


































Food Science and Nutrition Studies 

ISSN 2573-1661 (Print) ISSN 2573-167X (Online) 

Vol. 1, No. 2, 2017 

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104 
 

Phytochemicals Extraction and Nutraceuticals of Purple Corn 

W. Puminat1* & C. Teangpook2
 

1 Division of Food Chemistry and Physic, Institution of Food Research and Product Development, 

Kasetsart University, Kasetsart, Bangkok, Thailand 

2 Division of Food Processing and Preservation, Institution of Food Research and Product Development, 

Kasetsart University, Kasetsart, Bangkok, Thailand 

* W. Puminat, E-mail: ifrwnp@hotmail.com 

 

Received: October 19, 2017    Accepted: October 25, 2017    Online Published: November 3, 2017 

doi:10.22158/fsns.v1n2p104        URL: http://dx.doi.org/10.22158/fsns.v1n2p104 

 

Abstract 

Phytochemicals are naturally formed in plant of corn. Many kinds of flavonoids are the most 

important plant pigment. Purple corn is extracted with polar and nonpolar solvent and analyzed for 

flavonoids and antioxidants. The extract residue of purple corn is compared on extraction with various 

solvent groups. In the designed experiments, conditions of extraction depend on solvent, solvent ratio 

and evaporation temperature. The residue quantity of purple corn is respectively from extraction with 

three kinds of solvent such as acetone, chloroform: methanol (3:1) and pentane: hexane (1:1). All 

residue extract are eluted and with mobile phase and analyzed with diode array detector by HPLC. 

Anthocyanin, β-carotene and total tannin of purple corn were taken analysis an average (mean ± SD) 

in 100 g sample and shown as 198.42 ± 0.33 mg, 175.82 ± 0.17 mg and 273.75 ± 0.33 mg respectively. 

The efficiency of solvent extraction depends on the polarity of the substances. The extract by acetone 

can take the highest residue and mixture solvent take a high residue. The different polarities of solvent 

can make the difference of residue product in the extraction. Improving and development on the 

changes are useful for the best of raw material and products.  

Keywords 

purple corn, nutraceuticals, extraction and anthocyanin 

 

1. Introduction 

Flavonoids are naturally formed in plant. Flavonoids are widely distributed in plants, fulfilling many 

functions. Some flavonoids are phenolic substances that act in plant as antioxidants. Many kinds of 

flavonoids are the most important plant pigments. Plant pigments include a variety of different kinds of 

molecule, including porphyrins, carotenoids, anthocyanins and betalains. Purple pigments of purple 

corn have the properties of free radicals and reduce symptoms of cancer tumor types. They enhance the 

strengthen of body, increase an immunity antibodies and the red blood cells. They reduce the fat in 



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artery clogs, prevent heart disease and control blood sugar levels. Pigments color help anti-aging, the 

digestive system and deterioration of eye. 

Physical and chemical changes with growth, this is useful in helping to identify compounds in pigments. 

If we know the chemical properties of substances, we are likely to predict what kind of reaction. We see 

a chemical change based on certain chemical properties of pigments. These pigments are generally 

measured by determining the amount and wavelength of light that they absorb. The wavelength and 

amount of absorbed light can show the identify and content of pigments. All substances have properties 

that functional and identifying. we can utilize by separating them from one another by extraction with 

solvents and purity by chromatography.  

Purple corn has a rich composition of phytochemicals such as anthocyanins and phenolic compounds. 

The phytonutrients are natural compounds or substances found in certain plants which is believed to be 

beneficial to human health and help prevent various diseases. This class of phytonutrients includes 

pigments such as carotenoids (beta-carotene, lutein), flavonoids or phenolics, alkaloids, 

nitrogen-containing compounds and organosulfur compounds (indoles, glucosinolates). The color 

categories of phytonutrients are red, red purple (and blue), orange, orange-yellow, yellow-green, green 

and white-green. 

Liquid extraction also known as solvent extraction is a method to separate compounds based on their 

relative solubilities by preferentially dissolving that substance in a suitable solvent. Generally, polar 

solvents dissolve polar compounds and non-polar solvents dissolve non-polar compounds. Strongly 

polar compounds dissolve only in very polar, while strongly non-polar compounds dissolve only in 

very non-polar organic solvents. Polar and non-polar are not miscible with each other and will quickly 

separate into two layers even after being shaken well. The type of polarity, dipole moment, and 

hydrogen bonding of solvent is able to specify the dissolve. 

 

2. Materials and Methods  

2.1 The Characteristics and Physical Properties of Purple Corn 

This classification scheme will be based on their shapes and structures for morphological classification. 

