1 Optimization of Bioactive Compound Extraction from Rose Myrtle Fruit (Rhodomyrtus tomentosa,

2 (w.ait), Myrtaceae) from North Kalimantan, Indonesia as Antioxidant Source

3 Ismandari,T1*., Kumalaningsih, S2.,Wijana, S2., Mustaniroh, S.A2.

4 1
Agrotechnology Program Study, Faculty of Agriculture, Borneo Tarakan University, Amal Lama

5 Street No 1 Tarakan, North Kalimantan 77115, Indonesia

2
6 Department of Agro-industrial Technology, Brawijaya University, Veteran Street, Ketawanggede

7 Lowokwaru, Malang 65141, Indonesia

8 * Corresponding author : ismandarititik@yahoo.co.id

9 Author No.1: ORCID = https://orcid.org/0000-0001-9841-0136 (Ismandari, T)

10 Author No.2: ORCID = http://orcid.org/0000-0003-3891-2390 (Kumalaningsih, S)

11 Author No.3: ORCID = https://orcid.org/0000-0001-5898-9171 (Wijana, S)

12 Author No.4: ORCID = https://orcid.org/0000-0002-5840-1208 (Mustaniroh)

13

14 Abstract

15 Rose myrtle fruit (Rhodomyrtus tomentosa,(W.Ait),Myrtaceae) is one of fruits widely found

16 in North Kalimantan. This fruit contains bioactive compound that has a potential to be used as

17 antioxidant. The aim of this study was to obtain optimal temperature and time of extraction in

18 maintaining and protecting bioactive compound in rose myrtle fruit extract by using water as

19 solvent.

20 This research applied response surface method with central composite design for two

21 factors, namely X1 (temperature/°C) consisted of three levels: 70, 80, 90 °C and X 2 (time/minute)

22 which consisted of three levels of 60, 90, 120 minutes. Research parameters included total phenol

23 and antioxidant activity. Moreover, GC-MS was used for the characterization of chemical compound

24 component contained in rose myrtle fruit extract. Optimization of extraction condition resulted in

25 optimum temperature for extraction of 80.43 ºC and optimum time for extraction of 85 minutes
26 with optimum yield of total phenol of 73.77 mg/100 g fresh fruit and antioxidant activity of 1.0385

27 µg/ml with desirability of 0.892 or 89.2%, with a total of 33 bioactive compounds.

28

29 Keywords : antioxidant, extraction optimization, rose myrtle fruit, total phenol

30

31 1. Introduction

32 Rose myrtle fruit (Rhodomyrtus tomentosa,(W.Ait),Myrtaceae) is one of potential fruits in

33 North Kalimantan, particularly in Tarakan City. The production of rose myrtle fruit ranged of 1-1.3

34 ton/ha (Amarullah. 2011). Rose myrtle fruit contains bioactive compound which provides benefit for

35 health (Savithramma et al. 2011). Moreover, there are also 19 phenolic compounds and Gallic acid

36 which has a potential to be antioxidant source (Kwon et al. 2012). As mentioned by Lai et al. (2014),

37 150 gram of dry rose myrtle fruit contains food fiber (69.94-87.43%), α-tocopherol (38.90-51.87%

38 RDI), and linoleic acid (75.36% of total fatty acid).

39 Antioxidant is bioactive compound with ability to inhibit free radical and substrate oxidation

40 (Array et al, 2019). Free radical leads to chain reaction, resulting in cell damage, cellular aging,

41 chronic degenerative diseases such as cancer, diabetes, cardiovascular and neurovascular disease

42 (Masisi et al. 2016).

43 In general, fulfilment of antioxidant need for the body is done using synthetic antioxidant

44 like 4-Hexylresorcinol (Chen and Chen 2011), yet several researchers revealed that the use of

45 synthetic antioxidant succeeded in causing negative effect on health in addition to its toxicity

46 (Robledo et al. 2011). Therefore, natural antioxidant becomes one of alternatives urgently required.

