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Arabian Journal of Chemistry (2023) 16, 105003

King Saud University

Arabian Journal of Chemistry


www.ksu.edu.sa
www.sciencedirect.com

ORIGINAL ARTICLE

Micro-titer plate assay for measurement of total


phenolic and total flavonoid contents in medicinal
plant extracts
Kurnia Rahayu Purnomo Sari a,b, Zullies Ikawati c, Retno Danarti d,
Triana Hertiani e,*

a
Pharmaceutical Sciences Doctoral Study Program, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia
b
Faculty of Health, Universitas Jenderal Achmad Yani, Yogyakarta 55294, Indonesia
c
Department of Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta
55281, Indonesia
d
Department of Dermatology and Venereology, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah
Mada, Yogyakarta 55281, Indonesia
e
Department of Pharmaceutical Biology, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia

Received 28 February 2023; accepted 11 May 2023


Available online 18 May 2023

KEYWORDS Abstract Total phenolic content (TPC) measured by using the Folin-Ciocalteu method and Total
Microtiter plate assay; flavonoid content (TFC) based on a complex formation with AlCl3 assay are widely applied as part
Total Phenolic Content; of herbal extract quality control. Conducting the assay measurements in microtiter plates may save
Total Flavonoid Content; resources and time. The objective of this study was to evaluate the validity of the micro-titer plates
Medicinal plant extracts assay to determine the TFC and TPC in several medicinal plants’ extracts. To compare the devel-
oped TPC and TFC methods to the conventional assays, the confidence intervals of linear regres-
sion were used with a significance set as p < 0.05. Both of the standards showed good linearity in
the range of 10–60 mg L1 of gallic acid and 40–200 mg L1 of quercetin, (R2 > 0.999). Based on
the micro-titer plate assay, the limits of detection (LODs), and quantification (LOQs) for TPC ran-
ged between 1.19 mg L1 and 3.98 mg L1 whereas for TFC, they were 1.47 mg L1 and 4.90 mg
L1. The percent coefficient of variation (%CV) of the intra-day and inter-day assays were lower
than 1.46% and 1.91%, respectively, showing adequate precision. The recovery ranged between
101.25% and 105.93% for the TPC assay and between 96.78% and 101.28% for the TFC assay.
Twelve samples of medicinal extracts were analyzed by validated microplate assay for TPC and
TFC and then compared with the conventional method. Notably, there were no significant differ-
ences in the TPC and TFC content detected between the microplate and conventional methods for

* Correspondence author at: Department of Pharmaceutical Biology, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta 55281,
Indonesia.
E-mail address: hertiani@ugm.ac.id (T. Hertiani).
https://doi.org/10.1016/j.arabjc.2023.105003
1878-5352 Ó 2023 The Author(s). Published by Elsevier B.V. on behalf of King Saud University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
2 K.R.P. Sari et al.

all samples (p > 0.05). In conclusion, the microplate assay can be potentially used for TPC and
TFC determinations.
Ó 2023 The Author(s). Published by Elsevier B.V. on behalf of King Saud University. This is an open
access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction 2. Materials and methods

