ISSN 0975-2366

DOI: https://doi.org/10.31838/ijpr/2020.SP2.354
Research Article

Principal Component Analysis of Antioxidant Activities,

Total Phenolic contents, and Total Flavonoid contents
of Turmeric (Curcuma longa L.)
ABDUL ROHMAN1,2*, ADELIN THERESIA CAN1, IRNAWATI3, MOHAMAD RAFI4, ENDANG
LUKITANINGSIH1, NURRULHIDAYAH A. FADZILAH5
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gadjah Mada University, Yogyakarta,
55281, Indonesia
2
Center of Excellence, Institute for Halal Industry and System (IHIS), Gadjah Mada University,
Yogyakarta, 55281, Indonesia.
3
Faculty of Pharmacy, Halu Oleo University, Kendari, 93232, Indonesia
4
Department of Chemistry, Faculty of Mathematics and Natural Sciences, IPB University, Kampus IPB
Dramaga, Bogor, Indonesia.
5
International Institute for Halal Research and Training, International Islamic University of Malaysia,
Gombak, Malaysia
* Corresponding Author
Email ID: [email protected]
Received: 06.08.20, Revised: 13.09.20, Accepted: 08.10.20

ABSTRACT
Turmeric (Curcuma longa L.) is often used in the treatment of several diseases. Scientifically, turmeric has
been shown to have antioxidant activity and the group of compounds that play a role in this regard is the
curcuminoid, a compound belonging to the phenolic group. In addition, turmeric also contains secondary
metabolites, such as flavonoids, which are also shown to have antioxidant activity. This study aims to
determine the profile of total phenolic content, flavonoids, and antioxidant activity of turmeric extract from
various markets in Central Java, East Java, and Special District of Yogyakarta and to perform Principal
Component Analysis (PCA) for grouping the turmeric. Turmeric rhizome samples were prepared and
macerated with methanol. The methanolic extracts were determined for its total phenolic content by the
Folin-Ciocalteu method, flavonoids with AlCl3 reagent, and antioxidant activity by DPPH method, and then
subjected to chemometrics analysis using PCA dan Cluster Analysis (CA). The profiles of phenolic, flavonoid,
and IC50 values in each sample were different due to various factors that influence the content of secondary
metabolites. The results also revealed that PCA method did not succeed in grouping turmeric samples because
there was no correlation among variables, but CA method could provide the grouping of turmeric samples
into six groups based on the variables used.
keywords: Curcuma longa L., antioxidant, chemometrics, principal component analysis.

INTRODUCTION al., 1985). Curcuminoids have been shown to

Turmeric is a native plant from Southeast Asia have antioxidant activity in the presence of
and is widely grown in various countries, such as hydroxy phenolic groups and methoxy groups that
Bangladesh, China, the Philippines, India, act as antioxidants (Toda et al., 1985). In
Indonesia, Jamaica, Sri Lanka, and Taiwan addition to curcuminoids, turmeric also has other
(Hartati, 2013). Utilization of turmeric as a secondary metabolites, namely flavonoids in the
medicinal plant is often and commonly used to form of flavonol kaempferol and rutin
treat various types of diseases traditionally, such (Ghasemzadeh et al., 2012). Hydroxyl groups in
as fever, diarrhea, itchy skin, flu, and canker flavonoids donate hydrogen atoms and electrons
sores. The main content of turmeric is to free radicals to stabilize them and produce
curcuminoids (curcumin, demethoxicurcumin, stable flavonoid radicals (Heim et al., 2002). The
bisdemetoksikurkumin), essential oils, resins, fat, content of these compounds in each turmeric can
protein, calcium, phosphorus, and iron (Rahardjo vary due to many factors, one of which is the
and Rostiana, 2005). Curcuminoid, a phenolic geographical origin of the plant, where soil and
compound, has been shown to have antifungal environmental conditions, such as nutrients,
activity (Radwan et al., 2014), antibacterial weather, and temperature can affect the
(Rahmawati et al., 2014), anti-inflammatory formation of compounds and plant productivity
(Chainani-Wu, 2003), and antioxidants (Toda et (Sholehah et al., 2016).

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 Abdul Rohman et al / Principal Component Analysis of Antioxidant Activities, Total Phenolic contents,
and Total Flavonoid contents of Turmeric (Curcuma longa L.)

