Optimization Extraction time of Black Cincau (Mesona palustris BL) Based

Extraction on Pilot-Scale

Tri Dewanti Widyaningsih, Elok Waziiroh, Novita Wijayanti and Yulina L. M.

Department of Food Science and Technology, Faculty of Agricultural

Technology,Brawijaya University, Malang, Indonesia

*Corresponding author : tridewantiw@ub.ac.id

Address: Veteran, Malang, East Java, Indonesia, 65145

Telephone: +628125229021

Faximile: +62341-569214

E-mail: tridewantiw@ub.ac.id

Abstract

The objective of this study was to optimize the extraction time for black cincau

(Mesona palustris BL) based using historical-data response surface methodology

(RSM). Black cincau extraction development from laboratory scale to pilot-scale

needs adjustment and optimization of its critical parameters to obtain optimum

extract quality. The optimization was conducted based on its functional activity and

minimum extraction cost. Historical data was applied to determine the effect of

extraction time (min) on total phenolic content (TPC), antioxidant activity, IC 50, water

contentand extraction cost (IDR). The optimum extraction time were found to be

extraction time of 150 min. Under the optimized conditions, the experimental value

for TPC was66,378 + 1,45 mg/g CAE, antioxidant activity was74,54 + 0,63 %, IC50

was75,18+ 0,03 ppm, water content was4,99% and extraction cost was1.734 x 10 6
(IDR). Optimizing the extaction time in pilot-scale would give the similiar quality with

laboraty scale and feasible to commercialized.

Keyword: Black cincau (Mesona palustris BL), Pilot-Scale extraction, Extraction time,

Historiacal-data Response surface methodology (RSM).

Introduction

In recent years, there has been a great increase in consumer interest in the

active role of foods beyond its functional activity. Foods may provide health benefits

identified as food functionalities (Diplock et al., 1999). Among emergent

functionalities, antioxidant activity is the most used. Natural phenols, present in plant

food could exert their beneficial health effects mainly through their antioxidant activity

(Proteggente et al., 2002; Zhou and Yu, 2006). Moreover, these polyphenolic

compounds have been found effective in many health-related properties, such as

antioxidant, anticancer, antiviral and anti-inflammatory activities (Hyardin et al.,

2011).

Herbs have become more popular as a polyphenolic source in Indonesia in

recent years. Several common herbs and spices are now understood to exert many

benefical physiological effect (Srinivasan, 2005). Black cincau (Mesona palustris BL)

or grass jelly is a traditional food which is believed to have benefit as medicine. In

Indonesia it called cincau hitam, because of its color is translucent black, or Hsian-

tsao (Mesona procumbens Hemsl) in China or Taiwan. Black cincau extract

component consist of bioactive compound containing phenol, flavonoids and steroid.

Polyphenol compounds have been shown to act as imunomodulator; effectively able

to inhibit mitogen-stimulated proliferation of peripheral blood muscular cells, Ig

production, IL-2 and interferon gamma (IFN-γ) (Widyaningsih et al., 2013).

The availibility of polyphenolic compound in black cincau as antioxidant and

imunomodulator activity is ensured. However, the economical feasibility of an

industrial process also require working in such a way that high extraction effeciency

is attained (Chan et al., 2009). Many factors have been established to influence the

extraction efficacy, such as extraction methods, particle size, solvent type, solvent

concentration, solvent-to solid ratio, extraction temperature, extraction time and pH

(Pinelo et al., 2005; Banik and Pandey, 2007; Silva et al., 2007; Chan et al., 2009).

Black cincau based extraction at laboratory scale had been done, furthermore

black cincau based supplement production were going to develop into pilot-scale

production. Pilot-scale production need adjustment of its critical parameter in order to

obtain the similiar product characteristic with the laboratory product. In this study,

extraction time become critical parameter in extraction condition which was need to

be optimize.

Material and Methods

Plant material

Black cincau (Mesona palustris BL) simplisia was purchased from farmer in

Magetan, Middle Java, Indonesia and red ginger (Zingiber officinale var. rubrum)

simplisia from PT ASIMAS Lawang, East Java, Indonesia. Black cincau and red

ginger simplisia were grind into powder to obtain intensive contact area between

solvent and solid (herbs).

Chemical reagent

The solvent and chemical were used for analytical purposes. Reagent 2,2-

diphenyl-1-picrylhydrazyl/DPPH (Sigma) used for analyze antioxidant activity and

reagent Folin Ciocalteau, etanol 96% and Na 2CO3 used for analyze TPC.

