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New design of experiment combined with UV–Vis spectroscopy for extraction and estimation of polyphenols from Basil seeds, Red seeds, Sesame seeds and Ajwan seeds
Accepted Manuscript New design of experiment combined with UV–Vis spectroscopy for extraction and estimation of polyphenols from Basil seeds, Red seeds, Sesame seeds and Ajwan seeds
Fazal Mabood, Syed Abdullah Gilani, Javid Hussain, Sulaiman Alshidani, Said Alghawi, Mohammed Albroumi, Saif Alameri, Farah Jabeen, Zahid Hussain, Ahmed Al-Harrasi, Zahra K.M. Al Abri, Saima Farooq, Zakira Naureen, Ahmad Hamaed, M. Rasul Jan, Jasmin Shah PII: DOI: Reference:
S1386-1425(17)30072-0 doi: 10.1016/j.saa.2017.01.051 SAA 14904
To appear in:
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Received date: Revised date: Accepted date:
13 November 2016 3 January 2017 24 January 2017
Please cite this article as: Fazal Mabood, Syed Abdullah Gilani, Javid Hussain, Sulaiman Alshidani, Said Alghawi, Mohammed Albroumi, Saif Alameri, Farah Jabeen, Zahid Hussain, Ahmed Al-Harrasi, Zahra K.M. Al Abri, Saima Farooq, Zakira Naureen, Ahmad Hamaed, M. Rasul Jan, Jasmin Shah , New design of experiment combined with UV–Vis spectroscopy for extraction and estimation of polyphenols from Basil seeds, Red seeds, Sesame seeds and Ajwan seeds. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Saa(2017), doi: 10.1016/ j.saa.2017.01.051
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ACCEPTED MANUSCRIPT New Design of Experiment combined with UV-VIS Spectroscopy for Extraction and Estimation of Polyphenols from Basil Seeds, Red Seeds, Sesame Seeds and Ajwan Seeds
Fazal Mabood*a, Syed Abdullah Gilani*a, Javid Hussaina, Sulaiman Alshidania, Said Alghawia, Mohammed Albroumia, Saif Alameria, Farah Jabeenb, Zahid Hussainc, Ahmed Al-Harrasid,
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Zahra K. M. Al Abria, Saima Farooqa, Zakira Naureena, Ahmad Hamaeda, M.Rasul Jane, Jasmin
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Shahe,
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a) Department of Biological Sciences & Chemistry, College of Arts and Sciences, University of Nizwa, Sultanate of Oman, ([email protected],. [email protected])
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b) Department of Chemistry, University of Malakand, KPK, Pakistan. c) Department of Chemistry, Abdul Wali Khan University, KPK, Pakistan
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d) UoN Chair of Oman Medicinal Plants and Marine Products, University of Nizwa,
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Sultanate of Oman.
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e) Institute of Chemical Sciences, University of Peshawar, KPK, Pakistan
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Abstract
New experimental designs for the extraction of polyphenols from different seeds including basil Four variables the
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seed, red seed, Sesame seeds and ajwan seeds were investigated.
concentration and volume of methanol and NaOH solutions as well as the temperature and time
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of extraction were varied to see their effect on total phenol extraction. The temperature was varied in the range from 25ºC to 200ºC while the time in the range from 30 to 200 minutes. Response surface methodology was used to optimize the extraction parameters. The estimation of polyphenols was measured through phenols reduction UV-Vis spectroscopic method of phosphotungstic-phosphomolybdic acids (Folin-Ciocalteu’s reagent). Calibration curve was made by using tannic acid as a polyphenols standard in the concentration range from 0.1 to 10 ppm. The regression line obtained shows the value of correlation coefficient i.e. R =0.930 and Root mean square error of cross validation (RMSEC) value of 0.0654. The Basil seeds were found containing the highest amount of total phenols i.e. 785.76 mg/100g. While the Sesame
ACCEPTED MANUSCRIPT seeds having the least amount i.e. 33.08 mg/100g. The Ajwan seeds and the Red seeds are containing the medium amounts i.e. 379 mg/100g and 220.54 mg/100g respectively.
Keywords: Extraction of phenols, basil seed, red seed, Sesame seeds, ajwan seeds, Design of
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Experiment (DoE), UV-VIS spectroscopy
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1. Introduction
Plant polyphenols are secondary metabolites that may be extensively studied and used in
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different fields of studies such as chemotoxonomy, plant biochemistry, biogenesis etc. There are reports of more than 8,000 polyphenolic compounds, identified and isolated from plants sources
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(Pandey and Rizvi, 2009). These compounds have been reported from all plants parts such as roots, stem, leaves, flowers, fruits, and seeds.
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Plasma antioxidant capacity may be enhanced by using Polyphenol-rich foods (Pandey and Rizvi, 2009). Herbal teas in traditional medicines have been used to treat cardiovascular and
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cancer-related diseases (Khan et al., 2006). These herbal teas have also shown significant anticancer activities against human breast cancer cells (Li et al., 2016). Enormous reports on
available from seeds.
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studies of polyphenols from all plants parts have been reported, however, there are fewer reports
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The seeds are considered as rich sources for total phenol contents. Study of legume seeds such as chickpeas, field peas, faba beans, common vetch and lupins has shown that chickpeas had
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highest phenolic contents as compared to the other tested legumes (Magalhaes et al., 2017). Total phenolic contents of pigeon pea used in Oman for chronic diseases were tested for phenol contents (Al-Saeedi and Hossain, 2015). The results showed highest phenol contents in hexane extract followed by chloroform, methanol, butanol, ethylacetate, and water extracts. Alhakmani et al. (2014) studied phenol contents in seeds and fruits of Zizyphus spina-cristi in Oman which showed higher phenol contents in seeds than the fruits. Calibration model was applied using Fourier Transfer Near Infrared spectroscopy (FTNIR) in estimation of phenol contents in rapeseed-mustard genotypes (Bala and Singh, 2013).
ACCEPTED MANUSCRIPT Extraction and estimation of total phenol contents in Prunus dulcis seeds by warring blender method revealed that ethyl acetate fraction showed higher phenol contents and antioxidant acitivity (Dhingra et al., 2017). While comparing various methods of seeds oil extractions and fractions of Butia catarinensis seeds, Ethanol and ethanol-water by Soxhelt extraction exhibited higher total phenolic contents and antioxidant activities (Cruz et al., 2017). However, in another study,
soxhelt
extraction
method
was
not
significant,
where
Folch
method
of
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chloroform/methanol extraction from nutmeg, white mustard and coriander seed oils showed
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higher phenolic contents and 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) scavenging radicals than
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Soxhlet method of n-hexane fractions (Kozlowska et al., 2016). These studies show that uniform method of extraction may not be applied to all seeds of various plants species.