Physical characteristics are defining traits or features about general quality. The samples of purple corn 

are classified and weighted for determining an average of groups. A classification is provided the 

groups in order to understand the general standard of purple corn. 

2.1.1 Preparation of Purple Corn Powder and Drying 

Fresh purple corn is sliced in size 1-2 mm and crushed to a fine shred. Sample is taken drying by 

vacuum drying at 60°C for 7 hr. The samples are pulverized to a fine powder and stored in seal 

container for further analysis and the other process.  

2.1.2 Determination of Moisture Content   

Fresh purple corn is sliced in size 1-2 mm and dry with the vacuum oven. Moisture is determined by 

drying 1-3 g sample in a vacuum oven (25-100 mmHg) at 60°C for 7 hr. The moisture content of 



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sample is calculated as percentage of the different weight. 

2.1.3 Measurement the Colour of Purple Corn   

Sample is measured in three times and seven sampling. Colour measuring is taken with the Data Colour 

International Measurement model Colour Tools. CIE value (Commission Internationale de I’Eclaerage) 

display the colour value in CIELAB system L*(0 = black and 100 = white), a*(-a* = green and + a* = 

red) and b*(-b* = blue and + b* = yellow) at D65 10Deg (Light source Illuminant D). 

2.2 Analysis of Nutraceuticals Purple Corn   

2.2.1 Determination Total Anthocyanin by pH-Differential Method 

Total anthocyanin content is extracted and determined by UV-VIS spectroscopy. The extract is added to 

test tubes containing of buffers pH 1.0 and pH 4.5. They are thoroughly mixed by vortex mixer. 

Transperent sample mixer is measured at different pH with an absorbance at wavelength 530 nm and 

700 nm by UV-Visible spectrophotometer. Total anthocyanin content is calculated and determined as 

anthocyanin (cyanidin) in milligram/100 gram by the following equation % anthocyanin pigments 

(mg/l) = A* MW * DF * 1000 /  * l; A = (A530nm-A700nm) pH 1 - (A530nm-A700nm) pH 4.5. A is a 

difference of absorbance at pH 1and pH 4.5. MW. is molecular weight (cyanidin-3-glucoside = 449.2 

g/mol) and  is the molar absorptivity for cyanidin-3-glucoside ( = 26900 L/mol/cm). l is standard of 

pathlength1cm and DF is dilution factor.  

2.2.2 Determination Pigment Content and Pigments Extract 

Each sample is prepared by 5 g pasted purple corn immersed in 1-butanol 25 mL. They are extracted in 

an ultrasonic water bath for 2 hrs, and centrifuged at 4,500 rpm for 20 min. The upper layer is 

supernatant for pigment analysis by uv-visible spectrophotometer. The absorbance at 435 nm show 

pigment content of purple corn. 

2.2.3 Determination β-Carotene Extract by uv-visible Spectrophotometer 

Purple corn are extracted with hexane and acetone. Sample 1 g is added with 15 ml hexane: acetone 

(2:3). Mixture is soaked and shaken with vortex mixer for 10 minutes. They are allowed to stand by 

magnesia adsorber and centrifuged at 4500 rpm for 10 minutes. The extract is separated in vial with cap 

and measured by spectrophotometry at wavelength 436 nm. The determination of β-carotene is 

compared and calculated with standard solution. The standard curve of β-carotene is prepared at 

concentrations of 0.001-0.015 mg/ml. The concentrations are calculated using a linear regression of the 

five points in standard curve. 

2.2.4 Determination of Polyphenol or Total Phenolic Assay 

Total phenolic compounds are quantified as tannin content by using Folin Ciocalteu’s method. Sample 

is extracted with water and filtered by filter paper. Extract is formed a complex compound with 

Folin-Ciocalteu reagent. Polyphenol or tannin is analyzed by spectrophotometer. Blue colour 

compound is determined an absorbance at wavelength 760 nm. Blank is prepared by replacing the 

reagent on the same way by sample and standard reference. Samples are compared with standard 

calibration of tannin and calculated as total tannin content.  



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2.2.5 Determination of Amylose Content in Purple Corn 

The samples are dissolved with urea and dimethylsulfoxide (UDMSO) in a water bath at high 

temperature until changed in gel formulation. Contaminated fat is eluted with alcohol and dried in oven. 

Residues without ethanol are dissolved UDMSO again and adjusted the volume with water. Solution is 

formed the color with iodine in potassium iodide solution and measured the absorbance at 635nm 

wavelength. Absorbance is compared with standard reference and calculated for amylose content. 