47 The component of bioactive compound in plants has an ability to reduce free radical (Saeed et al.

48 2012). Furthermore, as mentioned by Maskam et al. (2014), rose myrtle fruit was able to inhibit

49 DPPH of free radical by 62.13%, this value was higher compared with antioxidant activity of

50 mangosteen peel of 2.496 μg/ml (Dugir, Dewa, Vanda 2014) and noni leaf (123.72 µg/m) (Rohman

51 and Sugeng 2017). However, the use of rose myrtle plant until now, particularly its fruit in North
52 Kalimantan is very limited. This condition is due to the limitation of information on the use technic

53 and benefit of the plant.

54 Until today, rose myrtle plant has not been maximally used and only considered as weed

55 with quite high growth rate, thus it exists as pest plant that is difficult to control. The benefit of rose

56 myrtle plant, especially its fruit, is not widely exposed, and this plant is commonly considered as

57 weed in order not to harm cultivated plants.

58 Concerning the chemical composition, bioactive compound content, and antioxidant

59 compound in rose myrtle fruit, it is necessary to conduct study on the use of rose myrtle fruit as

60 antioxidant source by not causing damage of bioactive compound through simple method. The

61 method applied was extraction using water as solvent. Water is an organic solvent that is safe,

62 inexpensive, easy to obtain, and never been used in the extraction of rose myrtle fruit. The aims of

63 this study were to obtain optimal condition in extraction process as well as to obtain antioxidant

64 compound without harming bioactive compound in rose myrtle fruits.

65

66 2. Materials and Methods

67 2.1. Materials

68 Materials used in this study were rose myrtle fruits (Rhodomyrtus tomentosa,

69 (W.Ait),Myrtaceae.) obtained from Tarakan City, North Kalimantan with criteria of physiologically

70 ripe fruit and water as solvent. Tools used included: water bath, thermocouple, digital scale, and

71 rotary evaporator.

72 2.2. Methods

73 The stage of this study was as follows: 1). Sorting and washing, 2). Size reducing, 3).

74 Extraction according to treatments, (4) Analysis of total phenolic content (GAE) and antioxidant

75 activity of rose myrtle fruit extract, and (5) analysis of biocative compounds using GC-MS. Sortation

76 of rose myrtle fruit was done by separating or sorting good fruits out of the damaged or defective
 77 fruit and other foreign object. Later, fruits were cleaned in running water, blended until smooth, and

78 prepared to be extracted.

79 2.3. Extraction of Bioactive Compound from Rise Myrtle Fruit.

80 Extraction of rose myrtle fruit bioactive compound was done using dekok method with

81 water as solvent. This process was started with sortation activity, followed by rose myrtle fruit

82 washing. Furthermore, clean fruits were weighed of 100 gram, thinly sliced at a size of ± 1 mm, and

83 blended. After that, extraction in water bath was carried out using water as solvent at ratio of 5:1

84 (solvent: sample) at temperature and time according to treatments. The product of rose myrtle fruit

85 extraction was filtered using vacuum filter to obtain filtrate and dregs. Removal of solvent in filtrate

86 was done using vacum rotary evaporator at temperature of 40 ºC for 1 hour in order to obtain

87 filtrate. The filtrate collected was further centrifuged at the speed of 5000 rpm for 10 minutes to

88 precipitate dirt, resulted in supernatant and pellet. The supernatant obtained was stored in

89 refrigerator to be analyzed further.

90 2.4. Experimental Design for Optimization of Bioactive Compound Extraction through Response

91 Surface Method (RSM).

92 Response Surface Method was used to assess total yield and total phenol produced from

93 extraction on two independent variables of extraction condition, namely temperature and time of

94 extraction. The composition of the two independent variables was designed using central composite

95 design. The point of temperature variable was 80 ºC, while the point obtained for variable of time

96 was 90 minutes. The model of mathematical equation of the central composite model with 2 factors

97 is shown below:

2 2
98 Y = β0 + ∑ β i X + ∑
2
i ∑ βu Xi X j (1)
i =1 i< j=1

99 In this term, Y is the response (yield), β0 is constant, βi, βii, βij are coefficients of

100 independent variable (X), X is independent variable without code (for variable of time of extraction:

101 temperature of extraction (X1) at level of 70, 80 and 90 ºC; time of extraction (X2) at level of 60, 90
102 and 120 minutes and ε is random error. Level of independent variable (temperature and time) in this

103 study is presented in Table 1 (Bharathi, J. Patterson, R. Rajendiran 2011).