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Therefore, new methods of extractions and quantifications will be required according to the seeds nature and properties.
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There are not only the extraction methods and species variations that affect the extraction of total phenolic contents but also the solvents have shown variable results. In above mentioned
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cases, hexane, ethylacetate and ethanol extract depending on species showed high phenol contents. In another study, methanol (as compared to ethanol, acetone and chloroform) was a
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significant solvent that showed highest total phenolic contents with Folin–Ciocalteu reagent (FC reagent) method and maximum antioxidant activity for the seed extracts of Origanum majorana
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(Dhull et al., 2016). Total phenolic compounds in seeds were in the range of 0.10–1.18 mg gallic acid equivalent/g dry weight basis (mg GAE/g dwb). HPLC study confirmed the presence
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of catechin, cinnamic acid, gallic acid and ascorbic acid. The antioxidant potential in seed extracts of Origanum majorana confirmed the presence of nutraceutical properties in them
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which will further be helpful in the preparation of various functional food products. Due to the complex nature of metabolic processes, statistical approaches are used to get meaningful results from the raw data that are also used when the aim is to develop any method or product. One of the statistical tests applied on development of methods is design of experiments (DoE). In the design of experiments, generally, Response Surface Methodology (RSM) is applied that reduces time, efforts on number of experiments, and helpful in getting the optimum results.
ACCEPTED MANUSCRIPT An objective of our study was to optimize the extraction and quantification of total phenols in seeds of different plants species using Folin–Ciocalteu reagent (FC reagent) method. To apply our new method of extraction and quantification of total phenolic contents, we had selected four seeds from different species belonging to four different families (Trachyspermum ammi, Family Apiaceae; Sesamum indicum, Family Pedaliaceae; Ocimum basilicum, Family Lamiaceae; and
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Lepidium sativum L.; Family Brassicaceae).
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The seeds of Basil plant (Ocimum basilicum) are known to have medicinal uses as diuretic, antipyretic, antispasmodic and stomachic, also Basil extracts have vast useful properties bactericidal,
anti-inflammatory,
antioxidative,
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including
antiulcer,
antidiarrheal,
chemopreventive, blood-sugar lowering, a nervous system stimulatory, radiation protection, and
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protective effect against oxidative DNA damage and mutagenesis.
The red seeds from Lepidium sativum in Sultanate of Oman, common name in Arabic is Hab el
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Rashaad or Thufa, belonging to the Cruciferae (Brassicaceae) family, are one of the natural sources of antioxidant. It is one of the herpes Pharmaceutical drugs which are widely used in
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gulf region as well as in alternative Pharmaceutical drugs. Lepidium sativum extracts have been
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screened for various biological activities like hypotensive, anti-microbial, bronchodilator, hypoglycemic and allelopathic activities.
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Sesame (Ocimum basilicum) is cultivated for its edible seeds, containing oil, considered as an antioxidant. It is used for skin freshness, anti-inflammatory agent, and as a home remedy for
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sunburns.
Ajwan seeds (Trachyspermum ammi) are commonly used in Indian curries and Middle East
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cuisines. It has been reported that thymol isolated from ajwan seeds successfully lowered blood pressure in rats (Aftab et al., 1995). Essential oils from the fruits of ajwan have significantly inhibited the production of aflatoxin B1 in chickpeas and wheat suggesting that it may be used as preservative in foods for longer shelflife (Kedia et al., 2015)
2. Materials and methods 2.1. Basil Seed Samples preparation
ACCEPTED MANUSCRIPT Four seeds i.e. basil seed, red seed, sesame seeds and ajwan seeds were purchased from local market in Sultanate of Oman. 34 beakers of 100 ml capacity were taken and to each backer 1.0 g of grinded seed sample was added. Similarly the different volumes and the concentrations of both Methanol and NaOH solutions were also added to each flask according to full factorial design as shown in Table 1. Each extraction mixture was prepared in duplicate order. So the total
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number of experimental run used was 34 for each seed.
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2.2. Design of Experiment & Statistical analysis
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For building the full factorial design the Unscrambler software was used. In this design four variable the concentration and volume of both methanol and NaOH solutions as well as the
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temperature and time were varied. The temperature was changed in the range from 25ºC to 200ºC while the time in the range from 30 min to 180 minutes. Two center points were also added to
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see the effect of curvature. Microsoft Excel 2010 and The Unscrambler version 9.0 by Camo were used for statistical analysis. The main effect plot, the interaction plots, the RSM model
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were also built to see the main effect as well as interaction and optimization of extraction conditions. Similarly ANOVA table was also built to find different statistical parameters for the
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concerned design.
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2.3. Quantification of Polyphenols by UV-Visible Spectroscopic analysis (Folin-Ciocalteu’s
Reagents
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reagent)
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A.R.Grade sodium tungstate, phosphomolybdic acid, ortho phosphoric acid, sodium carbonate, sodium hydroxide, tannic acid and distilled water were used. Solutions
a) Phosphomolybdate tungstic acid solution 50 g of sodium tungstate, 10g of phosphomolybdic acid and 25 ml of ortho phosphoric acid were added to 370 ml of distilled water. This solution was refluxed for two hours and then diluted upto the mark with distilled water in 500 ml volumetric flask. b) 5% NaOH solution
ACCEPTED MANUSCRIPT 15 g of sodium hydoxide was dissolved in a little of distilled water and diluted upto 250 ml volume in volumetric flask with distilled water. c) 1000 ppm Tannic acid solution 0.1g of tannic acid was dissolved in 10 ml of distilled water and diluted up to the mark in 100 mL volumetric flask with distilled water. Procedure
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Five mL of each sample as mentioned in sample preparation was taken in a 100 mL volumetric
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flask, to each flask 6 ml of phosphomolybdate tungstic acid solution and 20 ml of 5% NaOH
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solution were added. It is then kept for 10 minutes to develop blue color and then diluted up to the mark with distilled water. The same procedure was followed for the preparation of tannic
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acid standard solutions in the range of 0.1-40 ppm. Absorbance of the working standard solutions and samples was measured at 770 nm wavelength against blank using Thermo scientific UV-Vis
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using calibration plot shown in Figure 1.
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spectrophotometer England. The concentration of total phenol in each sample was determined
3. Results and discussion
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3.1. Full factorial experimental design for extraction of total phenol from seeds
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Table 1 shows the full factorial experimental design for the extraction of total phenol for those four seeds. In this design four variable the concentration and volume of both methanol and
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NaOH solutions as well as the temperature and time were varied. The temperature was changed in the range from 25ºC to 200ºC while the time in the range from 30 min to 180 minutes. Two
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center points were also added to see the effect of curvature.