To find out the difference between samples that Quantification of total phenolic contents
have chemical physical properties that are similar The measurement of total phenolic contents (TPC)
to variable variables is difficult to do with general was performed using a calibrated UV-Vis
statistical methods and often are subjective. spectrophotometer (Hitachi-Tokyo, Japan) as
Therefore, we need an analytical method that can described by Chun et al. (2003) with slight
effectively and accurately manage multivariate modifications. A 200 μL of turmeric extact (1
data. Chemometrics is used to manage and mg/mL; 1 mg dissolved in 1 mL methanol) was
handle multivariate data as well as in mixed with 0,4 mL of FCR, shaken up, and left for
experimental designs. PCA, one of the 5 minutes. Neutralization is performed by added
unsupervised pattern recognition, can be used for 4 mL of 7% Na2CO3, stirred, and added
grouping objects based on the principal aquabidest up to 10 mL. The test solution was
component (PC) obtained from multivariate data incubated at room temperature for 73 minutes
reduction as conducted by Widodo et al. (2019) and the absorbance was measured at wavelength
in the classification of medicinal plants using PCA 728 nm. TPC is expressed as mg gallic acid
based on antioxidant activity, total phenolic equivalent/g of the dry extract (mg GAE/g DW)
content, and total flavonoid content and through linear regression, which obtained from
Vallverdú-Queralt et al. (2015) in classifying gallic acid standard with various concentrations
several spice samples using PCA based on their (2, 4, 6, 8, 10, 12, and 14 ppm).
phenolic structure. The objective of this study was
Quantification of total flavonoid contents
to determine the profile of total phenolic content,
The measurement of total flavonoid contents
flavonoids, and antioxidant activity of turmeric
(TFC) was performed using a calibrated UV-Vis
extract from various markets in Central Java, East
spectrophotometer (Hitachi-Tokyo, Japan) and
Java, and Special District of Yogyakarta and to
according to Zou et al. (2004) with slight
perform Principal Component Analysis (PCA) for
modification. A 600 μL of the extract (1mg/mL in
grouping the turmeric.
methanol) was mixed with 4 mL of aquadest, 0,3
μL of NaNO2 10%, and left for 5 minutes. After
MATERIALS AND METHODS
Turmeric rhizome samples were collected from that, 0,3 μL of AlCl3 10% was added and left for 5
various markets of East Java (Mantingan, minutes again. The solution was added 4 mL of
Sumberpucung, Desa Sambi, Garum, Tugurante), NaOH 10% and aquadest up to 10 mL. The final
Central Java (Kraguman, Delanggu, Kartasura, solution was incubated for 22 minutes at room
Sidodadi Kleco), and Special District of temperature and the absorbance was measured
Yogyakarta (Kranggan, Wonosari). at wavelength 510 nm. TFC is expressed as mg
Authentification of samples were done by Faculty rutin equivalents/g of the dry extract (mg RE/g
of Pharmacy, Gadjah Mada University, DW) through linear regression which obtained
Yogyakarta. Methanol, NaNO2, NaOH, Folin- from rutin standard with various concentrations
Ciocalteau’s Phenol Reagen (FCR), Na2CO3, and (10, 20, 30, 40, 50, 60, and 70 ppm).
AlCl3 were purchased from E. Merck (Darmstadt, DPPH radical scavenging activity: DPPH radical
Germany). 2,2′-diphenyl-2-picrylhydrazyl (DPPH), scavenging activity was measured using a UV-Vis
gallic acid (purity 100%), and rutin (purity 95%) spectrophotometer with reference to the method
were obtained from Sigma (Aldrich, USA). All Kikuzaki et al. (2002). Various concentrations of
materials used were analytical grade. the sample (10-30 ppm) were added 1 mL of
Preparation of methanolic extract: Preparation DPPH 0.4 mM each and methanol up to 5 mL.
of methanolic extract was performed according to The test solution was incubated for 30 minutes at
Widodo et al. (2019) with slight modification. darkroom and the absorbance was measured at
Turmeric samples were washed, drained, oven wavelength 515 nm. The percentage of inhibition
(40°C), and grounded. Powdered samples were was calculated using [(A0 - A1) / A0] × 100, which
macerated using methanol (1:10 w/v) as is A0: the absorbance of the control and A1: the
extracting solvent. The samples were macerated absorbance of the extract or standard. The
twice with 60% volume of solvent within the first 3 inhibition curve was generated and the value of
days, then the solids were filtered and re- IC50 was calculated.
macerated with 40% volume of solvent for the Data Analysis: TPC, TFC, and IC50 were done
next 3 days. Extracts were concentrated using a with 2 replication and all of the data was
water bath. The methanolic extract obtained were expressed as mean ± standard deviation using
tested for antioxidant activity, determination of Microsoft Excel (Microsoft Inc., USA). The
phenolic contents, and flavonoid contents. chemometrics of principal component analysis
(PCA) and cluster analysis (CA) were performed