104 2.5. Analysis of Total Phenolic Content

105 Analysis of total phenol was measured through spectrophotometry method by using Folin-

106 Ciocalteau reagent (Mongkolsilp et al. 2004). Total phenolic compound in rose myrtle fruit is

107 expressed as Gallic Acid Equivalent (GAE). Gallic acid GAE is a common reference to measure the

108 amount of phenolic compound in a material. Gallic acid is used as a standard. Calculation of this total

109 phenol used the standard of concentration of 0; 1.5; 3; 6; and 8 ppm, with equation of standard

110 curve y = 0.0956x + 0.0029 with R2= 0.9989. Treatment of sample was similarly made using the

111 method of standard curve. Total phenol was determined based on the value of Gallic acid

112 equivalent.

113 2.6. Determination of Antioxidant Activity

114 Antioxidant activity of rose myrtle fruit extract was done in accordance with the method of

115 DPPH which is based on the ability of sample in reducing DPPH stable-free radical. 1. 1, 1-diphenyl-2-

116 picrylhydrazyl (Zou et al. 2004). One ml of 0.5 mM DPPH was put into test tube, added with 50 μl of

117 rose myrtle fruit extract at various concentrations. Further, 3.95 ml of ethanol was added. The

118 sample was homogenized using vortex and left for 30 minutes. The concentration of rose myrtle fruit

119 powder obtained was made until it reached the value of IC 50, that was the concentration that

120 produced radical capture concentration of 50% compared to control through a linear regression line

121 equation. Later, absorbance of the solution was read at wavelength of 517 nm. Read on the

122 absorbance of control solution, that was without the addition of vitamin E, was also done.

123 2.7. Analysis of Bioactive Compounds Using GC-MS.

124 Bioactive compounds of rose myrtle fruit extracts were analyzed using Gas Chromatograpy-

125 Mass Spectroscopy (GC-MS) Shimadzu QP 5000. A sample of 1 μL was injected into GC-MS which

126 was operated using a 25 m long glass column, 0.25 mm diameter and 0.25 μm thickness with a

127 stationary phase CP-Sil 5CB with oven temperature programmed between 70-270 ° C with a rate of
128 increase in temperature of 10 ° C / min, Helium carrier gas pressure of 12 kPa, a total rate of 30 mL /

129 min and a split ratio of 1:50

130 Statistical Analysis

131 Statistical analysis was performed using the software of MINITAB Release 14. This analysis

132 resulted in coefficient influenced the model and graph from the observed response in the form of

133 coefficient of regression, 3D response surface plot, and contour plot (using Design expert 7), to

134 examine the model concerning the optimum extraction process (Bezerra et al. 2008).

135

136 3. Results and Discussion

137 Observation result of the value of total phenol and antioxidant activity (IC 50) is presented in

138 Table 2.

139 Table 2. above presents the actual value and variable response of total phenol and

140 antioxidant. Total phenol of observation result ranged from 36.629 mg/g to 81.158 mg/g, while

141 antioxidant activity (IC50) ranged between 1.02 µg/ml and 2.0612 µg/ml. Response of each variable is

142 explained as follows.

143 3.1. Total phenolic content

144 Determination of total antioxidant of foods from plants can be done by measuring the

145 concentration of total phenolic content using Folin-ciocalteau reagent. Phenolic compound has an

146 important role in preventing oxidation (Kaur and Poonam 2014). Moreover, John, Sulaiman,

147 Satheesh, and Reddy (2014) said that test of total phenol was aimed to determine total phenolic

148 compound contained in sample, thus it was expected that high concentration of phenolic compound

149 content in sample resulted in high antioxidant activity. According to the analysis result of model

150 selection based on “Sequential Model Sum of Squares”, the selected model was quadratic for its

151 value of 0.0031 (<5%) which showed that the chance error of model was less than 5%, thus it

152 significantly affected the response of total phenol. Furthermore, based on the measurement of Lack