2.3 Extraction and Solvent Extract 

In the designed experiments on extraction model, conditions of extraction for the yield depend on 

solvent, solvent ratio and evaporation temperature. Control on the extract ratio is 1:7 wt/ml. Sample is 

approximately weighted in 10 g and mixed with 70 ml solvent extract. It is tightly closed the lid and 

shaken a mixture. Controls on the condition are the same temperature and shaker at speed 180 rpm for 

8 hour. The suspension is filtered through filter paper. All of extract liquids are done the evaporation of 

solvent by Buchi Syncorne Analyst in three stage of temperature at 40°C, 50°C and 60°C. The 

evaporation is controlled by temperature, vacuum pressure and speed controller at 150 rpm until 

dryness. Residue is blowed with nitrogen gas in order to evaporate the remained solvent. The specific 

substances are focused on weight of recovery residue. 

% Extracted = Total residue * 10 / wt.sample 

The extract in each experiment can be compared the differences and taken for the other analysis.  

2.4 Identification of Flavonoids and Antioxidants by HPLC 

2.4.1 Sample and Preparation Sample 

Analysis of anthocyanin is determined by HPLC (High Performance Liquid Chromatography). Residue 

is weighted and mixed with 25 ml ethanol. It is tightly closed the lid and shaken to dissolve by a vortex 

mixer. The sample is extracted with ultrasonic agitation for 15 min and shaken with a vortex mixer 

again. Solution is precipitated with centrifuge (Minicentrifuge C 1200). The upper layer of solution is 

followed by filter membrane ф 0.45 μm and injected for 1microL into the HPLC.  

2.4.2 Determining of Flavonoids and Total Flavonoids  

Conditions of analysis in grouping, mobile phase is acetonitrile: 5% formic acid: methanol: water; 75: 

10: 10: 5 and elute with column at flow rate 0.9 ml/min. Column is ODS Hypersil particle size 5 μm ID 

4.6 mm length 250 mm and detect the peak with diode array detector at a wavelength 280 nm. The 

concentration of cyanidin-3-glucoside standard reference is level 1.5-6 ng for determining as 

flavonoids and another series. 

2.4.3 Determining of Antioxidants and Total Antioxidants 

Antioxidant analysis, extracted sample is carried out by HPLC analysis with UV detector. Mobile phase 

is acetonitrile: methanol; 90: 10 and elute column at flow rate 0.7 ml/min. Both antioxidants and 

flavonoids analysis detected on the same wavelength and column. The analysis is determined at room 

temperature and identified the chromatogram in series of epicatechin. 

 



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2.5 Statistical Analysis 

The data is analysed by statistical program of ANOVA (Analysis of Variance). Statistical analysis of 

mean and variance in each treatment are taken with Duncan’s new multiple range test at the 

significance 0.0l. 

 

3. Results and Discussions 

3.1 Evaluation of Physical and Chemical Properties 

The physical and chemical characteristics are a defining of traits about qualification. Their identities 

are sometimes taken into consideration to evaluate certain corn. In the experiments, their properties are 

classified as either extensive or intensive. Extensive properties depend on the amount of matter in the 

sample such as colour, % silk or pollen, average weight per three pods. Intensive properties do not 

depend on the sample size or mass such as pigment, anthocyanin, tannin and amylose. Mean and 

standard deviation (Mean ± SD.) of % weight of purple corn are pod 46.16 ± 0.57%, silk or pollen 

19.88 ± 2.71%, peel 33.96 ± 2.55% and bulk weight per three pod 1.257 ± 0.015 kg. 

3.1.1 Moisture Content and Dry Sample  

An average (Mean ± SD.) moisture of fresh corn and silk is about 73.40 ± 1.01% and 24.47 ± 0.40 

respectively. Drying sample is prepared by heating in vacuum oven. The moisture is removed by 

evaporation of water. Moisture in the sample is measured by the difference of mass before and after 

drying. The moisture content of the sample is calculated as percentage of the different weight loss. An 

average moisture of drying powder is about 12.19 ± 0.02%. The samples are pulverized to a fine 

powder and stored in seal container for further analysis and process. 

3.1.2 Purple Colour of Corn and Colour Value (CIELAB) 

The pollen or silk has a high red colour but seed has a high blue colour. Colour in the CIELAB system 

(L*, a* and b*) of purple corn in seed and silk are different a statistical significance at α = 0.05. The 

relationships between the association of two variables are shown with Pearson’s correlation. Table 1 is 

shown Pearson’s correlation of colour and some characteristics of purple corn. In each part of corn pod 

and colour value in the table are compared the average and a variance on random analysis by statistics 

at significant level α = 0.05. 