Medicinal plant extracts are widely used as active ingredients in herbal- 2.1. Materials
based products. They contain metabolites, which differ in role and
function. The unique and specific composition and chemical properties
of each medicinal plant extract make them have high nutritional and Chromolaena odorata L (Kirinyu or Slam Weed) (leaves, roots,
medicinal values. A wide array of pharmacological activities were stems), Centella asiatica (Pegagan or Pennywort) leaves, Plan-
reported such as antibiotic (Oh & Jeon, 2015; Sanhueza et al., 2017), tago major L (Daun Sendok or Broadleaves Plantain) leaves,
antifungal (Ansari et al., 2013; Zabka & Pavela, 2013), antioxidant Chrysanthemum morifolium var. sheena and Chrysanthemum
(Nayak et al., 2015; Olas, 2018), anti-inflammatory (Ambriz-Pérez morifolium var. lamet (Krisan or Florist Daisy) flowers, Coleus
et al., 2016), anticancer (Roleira et al., 2015), and antiviral properties amboinicus Lour (Torbagun or Indian Borage) leaves, Brassica
and hence have applications in pharmaceutical, food, cosmetics, and oleraceae L.var. sabauda L. (Kubis Ungu or Purple Cabbage)
fine chemical industries (Balmus et al., 2016; Manach et al., 2004;
flowers, Brassica oleraceae L. var. capitata forma alba DC
Rajput et al., 2017; Tungmunnithum et al., 2018). Recent studies have
(Kubis Putih or White Cabbage) flowers, Folin-Ciocalteu
investigated that the antioxidant effects of plant products are mainly
attributed to phenolic compounds (Tungmunnithum et al., 2018; Xu (Merck chemicals), Sodium carbonate anhydrous (Merck
et al., 2017). chemicals), Methanol (Merck chemicals), aluminum chloride
There are more than 10,000 phenolic compounds that have been (AlCl3), quercetin (Sigma MCLS), gallic acid (Sigma MCLS),
isolated from plants so far. In general, phenolic metabolites are divided deionized water (WateroneÒ), Sodium acetate (Merck chemi-
into seven groups: coumarins, flavonoids, phenolic acids, lignans, lig- cals), and ethanol 70% (Genera Labora/local).
nin, stilbenes, and tannins (Huang et al., 2009). Many reports refer
to the synergistic effects of those groups of compounds in exerting 2.2. Instruments
pharmacological effects. Therefore, the determination of total phenolic
content (TPC) and total flavonoid content (TFC) in a medical plant
extract continues to be a valuable tool for the assessment of quality. An ultrasonic chamber (Cole Palmer) was used for the extrac-
Currently, the most common method used to measure the TPC of tion process of some samples, and the rotary evaporator (IKA-
all types of herbal samples is the Folin–Ciocalteu method. This partic- RV80) was used to concentrate the macerates. An ultraviolet
ular method is based on the reduction of the phosphomolybdate het- (UV)-Vis spectrophotometer (Thermo, Genesys 10S) was used
eropoly acids Mo(VI) center in the heteropoly complex to Mo(V), for the TPC and TFC measurement by the conventional
producing a blue coloration which is measured at around 750 nm method, and a microplate reader (Microlab 300) was used
(Bobo-Garcı́a et al., 2015). On the other hand, the TFC in plant for the determination by the microplate assay.
extracts is widely measured using an aluminum chloride colorimetric
assay. The method is based on the chelate formation of Al(III)-
flavonoids. The numerous oxo and hydroxyl groups contained in this
2.3. Sample preparation and extraction protocol
group’s compounds, contribute a great affinity of flavonoids to bind
metal ions such as Al(III), predominantly in a 1:1 ratio, depending Chromolaena odorata L, Centella asiatica, Chrysanthemum
on experimental conditions including pH value (Kasprzak et al., morifolium var. sheena and Chrysanthemum morifolium var.
2015; Pyrzynska & Pez kal, 2011; Shraim et al., 2021). lamet flowers were collected from Sleman, Yogyakarta,
The conventional methods for measuring the TPC and TFC are Indonesia. Brassica oleraceae L.var. sabauda L. flowers dan
time-consuming, labor-intensive, and use large quantities of reagents Brassica oleraceae L. var. capitata forma alba DC flowers were
(Johnson et al., 2022). The development of a valid high-throughput collected from Magelang, Jawa Tengah, Indonesia. Plantago
assay will be a valuable tool to overcome these downsides. In the case
major L and Coleus amboinicus Lour leaves were collected
of exploration for potential plant extracts as medicinal resources,
where a determination of TPC and TFC are needed as preliminary
from Bogor, Jawa Barat, Indonesia. All samples were rendered
screening steps, the filtrate extracted could be limited. Therefore, the and dried at the temperature of 50 °C.
development of this method is needed. Each dried sample was powdered to a particle size of 40
The assays in microplates have been reported with positive results mesh. Each sample was extracted with the extraction method
on a wide variety of samples such as apple, green tea, and grape seed as described in Table 1. Filtrates of each sample were rotary
extract for TPC and 2,2-diphenyl-1-picryl-hydrazl-hydrate (DPPH) evaporated to produced concentrated extracts and stored at
activity assay (Bobo-Garcı́a et al., 2015); sorghum for TPC and TFC 4 °C until analysis was performed. For stock solution, each
(Herald et al., 2012), seaweeds for TPC (Zhang et al., 2006), ginger extract was diluted in methanol and was sonicated to enhance
for TPC (Johnson et al., 2022), berries for TPC, TFC, and antioxidant the solubility of the phytochemical compound.
capacity (Horszwald & Andlauer, 2011) and Cyphostemma digitatum
for TPC and antioxidant capacity (Al-Duais et al., 2009). However,
no statistical comparison between the results obtained with the micro-
2.4. Conventional assay for total phenolic content (TPC)
plate and the conventional methods has been reported. In light of this
background, this study aimed to develop and validate the application The TPC is determined based on the reaction with the Folin–
of the Folin–Ciocalteu and AlCl3 micro-titer plate assays in compar- Ciocalteu reagent (Ribarova et al., 2005). In a 5 mL volumetric
ison to the conventional methods, and to implement the method in flask, 0.1 mL of the diluted extract and 0.1 mL of the Folin–
the determination of TPC and TFC of several plant extracts. Ciocalteu reagent was added and left to react for 5 min. To
Micro-titer plate assay for measurement of total phenolic and total flavonoid contents 3

Table 1 Sample identity and extraction process.