153 of Fit, the quadratic model was defined as “suggested”, which meant that the selected model
154 obtained a value of 0.2981 or it was not significantly different since the p value >5%, hence it was

155 concluded that the models were the appropriate models for the response of total phenol. The

156 equation of RSM from extraction process is as follows:

157 Y = - 10200.172 + 244.7916 X1 + 23.3496 X2 - 0.1153583 X1X2 - 1.448045 X12 -0.0801856 X22

158 .......................................................(2)

159 Y is total phenol, X1 is temperature of extraction, and X2 is time of extraction. The equation shows

160 that response of total phenol will increase, directly proportional, to the increasing temperature and

161 time of extraction, reflected by the positive constant value. The value of R-square (R2) = 0.81752

162 indicated that the data were able to support the model for about 81.75%, which included the factor

163 of temperature and time of extraction. The rest of 18.25% was affected by other factor excluded in

164 the model. Other factors that influenced total phenol were extraction method, solvent type, solvent

165 composition, and plant species. Contour plot and visualization of response surface produced from

166 the data of total phenol in extraction process that used response surface test are presented in Figure

167 1a. and 1b.

168 As shown in Figure 1a, x axis reflects the factor of temperature and y axis is for factor of

169 time. The line within the contour plot is the value of total phenol response. Concerning the contour

170 color, the red dot shows the highest value of total phenol response of 81.158 mg GAE/g, while the

171 lowest value is indicated by red color in contour plot of 36.629 mg GAE/g. The shape of response

172 surface produced from the interaction between these components is seen more clearly in the 3D

173 graph as shown in Figure 1b. In response surface figure (1b), it is seen in the treatment value of

174 extraction temperature and time that the total phenolic concentration of rose myrtle fruit extract

175 increased along with the increasing temperature and time of extraction, later it was stable and

176 tended to decrease afterward. According to Wazir et al. (2011), the use of high temperature in

177 extraction process will increase solubility of cell wall or the bound phenolic compound due to cell

178 element damage. Therefore, more phenolic compound is extracted. In addition, increase in

179 temperature causes the pores of solids to expand, thus water as solvent will easily diffuse into the
180 pores of solids of rose myrtle fruit extract and dissolve phenol. Hence, more phenol to interact and

181 leads to higher mass transfer of solute, namely from solid material to solvent.

182 Based on the analysis, it is known that the critical values for temperature and time were 80

183 °C and 90 minutes, respectively. In that point, total phenol was predicted to reach 81.158 µg/g.

184 However, at temperature above 80 °C, phenolic concentration decreased due to the high

185 temperature during the extraction process. Along with the increasing temperature, it is easier for

186 phenol to exit the cell of rose myrtle fruit. Heating during extraction process also has a function to

187 inactivate the enzyme of polyphenol oxidase (Tuminah 2004). The result above was in accordance

188 with the study conducted by Tan, Tan, and Ho (2013), that higher temperature applied in extraction

189 process will lead to higher inactivation of polyphenol oxidase enzyme, thus enzyme activity will be

190 lower, and phenol damage will be smaller. However, phenolic content will also be hampered due to

191 the increasing temperature of extraction, thus the amount of total phenol detected will reach the

192 maximum peak, and further will be constant and tended to decline. Moreover, as mentioned by

193 Sulaiman et al. (2017), phenolic compound will increase along with the increasing temperature and

194 time of extraction, yet total phenol will decrease at high temperature since several phenolic

195 compounds are sensitive to heat, hence increasing temperature will decrease phenol. Furthermore,

196 long extraction time may result in decomposition and phenolic oxidation (Naczk and Shahidi 2004 in

197 Tan, Tan, and Ho 2013).

198 In addition to the effect of temperature and time of extraction, the use of solvent also

199 determined the total phenol produced. In this study, extraction of rose myrtle fruits used water as

200 solvent. Water is one of solvents that is safe and has quite high solubility on the extracted substrate.

