The Effect of Extraction Condition On The Polyphenol Content and Antioxidant Activity of

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IJPST Volume 4, Nomor 2 , Juni 2017

THE EFFECT OF EXTRACTION CONDITION ON THE


POLYPHENOL CONTENT AND ANTIOXIDANT ACTIVITY OF
Curcuma zedoaria (Christm.) ROSCOE RHIZOME

Lia Marliani, Wempi Budiana, dan Yonara Anandari


Sekolah Tinggi Farmasi Bandung, Bandung, Indonesia

ABSTRAK
White turmeric (Curcuma zedoaria (Christm.) Roscoe) is one of Indonesian herbal medicine.
The extraction process to get polyphenol compound from natural product was influenced by
some factor such as solvent, temperature, time, and method of extraction. The objective of this
study was to determine the significant factor of extraction that is solvent, temperature and time
of extraction on the polyphenol content and antioxidant activity of white turmeric (Curcuma
zedoaria (Christm.) Roscoe) rhizome. Extraction was done by dynamic maceration method
with variations (23 factor variable design) of solvent (ethanol 96% and water), temperature
(25°C and 70°C), time (6 and 24 hours). The method of analysis of polyphenol content using
Folin Ciocalteu reagent, and the antioxidant activity using DPPH free radical reduction method.
The experiment design and data analysis using Design-Expert® Software Version 10. The result
showed that extraction using ethanol 96% at 70°C for 24 hours was gave high polyphenol
content and antioxidant activity. Data analysis was showed that polyphenol content and
antioxidant activity was influenced only by solvent of extraction. This study indicated that
solvent is significant extraction factor for polyphenol content and antioxidant activity of white
turmeric (Curcuma zedoaria (Christm.) Roscoe) rhizome.

Keywords: Antioxidant, Curcuma zedoaria (Christm.) Roscoe, extraction, polyphenol

Pengaruh Kondisi Ekstraksi terhadap Kandungan Polifenol dan Aktivitas


Antioksidan Rimpang Curcuma zedoaria (Christm.) Roscoe

ABSTRACT
Temu Putih (Curcuma zedoaria (Christm.) Roscoe) adalah salah satu obat herbal Indonesia.
Proses ekstraksi untuk mendapatkan senyawa golongan polifenol dipengaruhi oleh beberapa
factor seperti pelarut, suhu, waktu, dan metode ekstraksi. Tujuan penelitian ini adalah untuk
menentukan faktor ekstraksi (pelarut, suhu, dan waktu ekstraksi) yang signifikan terhadap
kandungan polifenol dan aktivitas antioksidan dari rimpang temu putih (Curcuma zedoaria
(Christm.) Roscoe) rhizome. Ekstraksi dilakukan dengan metode maserasi dinamik dengan
variasi (variasi desain faktorial 23) pelarut (etanol 96% dan air), suhu (25°C and 70°C), waktu
(6 dan 24 jam). Penetapan kadar polifenol menggunakan reagen Folin Ciocalteu dan aktivitas
antioksidan menggunakan metode reduksi radikal bebas DPPH. Design eksperimen dan analisis
data menggunakan Software Design-Expert® Version 10. Hasil penelitian menunjukkan bahwa
ekstraksi menggunakan ethanol 96% pada suhu 70°C selama 24 menghasilkan kandungan
poliphenol dan aktivitas antioksidan tertinggi. Analisis Data menunjukkan bahwa kandungan
Polifenol dan aktivitas antioksidan hanya dipengaruhi oleh pelarut ekstraksi. Penelitian ini
menunjukkan bahwa pelarut merupakan faktor ekstraksi yang signifikan terhadap kandungan
polifenol dan aktivitas antioksidan rimpang Temu Putih (Curcuma zedoaria (Christm.)
Roscoe).