Extraction condition

24 kg of black cincau simplisia and 1 kg red ginger was weighed and

extracted with 500 L of extracting solvent in double jacket-vacuum extractor. The

extraction condition were temperature extraction 40 0C, vacuum-pressure ± -45

cmHg, black cincau: red ginger ratio 24:1 and solvent-to-solid ratio 20:1 the mixture

was mixed by orbital paddle. Extraction was done after 5 h, after the extraction, the

extract was collected and dried into the oven for supplement production. The

extraction was replicated once.

DPPH radical-scavenging activity

Antioxidant activity was determined according to the method with slight

modification (Okawaet al., 2001). Sample was diluted with distilled water to obtain

concentration of 20, 40, 60 and 80 ppm, respectively. Each sample solution (4 ml)

was added to 1 ml DPPH solution (0.2 mM). The reduction of DPPH was measured

at 517 nm against a blank assay for 30 minutes. The percentage of radical inhibition

in medium was calculated as the different of absorbance of the blank and

absorbance of the sample divided by that of DPPH control at the same time

multiplied by 100.

It can be calculated based on this formula:

Ablank −Asampel
%inhibition= x 100 %
Ablank
 The value of sample concentration and inhibition percentage was

graphically plotted for equation of linear regression (y= ax + b). The equation was

used for calculating the IC 50 value (inhibitorconcentration 50%) from each sample.

The IC50 value is defined as the concentration of antioxidant (sample or control)

necessary to decrease the initial DPPH concentration (50%) and is expressed as

mg/ml.

Total Polyphenol Content Assay (TPC)

Concentration of total polyphenol was determined by Folin Ciocalteu method

with modifications(Andarwulan and Shetty, 2000). Blank solution was prepared by

adding 2 ml of 96% ethanol in a 10 ml test tube. Caffeic acid 1000 ppm was used as

stock solution. 50 mg tannic acid was diluted with 50 ml of 96% ethanol. The

sample was weighed approximately 1 mg and diluted with 2 ml of 96% ethanol in a

10 ml test tube. The standard solution and sample were added to 5 ml deionized

water, 0.5 ml of Folin Ciocalteu reagent (50% v/v) and incubated for 5 minutes.

Then, it was homogenized with 1 ml of sodium carbonate solution (5% v/v), and

incubated at room temperature and in the dark for 1 hour. After incubation, the

solution was homogenized again. Then the total phenol content was measured with

a spectrophotometer at a wavelength of 725 nm. The value of total phenolic content

is interpreted by milligram equivalents of caffeic acid per gram extract (mg/g

extract).
Industrial feasibility

Some variables were evaluate to determine industrial feasibility. The industrial

feasibilty variables were Break Even Point (BEP), Payback Period (PP), Net Present

Value (NPV), and Internal rate of Return (IRR).

Experimental design

The experimental design for this study was carried out using Historical-Data

RSM (Table 1). Historical data with single factor experiments were performed to

determine the extraction time for extract black cincau: red ginger. Single factor was

performed by fixing extraction temperatur (40 0C), ratio black cincau: red ginger

(24:1), and solvent-to-solid ratio (20:1). extraxtion was develop for 5 h and every 15

min the extract was evaluate for its phytochemical activity.

The experimental results were analyzed using Design Expert

Software(version 7.0). Optimal extraction time was obtained using linear model of

RSM. The suitability of the model equation for predicting the response value was

verified by conducting the extraction time under the recommended optimal time.

RESULT AND DISCUSSION

Laboratory extraction

Black cincau and ginger were extracted in laboratory scale to determine the

extract characteristic. The Laboratory-extraction condition was temperature 80 0C,

ratio black cincau:ginger 24:1, solvent-to-solid ratio 20:1 and extraction time 30-45

min. Table 2 was the extract characteristic under laboratory-extraction condition.

Optimization of extraction time in Pilot-Scale

Mathematical model for TPC, Antioxidant activity and Extraction cost response

Mathematical model under the optimal extraction time was performed by

Design Expert (Table 3). Mathematical model was specialized for each response.

The suitability of the model equation for predicting the response value was verified

by conducting the extraction under the recomended optimal condition (Chan et al.,

2009). The P-values were used as a tool for ensure the significance of each

coefficient, which in turn might indicated the interaction patterns between the

variable (Hou and Chen, 2008). The smaller the P-value, the more significant was

the corresponding coefficient.