201 Water is a polar molecule and the extract of rose myrtle fruit is also polar, thus extraction process

202 produced phenol in high amount (Wijngaard et al., 2009 in Dimcheva and Maria 2019). Total phenol in this study

203 was found to be higher than the result of previous study. In extraction of fresh rose myrtle fruit

204 conducted by Lai et al. (2015), solvent of acetone:water:acetic acid (50:49:1) resulted in total phenol

205 of 11.0 mg/100 g fresh fruit. Moreover, Zhao et al. (2017) mentioned that total phenol in rose myrtle
206 fruit dried using various drying methods and extracted using 80% acetone as solvent was 15.57

207 mg/100 g DW.

208 3.2. Antioxidant activity content

209 Antioxidant is a bioactive compound that can inhibit free radical and substrate oxidation in

210 which free radical may cause chain reaction that may result in cell damage, cellular aging, chronic

211 degenerative diseases, such as cancer, diabetes, cardiovascular and neurovascular disease (Masisi,

212 Beta, & Moghadasian 2016).

213 Result of Analysis of Variance of treatment on the antioxidant activity showed that the

214 quadratic model selected had the value of F compute of 43.962 and ρ value = 0.0001, temperature

215 obtained the value of F compute = 2.803 and ρ = 0.138, and time had the value of F compute = 0.502

216 and ρ = 0.501. The value of ρ < 0.05 indicated significant effect on response. Single factor of

217 temperature and time of extraction resulted in the value of ρ of temperature = 0.0001 and the value

218 of ρ of time = 0.0001 which meant that the value < 0.05, thus both treatments significantly affected

219 the response of antioxidant activity of rose myrtle fruit extract. The value of model deviation (lack of

220 fit) obtained was 0.0001.

221 Standard deviation obtained was 0.106 with R 2 = 0.969. The value of R-square (coefficient of

222 determination) in ANOVA of 0.969 reflected that the data were able to support the model for

223 96.90% which included the factor of temperature and time of extraction. The rest of 3.1% was

224 influenced by other factors excluded in the model. Other factors affected antioxidant activity were

225 extraction method, solvent type, solvent composition, and plant species. The value of R (Adj R-

226 Squared) in ANOVA of 0.947 reflected that coefficient of correlation of 94.7% was obtained. Equation

227 of RSM from extraction process is as follows:

228 Y = 36.52497 - 0.75309 X1 - 0.125881X2 + 0.000509 X1X2 + 0.004459 X12

229 + 0.0004779 X22 .........(3)

230 Y is antioxidant activity, X 1 is temperature of extraction, and X 2 is time of extraction. The equation

231 showed that antioxidant activity will increase, directly proportional to the increasing temperature
232 and time of extraction as shown by the positive constant value. The value of R-square (R2) obtained =

233 R2 = 0.969. The value of R-square (coefficient of determination) in ANOVA of 0.969 indicated that the

234 data were able to support the model for 96.90% which included the factor of temperature and time

235 of extraction. The rest of 3.1% was affected by other factors excluded in the model. Other factors

236 affected antioxidant activity were extraction method, extraction type, solvent composition, and

237 plant species. Contour plot and visualization of response surface from the data of antioxidant activity

238 in extraction process that applied response surface method can be seen in Figure 2.

239 In Figure 2a, red color of contour showed the highest value of antioxidant response of

240 2.0612. µg/ml. The blue color indicated the lowest response value of 1.02 µg/ml. The line consisted

241 of dots in the graph of contour plot showed a combination of both components at different amounts

242 which resulted in the same value of antioxidant response. The shape of response surface of the

243 interaction between components can be seen more clearly in a three-dimension graph as shown in

244 Figure 2b. In term of curve trend, higher addition of temperature and time of extraction increased

245 antioxidant value and further the value decreased after it passed the optimum treatment. This

246 finding was in line with the study carried out by Zhao et al. (2017) that increase in temperature and

247 time of extraction will both increase and decrease antioxidant activity. It was due to the reason that

248 heat will damage the cell tissue of plants extracted, thus increase the amount of active component

249 freed, yet the component will change and the amount will decrease along with the increasing

250 temperature and time.