Table 1. Full factorial experimental design for extraction of total phenol from basil seed #
NaOH % NaOH volume (mL) MEOH % MeOH volume (mL) Time (min) Tempt (οC)
1
2
10
2
10
30
25
2
2
10
2
10
30
25
3
10
10
2
10
120
25
10
2
10
120
25
5
2
50
2
10
120
200
6
2
50
2
10
120
200
7
10
50
2
10
30
200
8
10
50
2
10
30
200
9
2
10
10
10
120
200
10
2
10
10
10
120
200
11
10
10
10
10
30
200
12
10
10
10
10
30
200
13
2
50
10
10
30
25
14
2
50
10
10
30
25
15
10
50
10
10
120
25
16
10
50
10
10
120
25
17
2
10
2
50
30
200
18
2
10
2
50
30
200
19
10
10
2
50
120
200
20
10
10
2
50
120
200
21
2
50
2
50
120
25
22
2
50
2
50
120
25
23
10
50
2
50
30
25
24
10
50
2
50
30
25
25
2
10
10
50
120
25
26
2
10
10
50
120
25
27
10
10
10
50
30
25
28
10
10
10
50
30
25
29
2
50
10
50
30
200
30
2
50
10
50
30
200
31
10
50
10
50
120
200
32
10
50
10
50
120
200
33
6
30
6
30
75
112.5
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PT CE AC
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T
10
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4
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ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT 34
6
30
6
30
75
112.5
It was investigated that the optimum condition in case of Basil seeds for getting the highest antioxidant yield was obtained under 2% of NaOH and methanol concentration with 50ml of NaOH and 10 mL of methanol volumes at 200ºC and at 120min of time. While in case of red seeds higher extract yields was obtained under 10 % of NaOH and Methanol concentration with
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50 mL of NaOH and methanol volumes, at 200°C and at 120 mints of extraction time. In case of Ajwan seeds the highest amount of total phenols were obtained at 10 % of NaOH and 2 % of
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methanol concentration with 50ml of NaOH and methanol volumes at 25ºC of temperature and at 120 min of time. While in case of Sesame seeds the optimal conditions were obtained at 200ºC of
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10 mL of NaOH and 50 mL of methanol volume.
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temperature, and at 30 min of time, at 2 % of NaOH and Methanol concentration and including
3.2. Main Effect, Interaction plot
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The main effect plot for only Basil seeds is shown in Figure 1. It can be seen from Figure 1 that
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the main effect is 91.67% for temperature, that is the highest one while for NaOH volume is the least i.e. 8.33%. But we see the interaction effect of NaOH solution therefore we cannot ignore
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its significance from the design.
ACCEPTED MANUSCRIPT 91.67
Tempt (F=ADE=BCD
75.00
MeOH volume(D=A
Time(E=ABC=ADF)
NaOH %(A=BCE=DE
-0.020 -0.015 -0.010 Main Effect plot, * Y-var:
-0.005
0
0.005
0.010
0.015
0.020
0.025
0.030
Effects 0.035
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8.33
NaOH volume(B=A
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25.00
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MEOH %(C=ABE=BD
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41.67
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58.33
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Figure 1. Main Effect plot of polyphenol extraction from basil seed
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The main effects overview plot shows which effects are significant. From Figure 1 it can be seen that three effects are considered to be significant: Main effect of Temperature (F), Methanol
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volume (D) and percentage of methanol (C),But as it can be seen that % of NaOH is nonsignificant but due to Interaction effect (B) it was not excluded. As sodium hydroxide react with
process.
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phenol and covert it into salt that increase the solubility of phenols and ease the extraction
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Similarly the Figure 2 shows the regression curve for the full factorial experimental design for extraction of total phenol from basil seed only. The regression produces a double plot of both regression coefficients and predicted values. Regression coefficients are used to interpret the relationship between the Y- and X-variables. The plot of the predicted Y versus the measured Y indicates the predictive quality of the regression function.
ACCEPTED MANUSCRIPT 0.8
0.7
Predicted Y Elements: 34 Slope: 0.86567 Offset: 0.0700 Corre: 0.9304 R-Square: 0.920 RMSEC: 0.0566 SEC: 0.0575 Bias: -1.008e-08
6 19 31 12 9 30 18
0.6 8
0.5
26
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2
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0.2
0.1 0.3
0.4
0.5
0.6
0.7
Measured Y 0.8
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0.2 RESULT1, Y-var: %
25
3 21 13
0.3 1
33
7
27 28
22 4 15 16
14
34
29
32 11
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0.4
2324
17
10
5
20
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Figure 2. Prediction plot of DoE for the polyphenol extraction from basil seed
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It can be seen from Figure 2 that the design is having very good correlation value of 0.93 and
n
i 1
i
yˆ i ) 2
(1)
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RMSECV
(y
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with minimum RMSEC of 0.056. RMSECV is calculated using Eq. 1:
n
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where yi is the measured value (actual % of adulteration), yˆ i is the % of adulteration predicted by the model, and n is the number of segments left-out in the cross-validation procedure, which is equal to the number of samples of the training set. Smaller values of RMSECV are indicative of a better prediction ability of the model. Similarly ANOVA table was also built to find different statistical parameters for the concerned design and their interaction as shown in Table 2.
ACCEPTED MANUSCRIPT Table 2. ANOVA Table for DoE polyphenol extraction from basil seed DF
MS
F-ratio
p-value
0.704
15
4.69E-02
7.734
0
Intercept
0.606
1
0.606
99.854
0
NaOH % (A=BCE=DEF)
3.59E-03
1
3.59E-03
0.591
0.452
NaOH volume
1.06E-04
1
1.06E-04
1.75E-02
0.8963
MEOH %(C=ABE=BDF)
1.67E-03
1
0.274
0.6067
MeOH volume
4.10E-02
4.10E-02
6.759
0.0181
1
2.32E-02
3.816
0.0665
1
0.523
86.267
0
1
1.07E-02
1.761
0.2011
1
4.73E-04
7.79E-02
0.7834
2.05E-02
1
2.05E-02
3.371
0.0829
2.63E-02
1
2.63E-02
4.33
0.052
0.593
6
9.88E-02
16.288
0
2 Vars Int vs Main
7.48E-02
7
1.07E-02
1.762
0.1573
3 Vars Int vs 2 Vars Int
3.61E-02
2
1.80E-02
2.973
0.0766
Lack of Fit
1.50E-02
1
1.50E-02
2.702
0.1186
1
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(D=AEF=BCF) 2.32E-02
Tempt (F=ADE=BCD)
0.523 1.07E-02
AC=BE
4.73E-04
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AB=CE
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Time (E=ABC=ADF)
AE=BC=DF
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Main vs Mean
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AD=EF
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1.67E-03
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(B=ACE=CDF)
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Model
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SS
It can be seen from ANOVA Table 2 that the R2 = 0.866 and R value is 0.930 having good correlationship of the designed variables. From this table it is also apparent that the variable
ACCEPTED MANUSCRIPT NaOH having significance interaction effect with other variables. Actually NaOH help to react with OH group of polyphenols and convert them into salt that increase the solubility of polyphenols. 3.3 Quantification of Polyphenols The estimation of polyphenols was measured through phenols reduction UV-Vis spectroscopic
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method of phosphotungstic-phosphomolybdic acids (Folin-Ciocalteu’s reagent). Standard
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Calibration curve as shown in Figure 1 was made by using tannic acid as a polyphenols standard
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in the concentration range from 0.1 to 10 ppm. The regression line obtained shows the value of correlation coefficient i.e. R =0.930 and Root mean square error of cross validation (RMSEC)
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value of 0.0654.