Graphical analysis of models was also performed by normal plots of the

residuals, as illustrated in Figure 1. It was obvious that residuals depict a normal

distribution in all of responses, generally the point follow a straight line. In addition,

they also showed that the models cannot be improved further through any change in

the transformation of the response because data point in Figure 1 were scattered

and they did not exhibit a “S-shaped” curve. Therefore, these figure can be

considered as the best models of the historical data RSM design ( Jeirani et al.,

2013)

Effect of extraction time on TPC in Pilot Scale

Based on Figure 2. Showed that in increase in extraction time from 15 – 120

min was accompanied by an increment of TPC from 1,427 – 1,770 mg CAE/gand

showed drecement after 120 min.Extraction time was main paramenter in the

extraction procedure. The extraction time can either be as short as few minutes or

very long up to 24 hours (Chan et al., 2009; Laponik et al., 2005; Lee et al., 2005).
 Either increment extraction time and TPC was well explained by Fick`s

second law of diffusion, which state that final equilibrium will be achieved between

the solute concentration in the solid matrix (plant matrix) and in the bulk solution

(solvent) after a certain time, hence, an excessive extraction time was not useful to

extract more phenolic antioxidant (Silva et al., 2007)

Based on Susanti (2008) relationship between TPC and extraction time

showed with parabolic curve, it means TPC would increase untill maximum condition

and would decrease. That phenomenon happened because of exposure of matrix

substances would make phenolic compound extraction easier. Furthermore,

prolonged extraction process might lead to phenolic oxidation due to light or oxigen

expossure (Chan et al., 2009)

Setback TPC at 210 min was explained by Prior et al. (2005) which stated that

the folin-Ciocalteu method suffers from a number of interfering subtances. The

interfering substances may also react with the Folin-Ciocalteu reagent to give

elevated apparent phenolic content. Therefore, considering the TPC an extraction

time of 120 min was selected for RSM optimization

Effect of extraction time on antioxidant activity in Pilot-Scale

As shown in Figure 3. Antioxidant activity of black cincau:ginger extract

showed signignificantly higher as during extraction time to 165 min. After 165 min,

the antioxidant activity showed decrecement. The literature showed that the effect of

thermal treatment on the potential antioxidant activity of vegetables was different

depending on several factors such as species, intensity and modality of the

treatment. Analyzing the influence of heat treatment on the antioxidant activities and
polyphenolic compounds. Choi et al. (2006) found that polyphenolic compound

increased as heating temperature and time increased.

Roy et al. (2007) concluded that normal cooking temperatures (75°-100ºc, 10-

30 min) were detrimental to the phenolic content and to the antiradical and

antiproliferative activities of many vegetable juices; however mild heating (50ºc, 10-

30 min) preserved 80-100% of the phenolic content.

Effect of extraction time on cost extraction in Pilot Scale

Price of raw material, cost extraction for 15 min and labour salary were

parameters to evaluate industrial feasibility. As shown in Figure 4 increment of

extraction cost accompanied by extraction time. Cost extraction would influence

production cost and selling price respectively. Production cost showed significant

influence on selling price. Selling price was influenced by production cost 92 % and

8% for the other parameters such as market demand, market target, market

competition and consumers taste.

Optimization

Some steps were taken prior to optimize in order to identify the criteria of the

numerical optimization. The goal factor for extraction time was set to within in range,

TPC and antioxidant activity were set to maximize, and minimize for extraction cost

in the Design Expert Software. Table 4 showed the summary of the optimization.

A series solutions were generated and the solution to be employed for the

verification would be selected based on its desirability and suitability. The predicted

optimum extraction time was estimated to be 153,65 min or equal with 2,5 h. At this

optimum extraction time the corresponding predicted TPC, Antioxidant activity and

extraction cost were 1,636 + 0,12 mg CAE/g, 49,57 + 12,26 (%) and 1,733 + 0,0004
(IDR) respectively. After verification the responses value for TPC was 66,378 + 1,45

mg/g CAE, antioxidant activity was 74,54 + 0,63 % and extraction cost was 1,734 x

106 (IDR). These verification value was within the prediction internal range.

Therefore, the historical data RSM design was considered as an efficient statistical

technique to predict the optimum extraction time.

The optimum extraction time showed industrial feasibitily. The parameters for

Industrial feasibity were R/C ratio 1.97, NPV Rp 24.610.444.769 (IDR), IRR 61.11%,,

payback period 5.8 years.