251 In the 3D graph, it is seed that antioxidant activity continued to increase until the

252 temperature reached 80 °C with extraction time of 90 minutes. This increase was caused by the

253 solubility of active compound on the material which was possible due to cell wall damage resulted

254 from heating during extraction process (Khatun et al. 2006). Moreover, according to Wang et al.

255 (2008) in addition to heat, high antioxidant activity was caused by water as solvent in the extraction

256 process, in which antioxidant property in rose myrtle fruit is hydrophilic, thus the extracted bioactive
257 of antioxidant was quite high. Similar result was found by Tan, Tan, Ho (2011) that the use of polar

258 solvent would generate polar extract which reflected active antioxidant activity.

259 Treatment of high temperature may result in the damage of cell wall and sub-cellular of

260 plant cell to release large amount of active compound and produce strong free radical capturing

261 component compared to fresh material. Hence, extraction at high temperature will cause tissue

262 softening and the release of bound antioxidant component (Spigno et al. 2007).

263 Based on the analysis result using DPPH reagent, value of antioxidant activity of IC 50 had a

264 range between 1.02 µg/ml and 2.0612 µg/ml. This outcome showed that antioxidant value (IC 50) of

265 rose myrtle fruit extract for all treatment variations was included in the group of highly strong

266 antioxidant of < 50 Dg/ml. Compared to the result of Cui et al. (2013) who performed extraction

267 using TFA and methanol as solvent and produced IC 50 of 6.27 µg/m, Maskam et al., (2014) with

268 extraction using solvents of water:methanol:chloroform:petrolium ether and produced IC 50 of 107 -

269 250 µg/ml, as well as Pingping et al. (2015) with 95% ethanol as solvent that resulted in IC 50 value of

270 10.97 µg/ml. Therefore, the result of this study obtained better IC50.

271 3.3. Optimization of total phenolic response and antioxidant activity

272 Optimization result using Design Expert Ver. 9 Trial indicated that optimal condition

273 recommended in extraction process of rose myrtle fruit included extraction temperature of 80.43 ºC

274 and time of 85.00 minutes. This treatment produced a response prediction in the form of total

275 phenol of 73.77 mg/100 g fresh fruit, and antioxidant activity of 1.0385 µg/ml with desirability of

276 0.892 or 89.2%. Contour plot and response surface of prediction result are presented in Figure 3.

277 In Figure 3., it is seen that the value of desirability shown within the lines inside the contour plant.

278 Desirability indicates the desired scale for each response and determines the degree of desirability

279 for optimal solution result. The range of scale for desirability value was between 0-1, in which 0

280 reflected that the response was totally undesirable, while 1 showed that the response was

281 completely wanted (Bezzera et al. 2008). This study obtained desirability value of 0.892 or 89.2%, in

282 which if the number was closer to one, the desirability value for optimization will also increase.
283 Result of study on the parameter of total phenol and antioxidant activity has similar trend of

284 graph. At higher temperature and longer time of extraction, the value of both parameters will

285 increase, yet the value will decline along with the increasing temperature and time of extraction.

286 Effect of heating does not only increase total phenol and antioxidant activity, but also decrease it.

287 Declining total phenol and antioxidant activity occurred at temperature above 80 °C and time longer

288 than 90 minutes. Chew et al. (2011) mentioned that longer time extraction and higher temperature

289 will result in oxidation of phenolic compound and antioxidant activity.

290 Based on the result of research, dekok extraction method that used water as solvent has a

291 prospect to be further developed at larger scale since it has simple, safe, and easy to apply technic

292 (user friendly) (Vongsak et al. 2013). Several researches performed rose myrtle extraction using

293 supercritical carbon dioxide (Bimarks et al. 2011), reflux (Lai et al. 2014, and ultrasonic (Lai et al.

294 2015). However, besides some advantages are gained from the technic, particular and expensive

295 equipment are required before extraction was done, therefore it is not appropriate to be applied in

296 small and medium scale – industry (SMEs) Vongsak et al. (2013), particularly in North Kalimantan.

297 3.4. Identification of Bioactive Compounds in rose myrtle fruit

298 The results of the identification bioactive compounds in rose myrtle fruit using GC-MS are

299 presented in Figure 4 and Table 3.