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0.4
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0.35
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0.25 0.2 0.15
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Absorbance (A⁰)
0.3
0.1
AC
0.05 0
y = 0.0927x + 0.0058 R² = 0.9962
0
0.5
1
1.5
2 2.5 Concentration (ppm)
3
3.5
4
4.5
Figure 3. Standard calibration curve of tannic acids solutions The results for the amount of total phenol using the above standard calibration curve methods are shown in table 3.
ACCEPTED MANUSCRIPT Table 3. The amount of total phenols in different Seeds Red Seed
Sesame seed
Ajwan seed
(mg/100g)
mg/100g)
mg/100g)
mg/100g)
298.134
61.46321
20.287
139.0
2.
298.166
82.94343
23.687
88.8
3.
380.151
104.211
22.487
133.0
4.
354.261
89.11101
20.887
34.2
5.
785.761
125.6912
30.087
144.0
6.
736.138
173.9685
29.687
143.2
7.
567.853
132.0715
26.287
135.2
8.
464.293
134.8362
28.687
135.2
9.
595.901
166.7376
29.887
139.0
10.
675.728
150.5742
31.887
122.2
11.
701.618
188.8558
30.087
131.0
12.
570.011
184.6023
28.087
138.2
13.
388.781
124.4151
31.687
54.6
14.
293.851
97.83071
26.687
178.4
15.
343.474
163.3348
26.287
157.8
16.
345.631
168.8643
27.687
54.6
17.
623.948
182.9009
33.087
136.8
580.798
180.5615
30.287
131.6
697.303
218.843
27.887
120.8
736.138
192.684
29.087
139.0
390.939
154.8277
24.687
145.4
22.
354.261
140.5785
25.487
1062
23.
488.026
168.2263
23.487
148.4
24.
500.971
166.5249
20.087
1346
25.
632.578
127.3926
21.887
108.4
26.
319.741
86.98426
16.687
94.6
20. 21.
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19.
AC
18.
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1.
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Basil Seed
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S#
466.451
81.66737
26.087
85.8
28.
470.766
96.55466
29.487
92.4
29.
654.153
174.8192
28.487
379.0
30.
574.326
159.5066
28.887
274.2
31.
639.051
220.5444
24.287
328.8
32.
705.933
217.7797
14.687
107.0
33.
669.256
205.8698
28.287
365.0
34.
541.963
205.8698
20.287
372.4
CR
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27.
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ACCEPTED MANUSCRIPT
It can be seen from Table 3 that Basil seeds containing the highest amount of total phenols i.e.
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785.76 mg/100g. While the Sesame seeds having the least amount i.e. 33.08 mg/100g. The Ajwan seeds and the Red seeds are containing the medium amounts i.e. 379 mg/100g and 220.54
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mg/100g respectively.
4. Conclusion
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The proposed DoE method provides better extraction efficiency as compared to the conventional extraction methods for phenolic compounds extraction. It has also the advantage of acceptable
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accuracy and precision. This method is also easier and cheaper to perform than HPLC separations and do not require expansive reagents and organic solvents. The results of the study
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also show that, though seeds are used in very small quantity in food, they are potential sources of antioxidants. It is concluded that the basil seeds is the best source of antioxidant because it
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containing the highest amount of total phenols i.e. 785.76 mg/100g , while the Sesame seeds having the least amount i.e. 33.08 mg/100g. The Ajwan seeds and the Red seeds are containing the medium amounts i.e. 379 mg/100g and 220.54 mg/100g respectively.
References 1. AlHakmani, F., S. A. Khan, and A. Ahmed. 2014. Determination of total phenol, in-vitro antioxidant and anti-inflammatory activity of seeds and fruits of Zizyphus spina-christi grown in Oman. Asian Pacific Journal of Tropical Biomedicine, 4, S656–S660.
ACCEPTED MANUSCRIPT 2. Al-Saeedi, A. H., and M. A. Hossain. 2015. Total phenols, total flavonoids contents and free radical scavenging activity of seeds crude extracts of pigeon pea traditionally used in Oman for the treatment of several chronic diseases. Asian Pacific Journal of Tropical Disease, 5, 316–321. 3. Bala, M., and M. Singh. 2013. Non destructive estimation of total phenol and crude fiber content in intact seeds of rapeseed–mustard using FTNIR. Industrial Crops and Products, 42,
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357–362.
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4. Cruz, P. N., T. C. S. Pereira, C. Guindani, D. A. Oliveira, M. J. Rossi, and S. R. S. Ferreira.
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2017. Antioxidant and antibacterial potential of butia (Butia catarinensis) seed extracts obtained by supercritical fluid extraction. The Journal of Supercritical Fluids, 119, 229–237.
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5. Dhingra, N., A. Kar, R. Sharma, and S. Bhasin. 2017. In-vitro antioxidative potential of different fractions from Prunus dulcis seeds: Vis a vis antiproliferative and antibacterial
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activities of active compounds. South African Journal of Botany, 108, 184–192. 6. Behbahani, B. A., F. Tabatabaei Y. F. Shahidi, M. A. Hesarinejad, S. A. Mortazavi, and M.
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Mohebbi. 2017. Plantago major seed mucilage: Optimization of extraction and some physicochemical and rheological aspects. Carbohydrate Polymers, 155, 68-77.
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7. Granato D., and V. M. A. Calado, in Mathematical and Statistical Methods in Food Science and Technology, ed. D. Granato and G. Ares, 2014, Chap. 1, John Wiley & Sons, Ltd. USA,
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1–18.
8. Khan, N., F. Afaq, M. Saleem, N. Ahmad, and H. Mukhtar, Cancer Res., 2006, 66, 2500–
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2505.
9. Li, W., N. He, L. Tian, X. Shi, and X. Yang. 2016. Journal of Food and Drug Analysis, 2016,
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24, 156 527–538.