Characteristic of Functional Drink on Pilot-scale to Laboratory Scale

The characteristics of the black cincau based extract on pilot-scale and

laboratory scale were summarized in Table 5. Generally, the parameters value

between laboratory and pilot scale were not different. This condition showed that

extraction time for black cincau based extraction was critical parameters which gave

influence on phytocemical of extract quality

Conclusion

In this study confirmed the advantages of historical-data RSM in optimising

the extraction time of black cincau based. The result showed that optimal extraction

time was 2.5 h and the responses value for TPC was 66,378 + 1,45 mg/g CAE,

antioxidant activity was 74,54 + 0,63 % and extraction cost was 1,734 x 106 (IDR).

The response value in laboratory and pilot-scale generally were no difference. This

condition showed that extraction time was critical parameter in pilot-scale extraction.

The optimum extraction time showed industrial feasibitily to commercialized.

 Acknowledgements

The authors gratefully acknowledge to DP2M-DIKTI and Brawijaya University

for financial support through “RisetUnggulan Perguruan Tinggi 2013”.

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Table 1.The experimental design using Historical-Data RSM with the responses value
Factor Responses

Extraction
No. Extraction time TPC Antioxidant
cost
(min) (mg CAE/g) activity (%)
(X 106) (IDR*)

1 15.00 1.435 23.13 1.027

2 30.00 1.148 33.46 1.103

3 45.00 1.427 37.35 1.180

4 60.00 1.507 1.140 1.256

5 75.00 1.507 53.18 1.333

6 90.00 1.458 43.86 1.408

7 105.00 1.623 50.90 1.485

8 120.00 1.770 53.31 1.562

9 135.00 1.732 26.29 1.639

10 150.00 1.582 56.87 1.715

11 165.00 1.612 65.19 1.792

12 180.00 1.538 61.70 1.868

13 195.00 1.659 63.04 1.945

14 210.00 1.901 61.50 2.021

15 225.00 1.880 58.35 2.098

16 240.00 1.838 61.03 2.174

17 255.00 1.916 57.54 2.251

18 270.00 1.884 62.30 2.327

19 285.00 1.762 68.00 2.404

20 300.00 1.751 64.65 2.481

Note: 1 $ = 12.977 (kurs April. 22th 2015)

 Table 2. The extract characteristic (Laboratory-scale)

TPC Antioxidant Water content

IC50 (ppm)
(mg CAE/g) activity (%) (%)

62.509 + 1.13 69.86 + 2.04 78.20 + 0.96 3.96 + 0.73

(a) (b)

(c)

Figure 1. Normal plots of the residuals of (a) TPC; (b) antioxidant activity; and (c)

extraction cost.
 TPC
2.5

2
TPC (mg CAE/g)
1.5

0.5

0
0 50 100 150 200 250 300 350

Extraction time (min)

Figure 2.Effect of extraction time on TPC value

Antioxidant activity
80
70
Antioxidant activity (%)

60
50
40
30
20
10
0
0 50 100 150 200 250 300 350

Extraction time (min)

Figure 3. Effect of extraction time on antioxidant activity value

 Extraction cost

Extraction cost (x106) (IDR)

3

2.5

1.5

0.5

0
0 50 100 150 200 250 300 350

Extraction time (min))

Figure 4. Effect of extraction time on extraction cost

Table 3. Mathematical model for the responses

Responses Variables

Mathematical model Model Significance

(p<0.05)

TPC TPC = +1.35212 + 1.85193E-003 * Time < 0.0001

Antioxidant Antioxidant activity = +26.95295 + 0.14725 < 0.0001

activity * Time

Extraction Extraction cost = +9.4996 + 5.10150E-003 < 0.0001

cost *Time
 Table 4. Summary of optimization data

Response Goal Experimental region

Lower limit Upper limit

Extraction time Is in range 15 300

TPC maximize 1.148 1.916

Antioxidant activity maximize 1.14017 68.008

Extraction cost minimize 1.027 2.481

Table 5. Characteristic extract on laboratory and pilot-scale

Parameter Laboratory-scale Pilot-scale

TPC (mg CAE/g) 62.509 + 1.13 66.378 + 1.45

Antioxidant activity (%) 69.86 + 2.04 74.54 + 0.63

IC50 (ppm) 78.20 + 0.96 75.18 + 0.03

Water content (%) 3.96 + 0.73 4.99 + 0.08

Yield (%) 25.03 26.01