Sample Sample name Part of Extraction Extraction method
Code plants Solvent
CoL Chromolaena odorata L Leaves Ethanol 70% Maceration 1:8, 3 days, room
temperature
CoR Chromolaena odorata L Roots Ethanol 70% Maceration 1:8, 3 days, room
temperature
CoS Chromolaena odorata L Stems Ethanol 70% Maceration 1:8, 3 days, room
temperature
CaLE70 Centella asiatica Leaves Ethanol 70% UAE 1:10, 45 °C, 20 min
CaLE96 Centella asiatica Leaves Ethanol 96% UAE 1:10, 50 °C, 20 min
CaLEA Centella asiatica Leaves Ethyl acetate UAE 1:10, 50 °C, 20 min
PMLE50 Plantago major L Leaves Ethanol 10% UAE 1: 15, 50 °C, 40 min
CmS Chrysanthemum morifolium var. sheena Flowers Ethanol 70% Maceration 1:10, 3 days, room
temperature
CmL Chrysanthemum morifolium var. lamet Flowers Ethanol 70% Maceration 1:10, 3 days, room
temperature
CaL Coleus amboinicus Lour. Leaves Ethanol 70% Maceration 1:10, 3 days, room
temperature
BoS Brassica oleraceae L. var. sabauda L. Flowers Ethanol 70% Maceration 1:10, 3 days, room
temperature
BoA Brassica oleraceae L. var. capitata forma alba Flowers Ethanol 70% Maceration 1:10, 3 days, room
DC temperature
UAE, ultrasound-assisted extraction.

complete the reaction, 1 mL of a 7% sodium carbonate solu- 200 lL of a 10% AlCl3 solution. After incubating for 3 min
tion was added, and the volumetric flask was filled to its vol- at room temperature, 200 lL of a 1 M CH3COONa was added
ume with deionized water. After 120 min in the darkness at to the mixture. The final volume was adjusted to 5 mL with
room temperature, the absorbance of the samples at 750 nm methanol. After 40 min of incubation in the darkness at room
was measured in the UV–Vis spectrophotometer. The mea- temperature, the absorbance of this preparation was measured
sured absorbance of the same reaction with water instead of at 430 nm. A calibration curve was created in parallel under
the extract or standard was subtracted from the absorbance the same operating conditions using quercetin. Flavonoid con-
of the reaction with the sample. The phenolic content was tents are expressed in milligram equivalent to quercetin per
expressed in gallic acid equivalents (GAE) per gram after the gram of dry extract (mg QE/g).
preparation of a standard curve of gallic acid from 10 to
60 mg L1. 2.7. Microplate assay for TFC

2.5. Microplate assay for TPC To microplate, a TFC assay was conducted by applying the
aforementioned TFC method with some modifications. A total
The microplate TPC method was conducted by applying the of 50 lL of the sample extract or standard solution and 100 mL
aforementioned Folin–Ciocalteu methods with some modifica- methanol were added to each well. One well on the plate was
tions. A total of 20 lL of the diluted extract were mixed with used as a blank for the microplate absorbance reading and
20 lL Folin–Ciocalteu reagent and shaken for 1 min. The mix- filled up with 150 lL methanol. Following the addition of
ture was left for 5 min and then 200 lL of 7% sodium carbon- 20 lL AlCl3 10% to each well, the plate was gently shaken five
ate solution and 10 lL deionized water were added and the times each up and down and left to right in the horizontal
mixture was shaken at medium-continuous speed for 1 min. plane. The plate was kept in incubation for three minutes.
After 120 min in the darkness at room temperature, the absor- After the incubation period, 20 mL CH3COONa 1 M the added
bance was measured at 750 nm using the microplate reader. and followed by 60 lL Methanol. The plate was then incu-
The absorbance of the same reaction with water instead of bated in the darkness at room temperature for 40 min and then
the extract or standard was subtracted from the absorbance the absorbance was measured at 430 nm using a microplate
of the reaction with the sample. Gallic acid dilutions (10– reader.
60 mg L1) were used as standards for calibration. Phenolic
contents are expressed in milligram equivalent of gallic acid 2.8. Method validation
per gram of dry extract (mg GAE/g).
A validation study was conducted to demonstrate the applica-
2.6. Conventional assay for total flavonoid content (TFC) bility of this analytical approach to assess the medicinal plant
extracts’ quality. Validation comprised the assessment of speci-
The TFC is determined based on the reaction with the alu- ficity/selectivity, linearity, recovery, precision, the limits of
minum trichloride reagent (Shraim et al., 2021). 500 lL of detection (LODs), and quantification (LOQs). To evaluate
the diluted extract was mixed with 2 mL methanol and the linearity of the method, the calibration curves were plotted
4 K.R.P. Sari et al.