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 Abdul Rohman et al / Principal Component Analysis of Antioxidant Activities, Total Phenolic contents,
and Total Flavonoid contents of Turmeric (Curcuma longa L.)

using software Minitab version 19 (Minitab Inc., dan Siradz, 2005; Laitinen et al., 2005; SMD and
USA). Nurhayati, 2007; Nica Dewi et al., 2016;
Sholehah et al., 2016). Classification of turmeric
RESULTS AND DISCUSSION methanolic extracts was carried out using
TPC was measured using the Folin-Ciocalteu chemometrics of PCA, one of the unsupervised
method, which is based on the colorimetric pattern recognition techniques. The variables
principle. Phenolic compounds in an alkaline used were IC50, TPC, and TFC. The eigenvalue
state will become phenolic ions and then will shows that PC1, PC2, and PC3 contributed fairly
react with the Folin-Ciocalteu reagent (FCR) which towards overall PC, with 36.4%, 34.8%, and
forms a blue solution (Sánchez-Rangel et al., 28.9%. The score plot of PCA (Figure 1) places
2013). Among turmeric samples, turmeric from samples based on first principal component (PC1)
Sumberpucung Market, East Java (sample code: and second principal component (PC2) value. The
5) has the highest TPC value, which is 209.673 ± score plot of PCA of 14 methanolic extract
40.483 mg GAE/g DW and turmeric that has the turmeric shown in Figure 1, unable to classified
lowest TPC value is turmeric from Sambi Market, samples into a group because of the distribution
Kediri, East Java (code sample: 6) that is 164.943 of the samples didn’t show any pattern of group.
± 15.173 mg GAE/g DW. (Table 1)TFC was Loading plots of PCA (Figure 2) project the
measured using AlCl3 reagent. Flavonoids will burden or contribution of variables to PC1 and
experience nitration in an alkaline atmosphere PC2, and to illustrate the correlation between
with the addition of NaNO2, which will form a variables. The angle between variables can
yellow solution with AlCl3, and then the addition determine the correlation between the two
of NaOH will form a red solution (Pękal and variables. If the variables form a small angle,
Pyrzynska, 2014). Turmeric from Sidodadi Kleco there is a positive correlation between the two
Market (III), Central Java (sample code: 14) has variables, if it forms an angle about 90o, no
the highest TFC value, which is 616.473 ± 76.81 correlation found between variables, and if it
mg RE/g DW, while turmeric that has the lowest forms a large angle that is close to 180o, both
TFC value comes from Garum Market, Java East variables have a negative correlation (Widodo et
(sample code: 7) with 463.448 ± 52.125 mg al., 2019). All angles between variables show
RE/g DW (Table 2). The DPPH method is an easy angles close to 90o, which indicates that there is
method to implement and can have accurate no correlation between variables. This is ensured
results when compared to other methods in by the scatter plot between variables (Figures 3,
determining antioxidant activity (Buenger et al., 4, 5), having a correlation coefficient (r) less than
2006). Plants that have antioxidant compounds 0.2 which means there is no correlation between
will reduce DPPH which causes the purple color of these variables. The absence of correlation
the DPPH radical turn into a pale yellow solution. between the variables used is the cause of the
The antioxidant strength of a compound is PCA method is not success in classifying the
expressed as IC50 which is the substrate sample. PCA method can only be used if the
concentration that causes a reduction of 50% in variables are correlated and have not always
the color of the DPPH reagent and is obtained been successful in displaying several sample
through the regression equation of percent groups (Rohman, 2014). The failure of PCA in
inhibition (y-axis) with sample concentration (x- grouping samples causes another analysis is
axis). The smaller value of IC50, the greater the needed, cluster analysis (CA), a method for
antioxidant activity of the sample (Kedare and dividing samples/objects that have similarities
Singh, 2011). From the whole sample evaluated, based on the variables into clusters (Miller and
it was found that all samples had a very strong Miller, 2005). The variables used in this analysis
anti-oxidant activity which refers to Blois (2005) are TPC, TFC, and IC50, and grouping are done
classification (<50 ppm) (Table 3). Turmeric from based on similarity levels of 90. Based on the
Kraguman Market, Central Java (sample code: 9) dendrogram of CA (Figure 6), turmeric samples
has the smallest IC50 value, which is 17.716 ± were divided into 6 groups. Also, samples from
0.892 µg / mL, which indicates that turmeric has Kranggan (II) Market, Yogyakarta (sample code:
the highest antioxidant activity compared to other 2) and Sidodadi (II) Market, Central Java (sample
turmerics in this study. TPC and TFC in each code: 13) have the highest level of similarity
market are different because there are various based on the Euclidean distance between two
factors that can affect the content of secondary samples, 5,1324 (Figure 7). The closer the
metabolites in a plant, which is temperature, Euclidean distance is, the more similar the two
atmospheric environment (CO2, O2, and samples based on the variable used.
humidity), age of harvest, soil nutrients,
fertilization, and genotype of the plant (Nitisapto