10. Kozłowska, M., E. Gruczyńska, I. Ścibisz, and M. Rudzińska. 2016. Fatty acids and sterols composition, and antioxidant activity of oils extracted from plant seeds. Food Chemistry, 213, 450-456 11. Magalhaes, Sara C.Q., M. Taveira, A. R. J. Cabrita, A. J. M. Fonseca, P. Valentao, and P. B. Andrade. 2017. European marketable grain legume seeds: Further insight into phenolic compounds profiles. Food Chemistry, 215, 177–184. 12. Pandey, K, B., and S. I. Rizvi. 2009. Plant polyphenols as dietary antioxidants in human health and disease. Oxid. Med. Cell Longev., 2: 270 – 278.
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Graphical abstract
ACCEPTED MANUSCRIPT Highlights
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Extraction and Estimation of Polyphenols from Basil Seeds, Red Seeds, Sesame Seeds and Ajwan Seeds using New Design of Experiment & UV-Vis Spectroscopy
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Fazal Mabood, Syed Abdullah Gilani, Javid Hussain, Sulaiman Alshidani, Said Alghawi, Mohammed Albroumi, Saif Alameri, Farah Jabeen, Zahid Hussain, Ahmed Al-Harrasi, Zahra K.M. Al Abri, Saima Farooq, Zakira Naureen, Ahmad Hamaed, M. Rasul Jan, Jasmin Shah PII: DOI: Reference:
S1386-1425(17)30072-0 doi: 10.1016/j.saa.2017.01.051 SAA 14904
To appear in:
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Received date: Revised date: Accepted date:
13 November 2016 3 January 2017 24 January 2017
Please cite this article as: Fazal Mabood, Syed Abdullah Gilani, Javid Hussain, Sulaiman Alshidani, Said Alghawi, Mohammed Albroumi, Saif Alameri, Farah Jabeen, Zahid Hussain, Ahmed Al-Harrasi, Zahra K.M. Al Abri, Saima Farooq, Zakira Naureen, Ahmad Hamaed, M. Rasul Jan, Jasmin Shah , New design of experiment combined with UV–Vis spectroscopy for extraction and estimation of polyphenols from Basil seeds, Red seeds, Sesame seeds and Ajwan seeds. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Saa(2017), doi: 10.1016/ j.saa.2017.01.051
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ACCEPTED MANUSCRIPT New Design of Experiment combined with UV-VIS Spectroscopy for Extraction and Estimation of Polyphenols from Basil Seeds, Red Seeds, Sesame Seeds and Ajwan Seeds
Fazal Mabood*a, Syed Abdullah Gilani*a, Javid Hussaina, Sulaiman Alshidania, Said Alghawia, Mohammed Albroumia, Saif Alameria, Farah Jabeenb, Zahid Hussainc, Ahmed Al-Harrasid,
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Zahra K. M. Al Abria, Saima Farooqa, Zakira Naureena, Ahmad Hamaeda, M.Rasul Jane, Jasmin
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Shahe,
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a) Department of Biological Sciences & Chemistry, College of Arts and Sciences, University of Nizwa, Sultanate of Oman, ([email protected],. [email protected])
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b) Department of Chemistry, University of Malakand, KPK, Pakistan. c) Department of Chemistry, Abdul Wali Khan University, KPK, Pakistan
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d) UoN Chair of Oman Medicinal Plants and Marine Products, University of Nizwa,
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Sultanate of Oman.
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e) Institute of Chemical Sciences, University of Peshawar, KPK, Pakistan
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Abstract
New experimental designs for the extraction of polyphenols from different seeds including basil Four variables the
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seed, red seed, Sesame seeds and ajwan seeds were investigated.
concentration and volume of methanol and NaOH solutions as well as the temperature and time
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of extraction were varied to see their effect on total phenol extraction. The temperature was varied in the range from 25ºC to 200ºC while the time in the range from 30 to 200 minutes. Response surface methodology was used to optimize the extraction parameters. The estimation of polyphenols was measured through phenols reduction UV-Vis spectroscopic method of phosphotungstic-phosphomolybdic acids (Folin-Ciocalteu’s reagent). Calibration curve was made by using tannic acid as a polyphenols standard in the concentration range from 0.1 to 10 ppm. The regression line obtained shows the value of correlation coefficient i.e. R =0.930 and Root mean square error of cross validation (RMSEC) value of 0.0654. The Basil seeds were found containing the highest amount of total phenols i.e. 785.76 mg/100g. While the Sesame
ACCEPTED MANUSCRIPT seeds having the least amount i.e. 33.08 mg/100g. The Ajwan seeds and the Red seeds are containing the medium amounts i.e. 379 mg/100g and 220.54 mg/100g respectively.
Keywords: Extraction of phenols, basil seed, red seed, Sesame seeds, ajwan seeds, Design of
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Experiment (DoE), UV-VIS spectroscopy
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1. Introduction
Plant polyphenols are secondary metabolites that may be extensively studied and used in
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different fields of studies such as chemotoxonomy, plant biochemistry, biogenesis etc. There are reports of more than 8,000 polyphenolic compounds, identified and isolated from plants sources
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(Pandey and Rizvi, 2009). These compounds have been reported from all plants parts such as roots, stem, leaves, flowers, fruits, and seeds.
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Plasma antioxidant capacity may be enhanced by using Polyphenol-rich foods (Pandey and Rizvi, 2009). Herbal teas in traditional medicines have been used to treat cardiovascular and
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cancer-related diseases (Khan et al., 2006). These herbal teas have also shown significant anticancer activities against human breast cancer cells (Li et al., 2016). Enormous reports on
available from seeds.
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studies of polyphenols from all plants parts have been reported, however, there are fewer reports
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The seeds are considered as rich sources for total phenol contents. Study of legume seeds such as chickpeas, field peas, faba beans, common vetch and lupins has shown that chickpeas had
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highest phenolic contents as compared to the other tested legumes (Magalhaes et al., 2017). Total phenolic contents of pigeon pea used in Oman for chronic diseases were tested for phenol contents (Al-Saeedi and Hossain, 2015). The results showed highest phenol contents in hexane extract followed by chloroform, methanol, butanol, ethylacetate, and water extracts. Alhakmani et al. (2014) studied phenol contents in seeds and fruits of Zizyphus spina-cristi in Oman which showed higher phenol contents in seeds than the fruits. Calibration model was applied using Fourier Transfer Near Infrared spectroscopy (FTNIR) in estimation of phenol contents in rapeseed-mustard genotypes (Bala and Singh, 2013).