Kata kunci : Antioksidan, Curcuma zedoaria (Christm.) Roscoe, ekstraksi, polifenol

Korespondensi: Lia Marliani 57


l.marliani.pharm@gmail.com
IJPST Volume 4, Nomor 2 , Juni 2017

Introduction compound was extracted. The purpose of


White turmeric (Curcuma zedoaria this study was to determine the significant
(Christm.) Roscoe) is one of family
Zingiberaceae that usually used as one of extraction factors that influence the
Indonesian herbal medicine. Its rhizome polyphenol content and antioxidant activity
has antioxidant, anti-inflammatory, anti- of white turmeric (Curcuma zedoaria
cancer and anti-microbial activity 1. White (Christm.) Roscoe).
turmeric contains bioactive compounds Materials and Methods
such as flavonoids, polyphenols, Materials
curcuminoid, terpenoids and essential oils. White turmeric (Curcuma zedoaria
There are approximately 60 essential oil (Christm.) Roscoe) rhizome were collected
components and oleoresin in white turmeric from Purwakarta, West Java, Indonesia, at
with curzerenone, germacrone, camphor, Februari 2016. The specimen were
curcumenol as the largest component 2. identified at Herbarium Biology
The one of antioxidant compound Department, Padjadjaran University,
of white turmeric is polyphenols. Bandung, Indonesia. Sample was dried by
Polyphenols has been known as potent oven at temperature 50⁰C for 2 days. Dry
antioxidant. Polyphenols can scavenge material grinded and was sieved by mesh
radical species or inhibit some enzymes or 20 sieve.
chelating trace metals which involve in free Extraction
radical formation 3. Sample was extracted by dynamic
The extraction process aims to maceration method using 23 variable factor
obtain maximum active compounds and the (two levels of three factor) includes
best quality in activities. The yield of variation of solvent (ethanol 96% and
extraction is influenced by the type of water) in 200 mL, temperature (25oC and
extraction method, solvent, extraction time, 70oC) and time (6 hours and 24 hours). The
and temperature 4,5,6. There is no universal extraction process was done in an orbital
extraction method for all phenolic shaker at stirring speed of 30 rpm. Each
compounds because their solubility and extract was concentrated using Buchi®
physical characteristics varying as well as Rotavapor® R-215 to obtain spissum
varying form of phenolic compounds from extract.
simple to highly polymerized substances 3. Determination of total phenol content
Total phenol content and total flavonoid Total phenolic compounds contents
content of the extract are varying depend on were determined by the Folin-Ciocalteau
extraction solvent 7. The higher amounts of method 10. The 0.5 ml extract samples were
phenolic compound often extracted by mixed with 5 ml Folin Ciocalteu reagent for
more polar organic solvent such as 5 minute and Na2CO3(4 ml, 1 M) then
methanol and ethanol. The amount of added. The mixture was allowed to stand
polyphenols extracted are influenced by for 15 min and the phenols were determined
selecting the right solvent 8. According to by colorimetry at 765 nm. The standard was
Wissam et al, 2012 9, an increasing 20, 40, 50, 60, 80, and 100 µg/ml gallic
temperature of extraction will increases the acid. Total phenol values are expressed in
extraction efficiency by increases cell terms of gallic acid equivalent (µg/ml of
permeable, solubility and diffusion dry mass), which is a common reference
coefficients of the compounds. compound
μg
The antioxidant activities are 𝑚𝑔 𝐺𝑎𝑙𝑙𝑖𝑐 𝑎𝑐𝑖𝑑 𝑒𝑞𝑢𝑣𝑎𝑙𝑒𝑛𝑡 ( ) . 10−3
𝑇𝑜𝑡𝑎𝑙 𝑝ℎ𝑒𝑛𝑜𝑙 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 ( )= ml
𝐺 μg
𝑆𝑎𝑚𝑝𝑙𝑒 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (ml) . 10−6
depending on the level of antioxidant

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IJPST Volume 4, Nomor 2 , Juni 2017