 

Table 1. The Characteristics and Colour Value of Purple Corn and Its Silk 

Purple corn Total weight   Colour value   

 (kg) per 3 pod L* a* b* C 

Kenel and seed 1.257 ± 0.017 18.964 ± 3.924a 2.047 ± 0.746a,b 0.742 ± 0.480a 2.282 ± 0.523b 

Silk 0.250 ± 0.038 20.173 ± 3.49c 3.647 ± 2.052c 0.520 ± 0.585c,d 3.807 ± 1.784d 

a,c Correlation is significant at the 0.05 level (2-tailed) and b,d Correlation is significant at the 0.01 level 

(2-tailed). 



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In Table 1, a,c and b,d show the relationships according to Duncan’s multiple range test at α = 0.05 and 

α = 0.01. Colour value (CIELAB) in each column are significantly different at α = 0.05. 

3.2 Evaluation on Nutraceuticals of Purple Corn   

Anthocyanins are a group of naturally occurring flavonoid compounds, which play an important role in 

the colour quality of purple corn. In the experiments, the analysis of extract has respectively 

anthocyanin and pigment an average (Mean ± SD) 198.42 ± 0.33 mg/100 g and 204.52 ± 0.30 mg/100 

g. Tannin and β-carotene of purple corn are taken analysis and shown as fresh, frozen and drying 

sample in Table 2. 

 

Table 2. Some Phytonutients of Fresh Corn, Frozen Corn and Dry Corn 

Phytonutrients Fresh Corn (Mean ± SD) Frozen Corn (Mean ± SD) Dry Corn (Mean ± SD) 

Tannin (mg/100 g) 273.75 ± 0.33 259.55 ± 0.48 1274.32 ± 0.49 

β-carotene (mg/100 g) 175.82 ± 0.17 156.60 ± 0.02 496.54 ± 0.19 

 

Standard cruve of linear equation and R-squared value (tannin and β-carotene) in puple corn are 

respectively displayed as y = 0.2244x - 0.0066: R² = 0.9958 and y = 1.9256x - 0.0129: R2 = 0.9899. In 

the experiments, amylose of fresh purple corn is 121.75 ± 0.32 mg/100g and drying powder average 

834.35 ± 0.75 mg/100g. The calibration graph is shown a linearity, with a correlation coefficient of R² 

= 0.9986. The representative linear of regression equation for standard amylose is y = 0.1966x + 

0.0261. 

3.3 Solvent Extraction and Residue of Purple Corn with Various Solvent Extract 

 

 

Figure 1. The Extract Residues of the Various Solvent Polarities 



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3.3.1 Effect of Solvent on the Extraction of Nutraceuticals in Purple Corn 

All substances have properties as functionals and nutraceuticals. The crude extract is identified and 

analyzed for determination of flavonoids and antioxidants. The polarity of the substances are compared 

to the different extraction. The residue content is evaluated by comparing with various solvent extract. 

By separating them by extraction with solvents, difference of nutraceuticals are a process that 

substances dissolve selectively by one or more of the mixture of solvent. In the experiments, mixture 

solvent of chloroform and methanol is the best described system for partially polar and nonpolar can 

extract to high residue. The relationships of residue and solvent polarity in extraction of purple corn 

show on Figure 1. 

3.3.2 Residue Extract and Solvent Polarities in the Extraction of Purple Corn 

Figure 1 shows the different types of weight (%) of chemical composition by priority of polar 

extraction. They are found that the extract residue have higher yield than another extract solvent. If the 

dielectric constant of the extract is divided into three groups the difference of extract residue is from the 

solvent polarity. It is found that they have statistically a difference at significant level α = 0.05. 

 

Table 3. Capacity and Potential of Solvent Extract for the Extraction of Purple Corn 

Solvent extract Focus on Functional groups %Crude residue (g/ml) 

Acetone Pigments 10.682 

Chloform: Methanol; 3:1 The best partially polar and nonpolar 7.693 

Pentane +hexane; 1:1 Fat & Oil 7.644 

Ether + Petroleum ether; 1:1 Fat & Oil 5.635 

Butanol + Ether; 1:1 Pigments 5.635 

Hexane Non polar 5.586 

Ethanol Sugar 4.018 

Chloroform Soluble matter 2.205 

n-butanol Colour 0.763 

hexane + Acetone; 4:1 Fat & Oil 0.847 

n-butanol Colour 0.763 

n-heptane Unsaturated Fat 0.490 

 

In the experiments, the extract by acetone can take the highest residue and mixture solvent take a high 

residue. The different polarities of solvent can make the difference of residue product in the extraction. 