ACCEPTED MANUSCRIPT Extraction and estimation of total phenol contents in Prunus dulcis seeds by warring blender method revealed that ethyl acetate fraction showed higher phenol contents and antioxidant acitivity (Dhingra et al., 2017). While comparing various methods of seeds oil extractions and fractions of Butia catarinensis seeds, Ethanol and ethanol-water by Soxhelt extraction exhibited higher total phenolic contents and antioxidant activities (Cruz et al., 2017). However, in another study,
soxhelt
extraction
method
was
not
significant,
where
Folch
method
of
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chloroform/methanol extraction from nutmeg, white mustard and coriander seed oils showed
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higher phenolic contents and 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) scavenging radicals than
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Soxhlet method of n-hexane fractions (Kozlowska et al., 2016). These studies show that uniform method of extraction may not be applied to all seeds of various plants species.
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Therefore, new methods of extractions and quantifications will be required according to the seeds nature and properties.
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There are not only the extraction methods and species variations that affect the extraction of total phenolic contents but also the solvents have shown variable results. In above mentioned
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cases, hexane, ethylacetate and ethanol extract depending on species showed high phenol contents. In another study, methanol (as compared to ethanol, acetone and chloroform) was a
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significant solvent that showed highest total phenolic contents with Folin–Ciocalteu reagent (FC reagent) method and maximum antioxidant activity for the seed extracts of Origanum majorana
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(Dhull et al., 2016). Total phenolic compounds in seeds were in the range of 0.10–1.18 mg gallic acid equivalent/g dry weight basis (mg GAE/g dwb). HPLC study confirmed the presence
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of catechin, cinnamic acid, gallic acid and ascorbic acid. The antioxidant potential in seed extracts of Origanum majorana confirmed the presence of nutraceutical properties in them
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which will further be helpful in the preparation of various functional food products. Due to the complex nature of metabolic processes, statistical approaches are used to get meaningful results from the raw data that are also used when the aim is to develop any method or product. One of the statistical tests applied on development of methods is design of experiments (DoE). In the design of experiments, generally, Response Surface Methodology (RSM) is applied that reduces time, efforts on number of experiments, and helpful in getting the optimum results.
ACCEPTED MANUSCRIPT An objective of our study was to optimize the extraction and quantification of total phenols in seeds of different plants species using Folin–Ciocalteu reagent (FC reagent) method. To apply our new method of extraction and quantification of total phenolic contents, we had selected four seeds from different species belonging to four different families (Trachyspermum ammi, Family Apiaceae; Sesamum indicum, Family Pedaliaceae; Ocimum basilicum, Family Lamiaceae; and
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Lepidium sativum L.; Family Brassicaceae).
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The seeds of Basil plant (Ocimum basilicum) are known to have medicinal uses as diuretic, antipyretic, antispasmodic and stomachic, also Basil extracts have vast useful properties bactericidal,
anti-inflammatory,
antioxidative,
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including
antiulcer,
antidiarrheal,
chemopreventive, blood-sugar lowering, a nervous system stimulatory, radiation protection, and
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protective effect against oxidative DNA damage and mutagenesis.
The red seeds from Lepidium sativum in Sultanate of Oman, common name in Arabic is Hab el
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Rashaad or Thufa, belonging to the Cruciferae (Brassicaceae) family, are one of the natural sources of antioxidant. It is one of the herpes Pharmaceutical drugs which are widely used in
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gulf region as well as in alternative Pharmaceutical drugs. Lepidium sativum extracts have been
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screened for various biological activities like hypotensive, anti-microbial, bronchodilator, hypoglycemic and allelopathic activities.
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Sesame (Ocimum basilicum) is cultivated for its edible seeds, containing oil, considered as an antioxidant. It is used for skin freshness, anti-inflammatory agent, and as a home remedy for
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sunburns.
Ajwan seeds (Trachyspermum ammi) are commonly used in Indian curries and Middle East
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cuisines. It has been reported that thymol isolated from ajwan seeds successfully lowered blood pressure in rats (Aftab et al., 1995). Essential oils from the fruits of ajwan have significantly inhibited the production of aflatoxin B1 in chickpeas and wheat suggesting that it may be used as preservative in foods for longer shelflife (Kedia et al., 2015)
2. Materials and methods 2.1. Basil Seed Samples preparation
ACCEPTED MANUSCRIPT Four seeds i.e. basil seed, red seed, sesame seeds and ajwan seeds were purchased from local market in Sultanate of Oman. 34 beakers of 100 ml capacity were taken and to each backer 1.0 g of grinded seed sample was added. Similarly the different volumes and the concentrations of both Methanol and NaOH solutions were also added to each flask according to full factorial design as shown in Table 1. Each extraction mixture was prepared in duplicate order. So the total
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number of experimental run used was 34 for each seed.
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2.2. Design of Experiment & Statistical analysis
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For building the full factorial design the Unscrambler software was used. In this design four variable the concentration and volume of both methanol and NaOH solutions as well as the
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temperature and time were varied. The temperature was changed in the range from 25ºC to 200ºC while the time in the range from 30 min to 180 minutes. Two center points were also added to
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see the effect of curvature. Microsoft Excel 2010 and The Unscrambler version 9.0 by Camo were used for statistical analysis. The main effect plot, the interaction plots, the RSM model
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were also built to see the main effect as well as interaction and optimization of extraction conditions. Similarly ANOVA table was also built to find different statistical parameters for the
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concerned design.
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2.3. Quantification of Polyphenols by UV-Visible Spectroscopic analysis (Folin-Ciocalteu’s
Reagents
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reagent)
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A.R.Grade sodium tungstate, phosphomolybdic acid, ortho phosphoric acid, sodium carbonate, sodium hydroxide, tannic acid and distilled water were used. Solutions
a) Phosphomolybdate tungstic acid solution 50 g of sodium tungstate, 10g of phosphomolybdic acid and 25 ml of ortho phosphoric acid were added to 370 ml of distilled water. This solution was refluxed for two hours and then diluted upto the mark with distilled water in 500 ml volumetric flask. b) 5% NaOH solution
ACCEPTED MANUSCRIPT 15 g of sodium hydoxide was dissolved in a little of distilled water and diluted upto 250 ml volume in volumetric flask with distilled water. c) 1000 ppm Tannic acid solution 0.1g of tannic acid was dissolved in 10 ml of distilled water and diluted up to the mark in 100 mL volumetric flask with distilled water. Procedure
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Five mL of each sample as mentioned in sample preparation was taken in a 100 mL volumetric
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flask, to each flask 6 ml of phosphomolybdate tungstic acid solution and 20 ml of 5% NaOH
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solution were added. It is then kept for 10 minutes to develop blue color and then diluted up to the mark with distilled water. The same procedure was followed for the preparation of tannic
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acid standard solutions in the range of 0.1-40 ppm. Absorbance of the working standard solutions and samples was measured at 770 nm wavelength against blank using Thermo scientific UV-Vis
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using calibration plot shown in Figure 1.