by absorbance versus concentration of each standard. To pre- LOQ for the TPC microplate method were 1.19 and 3.98 mg
pare the standard solutions, gallic acid (10, 20, 30, 40, 50, and L1 GAE, respectively. In the case of the TFC microplate
60 mg L1) and quercetin (40, 80, 120, 160, and 200 mg L1) method, the LOD and LOQ were 1.47 and 4.90 mg L1 of
were dissolved in methanol. The linear regression equations quercetin equivalents, respectively (Table 2). The LOQ and
were calculated as y = ax ± b, where  was concentration LOQ both for TPC and TFC of the conventional method
and y was the absorbance of each standard. The acceptance are lower than the LOD and LOQ of the microplate method.
criteria for linearity are that the correlation coefficient (R2) This can demonstrate that the conventional method is more
should not be <0.995 over the working range of 80–120% sensitive and accurate than the microplate assay.
(Food and Drug Administration, 2020). The LODs and LOQs
were determined based on the standard deviation (SD) of the 3.2. Accuracy and precision
response and the slope, using the calibration curve data, and
calculated based on the SD of the intercept with y and the Accuracy was calculated and expressed as % recovery (%
slope of the calibration curve (S) according to the Eqs. (1) REC), which is the difference between the calculated value
and (2). of the standard obtained after running the microplate assay
LOD ¼ SD  3=S ð1Þ and the actual value of the prepared standard, expressed as a
percentage. The %REC values ranged from 101.30 to
LOQ ¼ SD  10=S ð2Þ 105.93% for the three concentrations of gallic acid and 96.78
to 101.28% for the concentration of quercetin tested, suggest-
Accuracy was evaluated by determining the method of
ing that the microplate assays can be reproduced with excellent
recovery. The mean percentage of recovery should be within
accuracy (Table 3). Similarly, the %CV values were <5% for
the following ranges. The precision of the intra- and inter-
all three concentrations tested, which is considered good preci-
day was evaluated by the repeated assay. The intra-day exper-
sion. The %CV values for inter-day variability and intra-day
iment was obtained by three plates for a single day, and the
reproducibility were 1.46% and 1.91%, respectively for gallic
inter-day was determined for three consecutive days. Analysis
acid. The %CV values for inter-day variability and intra-day
was performed in triplicate. The precision was expressed as the
reproducibility for quercetin were 1.48% and 0.89%, respec-
percent coefficient of variation (%CV) that calculated from
tively (Table 4).
SD compare to the Mean values of four repetitions of the anal-
ysis in each concentration.
3.3. Application of the validated microplate assay of TPC on
various medicinal plant extracts
2.9. Statistical analyses

A total of 12 extracts were analyzed in four replicates using the


Statistical comparisons of TPC and TFC between conven-
optimized microplate TPC method and then compared to the
tional and microtiter methods were performed based on
conventional method. The average coefficient of variation on
Levene’s test value for equality of variance and t-test value
each extract was a range of 0.34–2.29% which is considered
for equality of means of each sample extract. Both methods
good in precision. In comparison, the %CV obtained using
are considered statistically equal with a 95% confidence inter-
the conventional TPC method is approximately 0.75–2.43%.
val (CI), while the p-values of Levene’s test and t-test have to
Table 5(a) shows that the TPC levels of each extract sample
be >0.05. This statistical analysis was performed using Mini-
varied. The total phenolic of sample extract determined using
tab 17 Statistical Software.
microplate assay ranged from 8.48 to 28.72 mg/g GAE. The
highest TPC was obtained in Centella asiatica leaves which
3. Results and discussion were extracted using 70% ethanol and the lowest TPC was
in Brassica oleraceae L. var. capitata f. alba DC extract. The
3.1. Calibration curves and limits of detection and quantification TPC of each plant part shows varying values, for example in
Chromolaena odorata L, the part of the plant that contains
The standard calibration curves for the microplate methods the highest TPC is the leaf part while the root part contains
are presented in Fig. 1. The curves are linear when the concen- the smallest TPC. In addition, it can be seen that the solvent
tration of gallic acid is in the range of 10–60 mg L1 used during plant extraction can also make a difference in
(R2 = 0.999) and the concentration of quercetin is in the range the TPC levels of the samples. It can be seen in Centella asiat-
of 40–200 mg L1 (R2 = 0.999). When comparing these curves ica, the 70% ethanol solvent gave the highest TPC value fol-
with those obtained by conventional methods, it can be seen lowed by 96% ethanol and then ethyl acetate. This can be
that the slight changes in the methodology had more influence possible due to the better solubility of phenolic compounds
on the slope of the TPC assay than on the slope of the TFC in polar solvents.
assay. The slope of the calibration curve for the TFC using To compare the TPC conventional and microplate meth-
the microplate method was 0.005, while the slope in the con- ods, the result from both methods were then statistically ana-
ventional study was 0.005; for the calibration curves of the lyzed using SPSS 23 (IBM Corp., Armonk, NY). Table 5(a)
TPC using the microplate and conventional method, the slopes shows the p-values of Levene’s test for equality of variances
were 0.013 and 0.022, respectively. However, comparing the and the p-value of the t-test for equality of means. In all cases,
slopes of this work, it can be seen that greater differences in the p-values for Levene’s test for equality of variance were
the TPC and TFC microplate methods had a significant impact >0.05, which indicates that the variances among the measure-
on the sensitivity of the methods. In this work, the LOD and ments for each sample (n = 4) were not significantly different
Micro-titer plate assay for measurement of total phenolic and total flavonoid contents 5

Fig. 1 Representative standard curve for 96-well assay: (a) total phenolic content, and (b) total flavonoid content.