Determination of antioxidant activity temperature and time that influence the


The free radical scavenging activity total phenols content, and antioxidant
of extract was determined using the stable activity.
radical DPPH (1,1‐diphenyl‐2‐ The mathematical models for each
10,11
picrylhydrazyl) . Different response were evaluated using a multiple
concentration of each extract (50-250 regression method. The response function
µg/ml for ethanol 96% extract and 500- applied was a linear polynomial equation,
1000 µg/ml for water extract) were added at given by equation (1):
equal volume (1:1) of freshly prepared
DPPH solution (60 µg/ml) in methanol in
each test tube and mixed. The absorbance …..(1)
was measured after 30 min incubation at In equation (1), Y is the dependent
516 nm (λmax of DPPH solution). The variable; β0 is the constant term; k number
capability to scavenge the DPPH radical of variables; βi represents the coefficients
was calculated using the following of linear parameters; βij represents the
equation: coefficients of interaction parameters.
DPPH scavenged (%) = [(Ac – The significance of the equation
As)/Ac]x100 parameters for each response variable was
Ac is the absorbance of the control analyzed by F-test. Only the factors with
(DPPH solution) and As is the absorbance significance higher than or equal to 5%
of the extracts. The antioxidant activity of (p≤0.05) were considered. The model
the extract was expressed as IC50. The adequacy was checked accounting for the
IC50 value was determined from linear coefficient of determination (R2). The
regression analysis with Concentrations of response and result of data analysis by
extract as X axis and DPPH scavenged (%) Design-Expert® Software Version 10 was
as Y axis. showed by Interaction plot graphic (2D).
Data analysis Results and Discussion
The results were analyzed by The result of total phenol content
Factorial analysis. ANOVA/ Response determination showed that the highest
Surface Methodology (RSM) on the content was obtained by extraction using
experimental data was performed using ethanol 96% at 24 hours and temperature
using Design-Expert® Software Version 10 70ºC (see Table 1).
to determine which factors of solvent,
Table 1 Yield and Total Phenol Content of Extract
Extraction factor variation Total Phenol
Temperature Time Yield (%) Content
Solvent
(⁰C) (hours) (mgGAE/G)
Water 25 6 29.40±0.990 20.869 ± 3.648
Water 25 24 28.75±1.060 20.376 ± 2.079
Water 70 6 31.95±2.758 19.945 ± 6.831
Water 70 24 26.08±0.813 23.463 ±2.010
Ethanol 96% 25 6 18.70±0.778 76.879 ± 2.803
Ethanol 96% 25 24 18.05±0.848 84.028 ± 3.834
Ethanol 96% 70 6 23.70±2.616 80.250 ± 5.821
Ethanol 96% 70 24 26.15±5.162 84.150 ± 4.142

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IJPST Volume 4, Nomor 2 , Juni 2017

This is accordance with Do et al. maybe the reason why ethanol 96% extract
research 7 which its result showed the gave the highest the total phenol content.
extract obtained by 100% ethanol has Temperature and extraction time are
highest phenolic content than other solvent enhancing the solubility and diffusion
and the water extract showed the lowest coefficient of solute 13. The result showed
phenolic content. Rhizome may contain that at 70⁰C and 24 hour extraction, both
varying phenolic compounds from simple water and ethanol extract gave the highest
to polymerized subtances. The other phenolic compound. The chance of
compound such as carbohydrates and denaturation and oxidation of phenolics is
proteins may also associated with phenolics increased by high temperature and long
compound 3. The complex polyphenols are extraction which decrease the yield of
more soluble in organic solvent, such as 7
phenolics in the extracts . The result
ethanol than water. The solubility of showed that at 70⁰C and 24 hour extraction,
nonphenol compounds like carbohydrate phenolic compound of white turmeric
and protein are higher in water than in (Curcuma zedoaria (Christm.) Roscoe)
ethanol 12. The water can extract other rhizome may not be denaturated or
compound of white turmeric such as oxidized.
amylum that also abundant in rhizome. This