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spectrophotometer England. The concentration of total phenol in each sample was determined
3. Results and discussion
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3.1. Full factorial experimental design for extraction of total phenol from seeds
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Table 1 shows the full factorial experimental design for the extraction of total phenol for those four seeds. In this design four variable the concentration and volume of both methanol and
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NaOH solutions as well as the temperature and time were varied. The temperature was changed in the range from 25ºC to 200ºC while the time in the range from 30 min to 180 minutes. Two
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center points were also added to see the effect of curvature.
Table 1. Full factorial experimental design for extraction of total phenol from basil seed #
NaOH % NaOH volume (mL) MEOH % MeOH volume (mL) Time (min) Tempt (οC)
1
2
10
2
10
30
25
2
2
10
2
10
30
25
3
10
10
2
10
120
25
10
2
10
120
25
5
2
50
2
10
120
200
6
2
50
2
10
120
200
7
10
50
2
10
30
200
8
10
50
2
10
30
200
9
2
10
10
10
120
200
10
2
10
10
10
120
200
11
10
10
10
10
30
200
12
10
10
10
10
30
200
13
2
50
10
10
30
25
14
2
50
10
10
30
25
15
10
50
10
10
120
25
16
10
50
10
10
120
25
17
2
10
2
50
30
200
18
2
10
2
50
30
200
19
10
10
2
50
120
200
20
10
10
2
50
120
200
21
2
50
2
50
120
25
22
2
50
2
50
120
25
23
10
50
2
50
30
25
24
10
50
2
50
30
25
25
2
10
10
50
120
25
26
2
10
10
50
120
25
27
10
10
10
50
30
25
28
10
10
10
50
30
25
29
2
50
10
50
30
200
30
2
50
10
50
30
200
31
10
50
10
50
120
200
32
10
50
10
50
120
200
33
6
30
6
30
75
112.5
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10
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4
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ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT 34
6
30
6
30
75
112.5
It was investigated that the optimum condition in case of Basil seeds for getting the highest antioxidant yield was obtained under 2% of NaOH and methanol concentration with 50ml of NaOH and 10 mL of methanol volumes at 200ºC and at 120min of time. While in case of red seeds higher extract yields was obtained under 10 % of NaOH and Methanol concentration with
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50 mL of NaOH and methanol volumes, at 200°C and at 120 mints of extraction time. In case of Ajwan seeds the highest amount of total phenols were obtained at 10 % of NaOH and 2 % of
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methanol concentration with 50ml of NaOH and methanol volumes at 25ºC of temperature and at 120 min of time. While in case of Sesame seeds the optimal conditions were obtained at 200ºC of
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10 mL of NaOH and 50 mL of methanol volume.
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temperature, and at 30 min of time, at 2 % of NaOH and Methanol concentration and including
3.2. Main Effect, Interaction plot
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The main effect plot for only Basil seeds is shown in Figure 1. It can be seen from Figure 1 that
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the main effect is 91.67% for temperature, that is the highest one while for NaOH volume is the least i.e. 8.33%. But we see the interaction effect of NaOH solution therefore we cannot ignore
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its significance from the design.
ACCEPTED MANUSCRIPT 91.67
Tempt (F=ADE=BCD
75.00
MeOH volume(D=A
Time(E=ABC=ADF)
NaOH %(A=BCE=DE
-0.020 -0.015 -0.010 Main Effect plot, * Y-var:
-0.005
0
0.005
0.010
0.015
0.020
0.025
0.030
Effects 0.035
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8.33
NaOH volume(B=A
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25.00
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MEOH %(C=ABE=BD
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41.67
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58.33
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Figure 1. Main Effect plot of polyphenol extraction from basil seed
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The main effects overview plot shows which effects are significant. From Figure 1 it can be seen that three effects are considered to be significant: Main effect of Temperature (F), Methanol
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volume (D) and percentage of methanol (C),But as it can be seen that % of NaOH is nonsignificant but due to Interaction effect (B) it was not excluded. As sodium hydroxide react with
process.
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phenol and covert it into salt that increase the solubility of phenols and ease the extraction
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Similarly the Figure 2 shows the regression curve for the full factorial experimental design for extraction of total phenol from basil seed only. The regression produces a double plot of both regression coefficients and predicted values. Regression coefficients are used to interpret the relationship between the Y- and X-variables. The plot of the predicted Y versus the measured Y indicates the predictive quality of the regression function.
ACCEPTED MANUSCRIPT 0.8
0.7
Predicted Y Elements: 34 Slope: 0.86567 Offset: 0.0700 Corre: 0.9304 R-Square: 0.920 RMSEC: 0.0566 SEC: 0.0575 Bias: -1.008e-08
6 19 31 12 9 30 18
0.6 8
0.5
26
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2
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0.2
0.1 0.3
0.4
0.5
0.6
0.7
Measured Y 0.8
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0.2 RESULT1, Y-var: %
25
3 21 13
0.3 1
33
7
27 28
22 4 15 16
14
34
29
32 11
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0.4
2324
17
10
5
20
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Figure 2. Prediction plot of DoE for the polyphenol extraction from basil seed
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It can be seen from Figure 2 that the design is having very good correlation value of 0.93 and
n
i 1
i
yˆ i ) 2
(1)
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RMSECV
(y
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with minimum RMSEC of 0.056. RMSECV is calculated using Eq. 1:
n
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where yi is the measured value (actual % of adulteration), yˆ i is the % of adulteration predicted by the model, and n is the number of segments left-out in the cross-validation procedure, which is equal to the number of samples of the training set. Smaller values of RMSECV are indicative of a better prediction ability of the model. Similarly ANOVA table was also built to find different statistical parameters for the concerned design and their interaction as shown in Table 2.