Table 2 Comparison of calibration results from conventional assay and microplate assay, both at room temperature.
Slope Intercept Pearson R2 LOD (mg/L) LOQ (mg/L)
TPC
Conventional assay 0.022 0.083 0.990 0.58 1.93
Microplate assay 0.014 0.05 0.999 1.19 3.98
TFC
Conventional assay 0.005 0.049 0.992 0.80 2.66
Microplate assay 0.005 0.043 0.999 1.47 4.90
LOD, limits of detection; LOQ, limits of quantification; TPC, total phenolic content; TFC, total flavonoid content.

at 95% CI. The lack of significant differences in the variance All the p-values for the p-value of the t-test for equality of
indicates that both methods are equally precise and allow the means were >0.05, which indicates that there were no signifi-
performance of the p-value of the t-test for equality of means. cant differences between the mean values for both methods at
6 K.R.P. Sari et al.

the p-value from all samples is above 0.05. This result indicated
Table 3 Accuracy of 96-well assays in determining total
that the variances of all samples are equal for both methods.
phenolic and total flavonoid contents using gallic acid and
Furthermore, the p-value from the t-test analysis shows that
quercetin standards.
in all samples for both methods are >0.05. So, it can be con-
Assay Std 1 Std 2 Std 3 cluded that there were no significant differences between the
1 1
Total phenolics 20 mg mL 30 mg mL 40 mg mL1 microplate and conventional method to determine TFC from
Calculated (mg 21.19 ± 0.27 31.28 ± 0.23 40.52 ± 0.20 the sample extracts.
mL1)
%REC 105.93 104.26 101.30 3.5. General observations
%CV 1.12 0.66 0.44
Total flavonoid 80 mg mL1 120 mg mL1 160 mg mL1
Calculated (mg 77.42 ± 1.38 121.46 ± 2.82 156.04 ± 2.45 The microplate assay uses fewer resources and provides a rapid
mL1) measurement in comparison to conventional assays. The
%REC 96.78 101.28 97.52 microplate assay reduced the time and manpower needed to
%CV 1.60 2.07 1.40 transfer the reactant solutions to the cuvettes to be manually
Values are expressed as mean ± standard deviation of five repli- read in the spectrophotometer and allowed for more replicates
cates. Gallic acid was used for total phenolic content and quercetin to be run on the same extracts than was possible with the con-
was used for total flavonoid content; %REC, percentage recovery; ventional methods. The need for numerous cuvettes, transfer
%CV, percentage. pipettes, and test tubes was eliminated. Instead, the only con-
sumables used were pipette tips and 96-well plates. The
amounts of the reagent required also decreased. In the conven-
tional TPC assay, the total volume used of FC reagents is
100 mL per reaction, whereas only 20 mL of FC reagent was
a 95% CI. Based on the p-values resulting from both statistical required per reaction for the microplate assay. In the TFC
tests, it can be said that the conventional and the microplate assay, the AlCl3 used for the microplate assay was only
methods were equivalent in the concentration ranges studied. 20 mL per reaction compared to conventional assay that used
200 lL AlCl3 per reaction.
3.4. Application of the validated microplate assay of TFC assay The gallic acid and quercetin absorbances of the two meth-
on various medicinal plants extracts ods showed a correlation with R2 = 0.9987 and R2 = 0.9964,
respectively. This linearity was slightly higher for the micro-
The TFC determination by using the optimized microplates plate methods than for the conventional methods, even though
assay was also performed on 12 different extracts. The results all %CV < 5% and indicated good reproducibility of the
of the TFC analysis of both microplate and conventional method when applied to real sample extracts. However, the
methods are summarized in Table 5(b). The %CV range for savings on time, the amounts of samples that can be run per
the conventional assay was 0.29–1.89% and 0.33–2.56% for day, and the reduced amount of solvent due to the use of the
the microplate assay. It indicated that the determination of microplate assays more than compensated for the slightly
TFC using the microplate method for all sample extracts is higher variability especially for detection of lots sample
considered to have good precision. The TFC of the extract variant.
ranges from 2.37 mg/g quercetin equivalents (QE), which is Previous studies using microplates for the determination of
found in Chromolaena odorata L roots extract, to 21.59 mg/g TPC in grape seed apple and green tea extracts by Gloria in
QE which is found in Chromolaena odorata L leaves extract. 2015 showed that the results of the microtiter and conventional
The diversity of TFC levels from each sample can be seen in assay methods were linearly correlated. (Bobo-Garcı́a et al.,
Table 5 (b). In Chromolaena odorata L, every part of the plant 2015). Herald’s research in 2012 highlighted that the results
that contains various TFC content with the highest content is of TPC determination using a microplate showed good preci-
the leaf part while the smallest TFC is in the root part. In TFC, sion with a %CV value <10% on various test samples in
the solvent used during plant extraction can also make a differ- the form of flour and brans (Herald et al., 2012). The research
ence in the samples. As can be seen in Centella asiatica, the conducted by Johnson in 2022 on TPC determination also
highest TFC was showed in 70% ethanol solvent, followed obtained validated method results with good reproducibility,
by 96% ethanol and then ethyl acetate. with CV < 5% (Johnson et al., 2022).
The statistical analysis conducted for both conventional However, several aspects need to be considered in this
and microplate methods for TFC determination shows that microtiter test to minimize some errors in the results such as