The extract residue of purple corn is from extraction with three kinds of solvent such as pentane: 

hexane (1:1), chloroform: methanol (3:1) and acetone.  

 

 



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3.4 Determination of Flavonoids and Antioxidants by HPLC 

3.4.1 Determination the Flavonoid and Antioxidant Compounds from Chemical Extract with the 

Different Solvent by HPLC  

Purple corn is extracted from polar and nonpolar solvent by eleven groups of solvent extraction. They 

are analysed and detected on flavonoids and antioxidants by HPLC. The extract is eluted by mobile 

phase for flavonoids (acetonitrile: 5% formic acid: methanol: water; 75: 10: 10: 5) and antioxidants 

(acetonitrile: methanol; 90: 10) scaned with wavelength at 280 nm. The different types of molecule 

weight by solvent separation are defined with chemical positions. These flavonoids are generally 

indicated by the identifying wavelength and amount absorbance of light. A qualitative property is one 

that defines something based on characteristics. The wavelength and amount of absorbed light can 

determine of flavonoid and antioxidant content. On the Table, estimate at peak area of flavonoids and 

antioxidants are analysed and compared with various solvent extract. 

 

Table 4. The Peak Area (Min-Max) Flavonoid and Antioxidant by the Different Eluent 

Moblie phase 
Series of Functional 

groups 

Peak area of epicatichin 

Min-Max (mAU) 

Total peak area 

Min-Max (mAU) 

ACN: 5%HCOOH: MeOH: 

H2O; 75: 10: 10: 5 
Flavonoids 2.32-226.21 11.34-1053.50 

ACN: MeOH; 90: 10 Antioxidants 8.97-214.07 27.32-1054.42 

 

3.4.2 Chromatogram and Chemical Fingerprints  

The pattern of the extract components of purple corn are shown some chemicals. By solvent extraction, 

chemical composition of different types are orderly separated by weight (%) with HPLC. Simultaneous 

measurement at the same wavelength is shown that UV detector is the ability to perform 

spectrophotometric scanning and precise absorbance readings of wavelengths. Chemical profile or 

chromatogram of fingerprint is compared on the different solvent in analysis of flavonoids and 

antioxidants pattern.  

 

 

 



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Figure 2. The Difference of Mobile Phase in Flavonoid and Antioxidant Analysis of Crude 

Residue 

 

In peak of chromatogram at 280 nm, fingerprints allow to show height and area of peak at the 

difference of retention time. They are used to identify a determination for flavonoids and antioxidants. 

The different polarity of the extracting solvent, in each crude residue is constructed and compared on 

the peak area. Scanning and comparing with the profile of reference, the pattern profile is from the 

characteristics of wavelength by mobile phase. It is indicated some chemical components of flavonoids 

and antioxidants in purple corn. 

 

 

Figure 3. The Comparison on Flavonoid and Antioxidant Content by Extraction with the 

Difference of Solvent Extract 

 



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On the statistic analysis, Figure 3 shows the comparison on the difference of mobile phase in flavonoid 

analysis. The relationships of peak area base on the different solvent of the extract. They have pearson 

correlation by 2-tailed level at 0.940 and have significantly a difference by statistic at the 0.01. 

 

4. Conclusion 

Purple corn has a rich composition of nutraceuticals and phytochemicals. All substances have 

properties as functionals that we can use to identify them. By the nature, substance characteristics base 

on properties of quality and unique identity. Improving and development on the changes are useful in 

helping to the best of raw material and products. 

 

References 

Alan, D., Mc., & Andrew, W. (1997). Flavonoids: Isoflavonoids and neoflavonoids. IUPAC 

Compendium of chemical terminology (2nd ed.). Oxford: Blackwell Scientific. 

AOAC. (2000). Official Methods of Analysis. The Association of Official Analytical Chemists, 

Arlington Virginia. 

Lee, J., Rennaker, C., & Wrolstad, R. E. (2008). Correlation of two anthocyanin quantification method: 

HPLC and spectrophotometric methods. Food Chemistry, 110, 782-786. 

https://doi.org/10.1016/j.foodchem.2008.03.010 

Puminat, W., & Teangpook, C. (2014). The acidity enhancement in extraction of anthocyanin and color 

of rambutan peel. The journal of food technology, 106, 220-226. 

 