300 In Figure 4 and Table 3. it can be seen that the results of GC-MS rose myrtle fruit extract

301 produced 33 compounds, with 7 aromatic compounds, 8 flavonoid compounds, 2 phenolic acid

302 compounds, 1 carotenoid compound, 3 triterpenoid compounds, 3 terpenoid compounds, 7

303 phloroglucinol compounds, and 2 other components. At the optimum treatment, which is a

304 temperature of 80.65 ºC and an optimum time of 87,886 minutes using a water solvent, it turns out

305 to produce more compounds when compared to extracting karamunting fruit using acetone: water:

306 acetic acid in comparison (50: 49: 1) produces 19 components phenol (Lai et al., 2014).

307

308
309 4. Conclusion

310 This study showed that Surface Response is an effective method to optimize the condition

311 of rose myrtle fruit extraction. The model of response surface was verified statistically through

312 ANOVA. Calculation result of all independent variables had significant effects (p <0.05) on all

313 responses. Value of R2 of total phenol = 0.81752, and R 2 of antioxidant activity = 0.969, this result

314 showed that the quadratic polynomial model applied was accurate in analyzing interaction of all

315 parameters in study. Optimum condition in protecting bioactive compound in rose myrtle fruit was

316 80.43 ºC of temperature and time extraction of 85.00 minutes. This treatment produced total phenol

317 of 73.77 mg/100 g fresh fruit, and antioxidant activity of 1.0385 µg/ml with desirability of 0.892 or

318 89.2%, in which the desirability of optimization will be higher if the desirability value is closer to one.

319 The results of the analysis using GC-MS obtained 33 bioactive compounds in rose myrtle fruit

320 extraction.

321

322 Conflict of Interest : in this article there are no conflicts of interest whatsoever

323

324 Acknowledgement

325 Thank you to the LPDP for funding this research, with grant number 20161141011787. Thank

326 you to the promoters and co-promoters, all lecturers, staff, and laboratory staff at the Faculty of

327 Agricultural Technology, Universitas Brawijaya and Borneo Tarakan University who have helped the

328 research and preparation of this scientific article.

329

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435

436
437

438

439

440 Figure 1. (a). Contour plot and (b) Surface response which showed the effect of temperature and

441 time of extraction on total phenol of rose myrtle fruit extract

442 Design-Expert® Software Total Fenol

Design-Expert® Software

Total Fenol
120.00
Design points above predicted value

443
Total Fenol 55.8047 55.8047 Design points below predicted value
Design Points 81.158
81.158 60.3258 82
36.629
36.629 105.00

444 X1 = A: Suhu 73

T o ta l F e n o l
X2 = B: Waktu
X1 = A: Suhu 64.8469
B : W a k tu

64
X2 = B: Waktu 64.8469

445
90.00 60.3258 5
55

46

446 75.00
55.8047
120.00 90.00
105.00 85.00

447
69.3679
51.2837 90.00 80.00
60.00 75.00 75.00
70.00 75.00 80.00 85.00 90.00
B: Waktu A: Suhu
60.00 70.00

448 A: Suhu

449 (a) (b)

450
451 Figure 2. (a) Contour plot, (b) Surface response which showed the effect of temperature and time of

452 extraction on antioxidant activity of rose myrtle fruit extract

453 Design-Expert® Software

120.00
Aktivitas antioksidan Design-Expert® Software
1.58677 1.96043
Aktivitas antioksidan Antioksidan (IC50)
1.7736
Design Points Design points above predicted value
2.0612 1.58677 Design points below predicted value
454 1.02 105.00
2.0612
2.2
1.02
A n t io k s i d a n ( I C 5 0 )

X1 = A: Suhu 1.9
X1 = A: SUHU
B : W a k tu

X2 = B: W aktu X2 = B: WAKTU

455 90.00 5 1.6

1.3

456 75.00
1
1.2131
1.58677

457
1.58677
1.7736 120.00 90.00
1.39993
60.00 105.00 85.00
70.00 75.00 80.00 85.00 90.00 90.00 80.00
75.00 75.00
B: WAKTU A: SUHU
458 A: Suhu
60.00 70.00

459 (a) (b)