Table 4 Reproducibility of 96-well assays in determining total phenolic and total flavonoid contents using gallic acid and quercetin
standard.
Assay Inter-day variability Intra-day variability
Mean %CV Mean %CV
Total phenolic (GAE) 54.07 ± 0.79 1.46 51.37 ± 1.10 1.91
Total flavonoids (QE) 111.469 ± 1.69 1.48 106.19 ± 1.06 0.89
%CV, percent coefficient of variation; GAE, gallic acid equivalent; QE, quercetin equivalent.
Micro-titer plate assay for measurement of total phenolic and total flavonoid contents 7

Table 5 Comparison of microplate assay and conventional methods in the determination of total phenolic content and total flavonoid
content in sample extracts.
Sample 96-well assay Conventional method Levene’s test t-test
Value ± SD %CV Value ± SD %CV p-value p-value
Total phenolic content (mg/g GAE)
CoL 25.66 ± 0.28 0.93 25.49 ± 0.22 0.75 0.99 0.38
CoS 13.31 ± 0.05 0.34 13.11 ± 0.18 1.22 0.13 0.07
CoR 10.96 ± 0.23 1.82 10.58 ± 0.25 2.02 0.90 0.08
CaLE70 28.72 ± 0.76 2.29 29.08 ± 0.39 1.16 0.14 0.43
CaLE96 26.31 ± 0.46 2.23 26.72 ± 0.75 2.43 0.14 0.38
CaLEA 18.56 ± 0.15 0.70 18.41 ± 0.27 1.27 0.17 0.36
PmLE50 25.79 ± 0.51 1.70 26.46 ± 0.51 1.66 0.97 0.11
CmS 11.07 ± 0.18 1.43 10.83 ± 0.16 1.31 0.91 0.10
CmL 12.32 ± 0.13 0.92 12.07 ± 0.20 1.43 0.23 0.08
CaL 15.64 ± 0.25 1.38 15.88 ± 0.33 1.79 0.58 0.29
BoS 10.626 ± 0.12 0.96 10.83 ± 0.15 1.16 0.71 0.06
BoA 8.48 ± 0.18 1.85 8.75 ± 0.14 1.41 0.65 0.06
Total flavonoid content (mg/g QE)
CoL 21.59 ± 0.08 0.33 21.68 ± 0.30 1.22 0.22 0.58
CoS 3.59 ± 0.08 1.87 3.67 ± 0.03 0.74 0.06 0.09
CoR 2.37 ± 0.04 1.28 2.44 ± 0.05 1.81 0.55 0.07
CaLE70 16.13 ± 0.19 1.03 16.37 ± 0.15 0.81 0.48 0.09
CaLE96 15.89 ± 0.26 1.39 15.76 ± 0.22 1.23 0.80 0.46
CaLEA 10.27 ± 0.19 1.64 10.54 ± 0.15 1.26 0.62 0.07
PMLE50 12.55 ± 0.24 1.67 12.68 ± 0.21 1.45 0.52 0.45
CmS 4.12 ± 0.04 0.87 4.09 ± 0.06 1.28 0.22 0.40
CmL 5.66 ± 0.01 1.52 5.53 ± 0.12 1.89 0.72 0.15
CaL 11.24 ± 0.33 2.56 11.49 ± 0.14 1.03 0.10 0.21
BoS 2.97 ± 0.03 0.77 2.95 ± 0.01 0.29 0.20 0.22
BoA 4.00 ± 0.11 2.38 4.10 ± 0.05 1.13 0.37 0.17