ACCEPTED MANUSCRIPT Table 2. ANOVA Table for DoE polyphenol extraction from basil seed DF
MS
F-ratio
p-value
0.704
15
4.69E-02
7.734
0
Intercept
0.606
1
0.606
99.854
0
NaOH % (A=BCE=DEF)
3.59E-03
1
3.59E-03
0.591
0.452
NaOH volume
1.06E-04
1
1.06E-04
1.75E-02
0.8963
MEOH %(C=ABE=BDF)
1.67E-03
1
0.274
0.6067
MeOH volume
4.10E-02
4.10E-02
6.759
0.0181
1
2.32E-02
3.816
0.0665
1
0.523
86.267
0
1
1.07E-02
1.761
0.2011
1
4.73E-04
7.79E-02
0.7834
2.05E-02
1
2.05E-02
3.371
0.0829
2.63E-02
1
2.63E-02
4.33
0.052
0.593
6
9.88E-02
16.288
0
2 Vars Int vs Main
7.48E-02
7
1.07E-02
1.762
0.1573
3 Vars Int vs 2 Vars Int
3.61E-02
2
1.80E-02
2.973
0.0766
Lack of Fit
1.50E-02
1
1.50E-02
2.702
0.1186
1
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(D=AEF=BCF) 2.32E-02
Tempt (F=ADE=BCD)
0.523 1.07E-02
AC=BE
4.73E-04
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AB=CE
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Time (E=ABC=ADF)
AE=BC=DF
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Main vs Mean
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AD=EF
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1.67E-03
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(B=ACE=CDF)
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Model
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It can be seen from ANOVA Table 2 that the R2 = 0.866 and R value is 0.930 having good correlationship of the designed variables. From this table it is also apparent that the variable
ACCEPTED MANUSCRIPT NaOH having significance interaction effect with other variables. Actually NaOH help to react with OH group of polyphenols and convert them into salt that increase the solubility of polyphenols. 3.3 Quantification of Polyphenols The estimation of polyphenols was measured through phenols reduction UV-Vis spectroscopic
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method of phosphotungstic-phosphomolybdic acids (Folin-Ciocalteu’s reagent). Standard
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Calibration curve as shown in Figure 1 was made by using tannic acid as a polyphenols standard
CR
in the concentration range from 0.1 to 10 ppm. The regression line obtained shows the value of correlation coefficient i.e. R =0.930 and Root mean square error of cross validation (RMSEC)
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value of 0.0654.
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0.4
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0.35
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0.25 0.2 0.15
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Absorbance (A⁰)
0.3
0.1
AC
0.05 0
y = 0.0927x + 0.0058 R² = 0.9962
0
0.5
1
1.5
2 2.5 Concentration (ppm)
3
3.5
4
4.5
Figure 3. Standard calibration curve of tannic acids solutions The results for the amount of total phenol using the above standard calibration curve methods are shown in table 3.
ACCEPTED MANUSCRIPT Table 3. The amount of total phenols in different Seeds Red Seed
Sesame seed
Ajwan seed
(mg/100g)
mg/100g)
mg/100g)
mg/100g)
298.134
61.46321
20.287
139.0
2.
298.166
82.94343
23.687
88.8
3.
380.151
104.211
22.487
133.0
4.
354.261
89.11101
20.887
34.2
5.
785.761
125.6912
30.087
144.0
6.
736.138
173.9685
29.687
143.2
7.
567.853
132.0715
26.287
135.2
8.
464.293
134.8362
28.687
135.2
9.
595.901
166.7376
29.887
139.0
10.
675.728
150.5742
31.887
122.2
11.
701.618
188.8558
30.087
131.0
12.
570.011
184.6023
28.087
138.2
13.
388.781
124.4151
31.687
54.6
14.
293.851
97.83071
26.687
178.4
15.
343.474
163.3348
26.287
157.8
16.
345.631
168.8643
27.687
54.6
17.
623.948
182.9009
33.087
136.8
580.798
180.5615
30.287
131.6
697.303
218.843
27.887
120.8
736.138
192.684
29.087
139.0
390.939
154.8277
24.687
145.4
22.
354.261
140.5785
25.487
1062
23.
488.026
168.2263
23.487
148.4
24.
500.971
166.5249
20.087
1346
25.
632.578
127.3926
21.887
108.4
26.
319.741
86.98426
16.687
94.6
20. 21.
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19.
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Basil Seed
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S#
466.451
81.66737
26.087
85.8
28.
470.766
96.55466
29.487
92.4
29.
654.153
174.8192
28.487
379.0
30.
574.326
159.5066
28.887
274.2
31.
639.051
220.5444
24.287
328.8
32.
705.933
217.7797
14.687
107.0
33.
669.256
205.8698
28.287
365.0
34.
541.963
205.8698
20.287
372.4
CR
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27.
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It can be seen from Table 3 that Basil seeds containing the highest amount of total phenols i.e.
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785.76 mg/100g. While the Sesame seeds having the least amount i.e. 33.08 mg/100g. The Ajwan seeds and the Red seeds are containing the medium amounts i.e. 379 mg/100g and 220.54
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mg/100g respectively.
4. Conclusion
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The proposed DoE method provides better extraction efficiency as compared to the conventional extraction methods for phenolic compounds extraction. It has also the advantage of acceptable
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accuracy and precision. This method is also easier and cheaper to perform than HPLC separations and do not require expansive reagents and organic solvents. The results of the study
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also show that, though seeds are used in very small quantity in food, they are potential sources of antioxidants. It is concluded that the basil seeds is the best source of antioxidant because it
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containing the highest amount of total phenols i.e. 785.76 mg/100g , while the Sesame seeds having the least amount i.e. 33.08 mg/100g. The Ajwan seeds and the Red seeds are containing the medium amounts i.e. 379 mg/100g and 220.54 mg/100g respectively.
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ACCEPTED MANUSCRIPT 2. Al-Saeedi, A. H., and M. A. Hossain. 2015. Total phenols, total flavonoids contents and free radical scavenging activity of seeds crude extracts of pigeon pea traditionally used in Oman for the treatment of several chronic diseases. Asian Pacific Journal of Tropical Disease, 5, 316–321. 3. Bala, M., and M. Singh. 2013. Non destructive estimation of total phenol and crude fiber content in intact seeds of rapeseed–mustard using FTNIR. Industrial Crops and Products, 42,
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5. Dhingra, N., A. Kar, R. Sharma, and S. Bhasin. 2017. In-vitro antioxidative potential of different fractions from Prunus dulcis seeds: Vis a vis antiproliferative and antibacterial
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activities of active compounds. South African Journal of Botany, 108, 184–192. 6. Behbahani, B. A., F. Tabatabaei Y. F. Shahidi, M. A. Hesarinejad, S. A. Mortazavi, and M.
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10. Kozłowska, M., E. Gruczyńska, I. Ścibisz, and M. Rudzińska. 2016. Fatty acids and sterols composition, and antioxidant activity of oils extracted from plant seeds. Food Chemistry, 213, 450-456 11. Magalhaes, Sara C.Q., M. Taveira, A. R. J. Cabrita, A. J. M. Fonseca, P. Valentao, and P. B. Andrade. 2017. European marketable grain legume seeds: Further insight into phenolic compounds profiles. Food Chemistry, 215, 177–184. 12. Pandey, K, B., and S. I. Rizvi. 2009. Plant polyphenols as dietary antioxidants in human health and disease. Oxid. Med. Cell Longev., 2: 270 – 278.
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Graphical abstract
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Extraction and Estimation of Polyphenols from Basil Seeds, Red Seeds, Sesame Seeds and Ajwan Seeds using New Design of Experiment & UV-Vis Spectroscopy
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