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 Abdul Rohman et al / Principal Component Analysis of Antioxidant Activities, Total Phenolic contents,
and Total Flavonoid contents of Turmeric (Curcuma longa L.)

CONCLUSION to genotype, environment, and ontogeny. J.

All of the turmeric samples shows very strong Chem. Ecol. 31: 697–717. doi:10.1007/s10886-
antioxidant activity (<50 ppm). PCA was not 005-3539-7
successful in terms of grouping samples because 10. Miller, J.C., Miller, J.N., 2005. Chemometrics for
there is no correlation between the variables. Analytical, Fifth edit. ed. Pearson Education
Therefore, CA successfully divided the turmeric Limited, Essex, England.
samples into 6 groups according to phenolic 11. Nica Dewi, P., Hartiati, A., Mulyani, S., 2016.
contents, flavonoid contents, and antioxidant Influence of Harvest Age and Maceration Rate on
activity. the Content of Curcumin and Antioxidant
Activity of Turmeric Extract (Curcuma
Conflict of Interest: We have no conflict of Domestica Val.). J. Rekayasa Dan Manaj.
interest during this study. Agroindustri 4: 105–115.
12. Pękal, A., Pyrzynska, K., 2014. Evaluation of
ACKNOWLEDGEMENT Aluminium Complexation Reaction for Flavonoid
We thank to the Ministry of Research and Content Assay. Food Anal. Methods 7: 1776–
technology scheme World Class Research 2020 1782. doi:10.1007/s12161-014-9814-x
with contract number of 869/UN1/DITLIT/DIT- 13. Radwan, M.M., Tabanca, N., Wedge, D.E.,
LIT/PT/2020. Tarawneh, A.H., Cutler, S.J., 2014. Antifungal
Compounds from Turmeric and Nutmeg with
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and Total Flavonoid contents of Turmeric (Curcuma longa L.)

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Fig.1: Score plot of PCA : 1 = Kranggan(I); 2 = Kranggan(II); 3 = Wonosari; 4 = Mantingan; 5 =

Sumberpucung; 6 = Pasar Desa Sambi; 7 = Garum; 8 = Bendo, Tugurante; 9 = Kraguman; 10 =
Delanggu; 11 = Kartasura (Batu); 12 = Sidodadi (I); 13 = Sidodadi(II); 14 = Sidodadi(III)

Fig.2: Loading plot of principal component analysis (PCA) for classification of turmeric samples
from different regions

Fig.3: The scatter plot for the correlation between IC50 (x-axis) and total flavonoid contents (y-
axis)

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 Abdul Rohman et al / Principal Component Analysis of Antioxidant Activities, Total Phenolic contents,
and Total Flavonoid contents of Turmeric (Curcuma longa L.)

Fig.4: The scatter plot for the correlation between IC50 (x-axis) and total phenolics content (y-
axis).