the accuracy or pipetting technique of the researcher and the This micro-titter plate method can be applied at the initial
solubility of the sample in the solvent. If the extract or sample screening process stage for the exploration of efficacious natu-
is partly insoluble in the solvent used, it will cause unreliable ral plants, for example, in the process of extraction optimiza-
results because the particles in the sample can interfere with tion which could obtain an extract with limited yield. In this
the intensity reading on the microplate reader causing it to case, this method will be useful because it can minimize the
become unstable and even higher, and this also applies in the resources needed.
determination with conventional methods. Therefore, the
selection of the sample solvent is a critical point in this test.
4. Conclusions
Determination of total flavonoids with AlCl3 reagent will
react with the C4 keto group and between the C3 or C5 OH
The microplate method developed in this study showed several ben-
groups of flavonoids. In addition, aluminum chloride can also
efits not only can handle large number of samples in one experi-
form complexes with ortho-dihydroxyl groups in the A or B ment but also reduces the amount of sample and reagent
rings of flavonoid compounds. The method for determining requirements for total phenolic and total flavonoid content. Fur-
total flavonoids using aluminum chloride reagent is more thermore, it showed acceptable repeatability and reproducibility.
specific for flavones and flavonols, so it is necessary to develop The repeatability, reproducibility, and percentage of recovery for
a microtiter assay method for determining total flavonoids the TPC and TFC microplate methods showed a precision below
using other methods, such as using the 2,4- 5% and accuracy between 96.78 and 105.93%. For the application
dinitrophenylhydrazine reagent, which is more stable for the for several extract, the microplate and the conventional methods
determination of flavanones (Chang et al., 2002). are equal based on statistical analysis at a 95% confidence level
These microplate assays can be used for routine screening of a
The extract samples used in this study are diverse to represent
large number of samples because it was demonstrated to be as
different types of plants both in terms of species but also plant
reproducible, efficient, accurate, and precise as the conventional
parts (roots, leaves, stems, to flowers). This is to get an idea of method for determining total phenolic content and total flavonoid
the breadth of application of this microtiter method when content in several extracts.
applied to herbal extracts which contain phytochemical com-
pounds with various physical and chemical properties. The con-
tent of these phytochemical compounds is of course also Declaration of Competing Interest
influenced by the type of raw material since when using colored
flower parts there will be various dyes, and in leaves containing The authors declare that they have no known competing
chlorophyll, or roots and stems containing lignin because these financial interests or personal relationships that could have
compounds may interfere with the analysis process. appeared to influence the work reported in this paper.
8 K.R.P. Sari et al.