460

461 Figure 3. Graph of contour plot (a) and response surface (b) value of desirability

462 Design-Expert® Software

120.00
Desirability
0.297
Design-Expert® Software

Desirability 0.446 Desirability

Design Points 0.446 1

463
0.743
1
0
0 105.00 0.900
X1 = A: Suhu

464 X1 = A: Suhu 0.594

X2 = B: Waktu
0.675
B : W a k tu

X2 = B: Waktu
D e s ira b ility

0.594
90.00 5 0.450
Prediction 0.892

465 0.225

466
75.00 0.000

0.446
120.00 90.00

467 60.00
70.00 75.00 80.00 85.00 90.00
105.00
90.00 80.00
85.00

75.00 75.00
B: Waktu A: Suhu
60.00 70.00
A: Suhu
468
469 (a) (b)
470

471

472 Figure 4. GC-MS Bioactive Compounds profile of a crude extract from rose myrtle fruit sample
473

474

475

476

477

478

479

480

481

482

483

484 Table 1. Level of independent variable, code and optimized value

Independent variable Unit -1 +1 -α +α

Temperature (X1) º Celcius 70 90 65.9 94.14
Time (X2) Minute 60 120 47.57 132.4
485

486 Table 2. Matrix of factor and level in optimization of rose myrtle fruit extraction using central

487 composite design

488
Coded Factor Value Actual Factor Value Total Phenolic Antioxidant
Temperature of Time of extraction (X1) (X2) Response (mg Response (IC50)
extraction (ºC) (minute) GAE/g) µg/ml
0 0 80 90 74.151 1.02
-1 -1 70 60 49.845 1.872
-1 +1 70 120 57.726 1.57
0 0 80 90 81.158 1.034
-1.41 0 65.9 90 36.629 1.964
0 -1.41 80 47.57 57.538 1.908
+1 +1 90 120 57.377 2.059
0 + 1.41 80 132.4 50.879 2.054
0 0 80 90 78.764 1.031
0 0 80 90 69.087 1.028
 0 0 80 90 65.367 1.03
+1 -1 90 60 63.339 1.75
+1.41 +1 94.14 90 42.733 2.0612
489
490

491

492 Table 3.Bioactive Compounds detected by GC-MS in a rose myrtle fruit

Peaks Name of compound Compound class % RA

1 p Cymene aromatik 0,85149
2 β Pinene aromatik 0,96152
3 Limonene aromatik 0,96152
4 Myrcene Flavonoid 1,48947
5 Myrtenal Flavonoid 1,58175
6 Rosefuran aromatik 1,05802
7 Caffeic acid Phenolic acid 2,21473
8 β Ionone karotenoid 0,94068
9 Ferulic acid Phenolic acid 1,89295
10 β Caryophyllene Aromatik 2,02761
11 β Sesquiphellandrene Aromatik 1,41364
12 β Elemene aromatik 0,93689
13 Kaempferol Flavonoid 4,53045
14 Quercetin Flavonoid 3,97659
15 Myricetin Flavonoid 1,58164
16 Campesterol Flavonoid 3,07878
17 Stigmasterol Flavonoid 2,55941
18 β Sitosterol Flavonoid 2,07134
19 β Amyrin Triterpenoid 4,98656
20 Taraxerol Komponen lain 3,17736
21 Lupeol Terpenoid 4,03251
22 Friedelin Terpenoid 6,66648
23 Rhodomyrtoxin Phloroglucinol 2,84213
24 ψ Rhodomyrtoxin Phloroglucinol 3,79533
25 Rhodomyrtoxin B Phloroglucinol 6,15375
26 Rhodomyrtoxin C Phloroglucinol 3,05528
27 β Amyrenonol Triterpenoid 2,38998
28 Rhodomyrtosone A Phloroglucinol 7,31293
29 Rhodomyrtosone B Phloroglucinol 6,43381
30 Rhodomyrtone Phloroglucinol 3,76308
31 Betulin Terpenoid 5,17220
32 3 β 21 α 22(30) hopene 3,29 diol 3,07757
33 Betulin monoacetate Triterpenoid 2,84125
493

494