Fig.5: The scatter plot for the correlation between total phenolics content (x-axis) and Total
flavonoid contents (y-axis)

Fig.6: Dendogram of cluster analysis; 1 = Kranggan(I); 2 = Kranggan(II); 3 = Wonosari; 4 =

Mantingan; 5 = Sumberpucung; 6 = Pasar Desa Sambi; 7 = Garum; 8 = Bendo, Tugurante; 9 =
Kraguman; 10 = Delanggu; 11 = Kartasura (Batu); 12 = Sidodadi (I); 13 = Sidodadi(II); 14 =
Sidodadi(III)

Fig.7: Amalgamation Steps during cluster analysis of turmeric samples

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 Abdul Rohman et al / Principal Component Analysis of Antioxidant Activities, Total Phenolic contents,
and Total Flavonoid contents of Turmeric (Curcuma longa L.)

Table 1: Total phenolics contents (TPC) of methanolic extracts of turmeric samples

Code Sample Province TPC ± SD (mg GAE/g DW)
1 Kranggan (I) Special District of Yogyakarta 183.01 ± 6.757
2 Kranggan (II) Special District of Yogyakarta 184.702 ± 21.851
3 Wonosari Special District of Yogyakarta 184.557 ± 23.642
4 Mantingan East Java 203.416 ± 29.051
5 Sumberpucung East Java 209.673 ± 40.483
6 Pasa Desa Sambi, Kediri East Java 164.943 ± 15.173
7 Garum East Java 175.562 ± 9.909
8 Bendo, Tugurante East Java 171.023 ± 3.019
9 Kraguman Central Java 197.987 ± 16.525
10 Delanggu Central Java 183.804 ± 17.383
11 Kartasura (Batu) Central Java 177.9 ± 24.578
12 Sidodadi Kleco (I) Central Java 188.556 ± 14.804
13 Sidodadi Kleco (II) Central Java 184.747 ± 7.357
14 Sidodadi Kleco (III) Central Java 176.644 ± 10.979
Table 2: Total flavonoid contents (TFC) of methanolic extracts of turmeric samples
Code Sample Province TFC ± SD (mg RE/g DW)
1 Kranggan (I) Special District of Yogyakarta 542.576 ± 43.288
2 Kranggan (II) Special District of Yogyakarta 510.509 ± 61.093
3 Wonosari Special District of Yogyakarta 609.520 ± 49.674
4 Mantingan East Java 581.157 ± 120.755
5 Sumberpucung East Java 497.994 ± 58.977
6 Pasa Desa Sambi, Kediri East Java 493.456 ± 14.485
7 Garum East Java 463.448 ± 52.125
8 Bendo, Tugurante East Java 498.562 ± 22
9 Kraguman Central Java 504.101 ± 21.33
10 Delanggu Central Java 513.514 ± 12.347
11 Kartasura (Batu) Central Java 553.415 ± 7.222
12 Sidodadi Kleco (I) Central Java 507.758 ± 53.793
13 Sidodadi Kleco (II) Central Java 506.15 ± 6.847
14 Sidodadi Kleco (III) Central Java 616.473 ± 76.81
Table 3: IC50 value of DPPH radical scavenging of methanolic extracts turmeric samples
Code Sample Province IC50 ± SD (µg/mL)
1 Kranggan (I) Special District of Yogyakarta 22.132 ± 1.010
2 Kranggan (II) Special District of Yogyakarta 25.359 ± 1.440
3 Wonosari Special District of Yogyakarta 22.117 ± 1.037
4 Mantingan East Java 21.496 ± 1.414
5 Sumberpucung East Java 22.1 ± 1.019
6 Pasa Desa Sambi, Kediri East Java 20.499 ± 1.039
7 Garum East Java 19.537 ± 1.747
8 Bendo, Tugurante East Java 24.723 ± 0.603
9 Kraguman Central Java 17.716 ± 0.892
10 Delanggu Central Java 19.149 ± 0.936
11 Kartasura (Batu) Central Java 18.467 ± 1.512
12 Sidodadi Kleco (I) Central Java 18.095 ± 0.130
13 Sidodadi Kleco (II) Central Java 22.650 ± 1.710
14 Sidodadi Kleco (III) Central Java 20.670 ± 1.062

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