Acknowledgments Kasprzak, M.M., Erxleben, A., Ochocki, J., 2015. Properties and
applications of flavonoid metal complexes. RSC Adv. 5 (57),
45853–45877. https://doi.org/10.1039/C5RA05069C.
The author is grateful to the Center for Education Financial
Manach, C., Scalbert, A., Morand, C., Rémésy, C., Jiménez, L., 2004.
Services and Indonesia Endowment Funds for Education for Polyphenols: food sources and bioavailability. Am. J. Clin. Nutr.
financial support as Doctoral Scholarship and the staff at Kli- 79 (5), 727–747. https://doi.org/10.1093/ajcn/79.5.727.
nik Bahasa, Office of Research and Publication, Faculty of Nayak, B., Liu, R.H., Tang, J., 2015. Effect of processing on phenolic
Medicine, Public Health, and Nursing, Universitas Gadjah antioxidants of fruits, vegetables, and grains—a review. Crit. Rev.
Mada, who kindly provided proofreading assistance. Food Sci. Nutr. 55 (7), 887–918. https://doi.org/10.1080/
10408398.2011.654142.
References Oh, E., Jeon, B., 2015. Synergistic anti-Campylobacter jejuni activity
of fluoroquinolone and macrolide antibiotics with phenolic com-
pounds. Front. Microbiol. 6, 1129.
Al-Duais, M., Müller, L., Böhm, V., Jetschke, G., 2009. Antioxidant
Olas, B., 2018. Berry phenolic antioxidants–implications for human
capacity and total phenolics of Cyphostemma digitatum before and
health? Front. Pharmacol. 9, 78. https://doi.org/10.3389/
after processing: use of different assays. Eur. Food Res. Technol.
fmicb.2015.01129.
228 (5), 813–821. https://doi.org/10.1007/s00217-008-0994-8.
Pyrzynska, K., Pez kal, A., 2011. Flavonoids as analytical reagents. Crit.
Ambriz-Pérez, D.L., Leyva-López, N., Gutierrez-Grijalva, E.P.,
Rev. Anal. Chem. 41 (4), 335–345. https://doi.org/10.1080/
Heredia, J.B., 2016. Phenolic compounds: Natural alternative in
10408347.2011.607077.
inflammation treatment. A Review. Cogent Food Agric. 2 (1),
Rajput, D.S., Dash, D.K., Sahu, A.K., Mishra, K., Kashyap P, & S, P.
1131412. https://doi.org/10.1080/23311932.2015.1131412.
M., 2017. Brief update on Indian herbs and spices used for diabetes
Ansari, M.A., Anurag, A., Fatima, Z., Hameed, S., 2013. Natural
in rural area of Chhattisgarh. In: International Journal of
phenolic compounds: a potential antifungal agent. Microbial
Pharmaceutical Chemistry and Analysis. academia.edu. https://
Pathogens Strateg. Combating Them: Sci. Technol. Educ. 1,
www.academia.edu/download/53241726/IJPCA_41_1-4.pdf.
1189–1195. https://doi.org/10.1155/2014/895193.
Ribarova, F., Atanassova, M., Marinova, D., Ribarova, F., Atanas-
Balmus, I.M., Ciobica, A., Trifan, A., Stanciu, C., 2016. The
sova, M., 2005. Total phenolics and flavonoids in Bulgarian fruits
implications of oxidative stress and antioxidant therapies in
and vegetables. JU Chem. Metal 40, 255–260.
Inflammatory Bowel Disease: Clinical aspects and animal models.
Roleira, F.M.F., Tavares-da-Silva, E.J., Varela, C.L., Costa, S.C.,
Saudi J. Gastroenterol.: Off. J. Saudi Gastroenterol. Assoc. 22 (1),
Silva, T., Garrido, J., Borges, F., 2015. Plant derived and dietary
3. https://doi.org/10.4103/1319-3767.173753.
phenolic antioxidants: Anticancer properties. Food Chem. 183,
Bobo-Garcı́a, G., Davidov-Pardo, G., Arroqui, C., Vı́rseda, P., Marı́n-
235–258. https://doi.org/10.1016/j.foodchem.2015.03.039.
Arroyo, M.R., Navarro, M., 2015. Intra-laboratory validation of
Sanhueza, L., Melo, R., Montero, R., Maisey, K., Mendoza, L.,
microplate methods for total phenolic content and antioxidant
Wilkens, M., 2017. Synergistic interactions between phenolic
activity on polyphenolic extracts, and comparison with conven-
compounds identified in grape pomace extract with antibiotics of
tional spectrophotometric methods. J. Sci. Food Agric. 95 (1), 204–
different classes against Staphylococcus aureus and Escherichia
209. https://doi.org/10.1002/jsfa.6706.
coli. PLoS One 12 (2), e0172273.
Chang, C.-C., Yang, M.-H., Wen, H.-M., Chern, J.-C., 2002.
Shraim, A.M., Ahmed, T.A., Rahman, M.M., Hijji, Y.M., 2021.
Estimation of total flavonoid content in propolis by two comple-
Determination of total flavonoid content by aluminum chloride
mentary colorimetric methods. J. Food Drug Anal. 10 (3). https://
assay: A critical evaluation. LWT 150,. https://doi.org/10.1016/j.
doi.org/10.38212/2224-6614.2748.
lwt.2021.111932 111932.
Food and Drug Administration, 2020. Methods, Method Verification
Tungmunnithum, D., Thongboonyou, A., Pholboon, A., Yangsabai,
and Validation II, 1–32.
A., 2018. Flavonoids and other phenolic compounds from medic-
Herald, T.J., Gadgil, P., Tilley, M., 2012. High-throughput micro plate
inal plants for pharmaceutical and medical aspects: An overview.
assays for screening flavonoid content and DPPH-scavenging
Medicines 5 (3), 93. https://doi.org/10.3390/medicines5030093.
activity in sorghum bran and flour. J. Sci. Food Agric. 92 (11),
Xu, D.-P., Li, Y., Meng, X., Zhou, T., Zhou, Y., Zheng, J., Zhang, J.-
2326–2331. https://doi.org/10.1002/jsfa.5633.
J., Li, H.-B., 2017. Natural antioxidants in foods and medicinal
Horszwald, A., Andlauer, W., 2011. Characterisation of bioactive
plants: Extraction, assessment and resources. Int. J. Mol. Sci. 18
compounds in berry juices by traditional photometric and modern
(1), 96. https://doi.org/10.3390/ijms18010096.
microplate methods. J. Berry Res. 1 (4), 189–199. https://doi.org/
Zabka, M., Pavela, R., 2013. Antifungal efficacy of some natural
10.3233/JBR-2011-020.
phenolic compounds against significant pathogenic and toxinogenic
Huang, W.-Y., Cai, Y.-Z., Zhang, Y., 2009. Natural phenolic
filamentous fungi. Chemosphere 93 (6), 1051–1056. https://doi.org/
compounds from medicinal herbs and dietary plants: potential
10.1016/j.chemosphere.2013.05.076.
use for cancer prevention. Nutr. Cancer 62 (1), 1–20. https://doi.
Zhang, Q., Zhang, J., Shen, J., Silva, A., Dennis, D.A., Barrow, C.J.,
org/10.1080/01635580903191585.
2006. A simple 96-well microplate method for estimation of total
Johnson, J.B., Mani, J.S., Naiker, M., 2022. Development and
polyphenol content in seaweeds. J. Appl. Phycol. 18 (3), 445–450.
Validation of a 96-Well Microplate Assay for the Measurement
https://doi.org/10.1007/s10811-006-9048-4.
of Total Phenolic Content in Ginger Extracts. In: Food Analytical
Methods (Vol. 15, Issue 2, pp. 413–420). Doi: 10.1007/s12161-021-
02127-9.

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