT

This research was supported by the Soonchunhyang University Research Fund.

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Sample		Contents

		Total polyphenol (mg • GAE1)/g)	Total flavonoid (mg • QE2)/g)

Means values± SD from triplicate separated experiments are shown. Means within a column followed by the same letter are not significant based on the DMRT (* p < 0.05). GAE; Gallic acid equivalents. QE; Quercetin equivalents.
DB 20°C	5 min	10.86 ± 0.33k	2.17 ± 0.09j
	15 min	11.28 ± 0.40k	2.60 ± 0.07j
	30 min	12.13 ± 0.45k	3.81 ± 0.09i

DB 80°C	5 min	20.18 ± 0.48j	7.67 ± 0.19g
	15 min	25.67 ± 0.36i	9.71 ± 0.41ef
	30 min	26.55 ± 0.44i	10.83 ± 0.38e

DB 100°C	5 min	25.46 ± 0.83i	9.28 ± 0.40ef
	15 min	32.06 ± 0.58h	17.12 ± 0.44c
	30 min	38.75 ± 0.48e	25.33 ± 0.41b

RB 20°C	5 min	27.20 ± 0.42hi	4.68 ± 0.41h
	15 min	34.73 ± 0.44g	7.60 ± 0.15g
	30 min	36.96 ± 0.41f	8.73 ± 0.05f

RB 80°C	5 min	39.49 ± 0.08e	14.31 ± 0.59d
	15 min	45.35 ± 0.28d	17.97 ± 0.21c
	30 min	47.51 ± 1.16d	19.41 ± 0.40bc

RB 100°C	5 min	51.42 ± 1.23c	18.67 ± 0.11bc
	15 min	77.31 ± 0.98b	24.41 ± 0.05b
	30 min	83.97 ± 2.02a	33.32 ± 0.25a

Sample	Contents

	Total polyphenol (mg • GAE1)/g)	Total flavonoid (mg • QE2)/g)

Means values± SD from triplicate separated experiments are shown. Means within a column followed by the same letter are not significant based on the DMRT (* p < 0.05). GAE; Gallic acid equivalents. QE; Quercetin equivalents.
Non-peeled DB	14.33 ± 0.45c	11.33 ± 0.17c
Non-peeled RB	53.65 ± 1.36a	20.09 ± 0.49a
Peeled DB	9.47 ± 0.39d	5.68 ± 0.19d
Peeled RB	22.15 ± 0.66b	13.63 ± 0.48b

Sample		Contents (mg/100 g)

		GA1)	CGA2)	CFA3)

Means values± SD form triplicate separated experiments are shown. Means within a column followed by the same letter are not significant based on the DMRT (* p < 0.05). GA; Gallic acid, CGA; Chlorogenic acid, CFA; Caffeic acid, –; no detectable.
DB 20°C	5 min	21.67 ± 0.09jk	7.83 ± 0.35n	–4)
	15 min	25.98 ± 0.32j	11.52 ± 0.27n	–
	30 min	31.40 ± 0.43i	16.44 ± 0.17m	0.32 ± 0.02m

DB 80°C	5 min	13.24 ± 0.44l	107.71 ± 0.22j	1.10 ± 0.04l
	15 min	16.70 ± 0.17k	129.87 ± 4.62h	1.55 ± 0.01l
	30 min	17.94 ± 0.50k	136.62 ± 1.68g	3.86 ± 0.05k

DB 100°C	5 min	13.63 ± 0.37l	133.98 ± 4.52gh	2.62 ± 0.03kl
	15 min	21.77 ± 0.41jk	160.78 ± 3.42e	8.43 ± 0.29j
	30 min	23.83 ± 0.39j	179.81 ± 4.42c	12.32 ± 0.32i

RB 20°C	5 min	74.82 ± 0.39h	87.15 ± 3.88l	17.35 ± 0.45h
	15 min	96.03 ± 3.11g	102.09 ± 2.72k	20.57 ± 0.28g
	30 min	116.51 ± 4.49f	111.42 ± 2.06i	25.04 ± 0.36f

RB 80°C	5 min	124.95 ± 4.76e	127.79 ± 2.92h	30.01 ± 0.32e
	15 min	146.33 ± 4.29d	145.87 ± 2.27f	32.38 ± 0.21d
	30 min	152.85 ± 3.28d	174.43 ± 3.14d	36.40 ± 0.39c

RB 100°C	5 min	170.82 ± 3.71c	158.56 ± 3.16e	33.14 ± 0.69d
	15 min	218.73 ± 3.66b	222.25 ± 3.86b	38.46 ± 1.01b
	30 min	257.13 ± 4.25a	242.16 ± 4.29a	45.79 ± 2.03a