Design-Expert® Sof tware


Factor Coding: Actual
Kadar Fenol (mg/G)
One Factor
X1 = A: Pelarut 100
Actual Factors
B: Suhu Ekstraksi = 44,4595
C: Waktu Ekstraksi = 15
80
Total phenol content
Kadar Fenol (mg/G)

60
(mgGAE/G)

40

20

Air Etanol
WATE Solvent ETHANO
R
Figure.1 : The graphic of solvent effect to A:total phenol L
Pelarut content (Design-Expert®)
content and ethanol 96% gave the highest
Although at different temperature total phenol content (figure 1).
and time of extraction showed different The result of antioxidant activity
phenol content, the data analysis by that determined by free radical DPPH
Design-Expert® showed only solvent scavenging activity showed the lowest IC50
variation ((significance higher than 5%, p value also was obtained by extraction using
p≤0.05) could affecting the total phenol ethanol 96% at 6 hours and temperature
70ºC (see Table 2).

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IJPST Volume 4, Nomor 2 , Juni 2017

Table 2 : IC 50 value of Extract


Extraction factor variation
Temperature Time IC50 (µg/ml)
Solvent
(⁰C) (hours)
Water 25 6 956.16±20.27
Water 25 24 754.80±244.38
Water 70 6 821.18±107.20
Water 70 24 756.13±77.83
Ethanol 96% 25 6 203.12±9.69
Ethanol 96% 25 24 194.70±0.40
Ethanol 96% 70 6 184.25±6.35
Ethanol 96% 70 24 210.12±5.13

The lowest IC50 value determine the white turmeric (Curcuma zedoaria
more potent antioxidant activity. This result (Christm.) Roscoe). Although at different
(Table 1 and 2) showed that the high total temperature and time of extraction showed
phenol content gave the most active different phenol content, but they are not
antioxidant. It is also determined the role of significant.
polyphenol as antioxidant compound of
Design-Expert® Sof tware
Factor Coding: Actual
Original Scale
One Factor
IC 50 (ppm)
1000
X1 = A: Pelarut

Actual Factors
B: Suhu Ekstraksi = 47,5
C: Waktu Ekstraksi = 15
800
IC 50 (µg/ml)
IC 50 (ppm)

600

400

200

WATE
Air
Solven ETHAN
Etanol

Figure.2 : The graphic R t A:to


of solvent effect IC50 valueOL
Pelarut (Design-Expert®)

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IJPST Volume 4, Nomor 2 , Juni 2017

The data analysis by Design- concentration, extraction time and


Expert® determines that only solvent extraction temperature on the
variation (significance higher than 5%, recovery of phenolic compounds
p≤0.05) could influence the antioxidant and antioxidant capacity of
activity (figure 2). The potent antioxidant Orthosiphon stamineus extracts.
showed by extract which obtain using International Food Research
organic solvent (ethanol 96%). Journal. 2011; 18: 1427-1435.
Conclusion 6. Ng L.Y., Ang Y.K., Khoo H.E. and
This study showed that solvent is Yim H.S. Influence of different
significant extraction factor for polyphenol extraction parameters on
content and antioxidant activity of white antioxidant properties of Carica
turmeric (Curcuma zedoaria (Christm.) papaya peel and seed. Research
Roscoe) rhizome than temperature and time Journal of Phytochemistry. 2012; 6:
of extraction. 61-74.
7. Do QD, Angkawijaya AE, Tran-
Acknowledgement Nguyen PL, Huynh LH, Soetaredjo
FE, Ismadji S, Ju YH. Effect of
This research was supported by an Internal extraction solvent on total phenol
Grant from Bandung School of Pharmacy, content, total flavonoid content, and
Bandung, Indonesia. antioxidant activity of Limnophila
aromatic. Journal of Food and Drug
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