Isolation, identification and chemical characterization of compounds from phenolic extracts of peels of Kufri Chipsona-3 and Kufri Jyoti potatoes having synergistic antioxidant interactions in combination

Abhishek Bhattacharya; Rabi Ranjan Chattopadhyay

doi:10.55976/fnds.22024123129-40

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Journal of Food, Nutrition and Diet Science, Volume 2 Issue 1, 2024 /

Original Research

Isolation, identification and chemical characterization of compounds from phenolic extracts of peels of Kufri Chipsona-3 and Kufri Jyoti potatoes having synergistic antioxidant interactions in combination

Abhishek Bhattacharya ¹ ^* , Rabi Ranjan Chattopadhyay ²

1. Agricultural and Ecological Research Unit, Indian Statistical Institute 203, Barrackpore Trunk Road, Kolkata -700108, India
2. Agricultural and Ecological Research Unit, Indian Statistical Institute 203, Barrackpore Trunk Road, Kolkata -700108, India

Correspondence: abhishekbhattacharya224@gmail.com

DOI: https://doi.org/10.55976/fnds.22024123129-40

Received

23 November 2023
Revised

26 February 2024
Accepted

06 March 2024
Published

01 April 2024

Potato peel waste Natural antioxidants Synergistic interactions TLC bioautography Spectrometric analysis Cytotoxicity study

Abstract

The study focused on the isolation, identification and characterization of the bioactive compounds that are responsible for synergistic antioxidant interactions in combination from the peel extract of Kufri Chipsona-3 and Kufri Jyoti potato varieties by Thin-Layer Chromatography (TLC) bioautography mediated isolation technique which was followed by isobologram analysis as well as characterization using different spectrometric methods. The results revealed that among the isolated seven antioxidant components of potato peel extracts (three components with Rf : 0.33, 0.52 and 0.57 from the peel extract of Kufri Chipsona-3 and four components with Rf : 0.14, 0.22, 0.36, 0.61 from the peel extract of Kufri Jyoti, only one component from Kufri Chipsona-3 (Rf : 0.57) and one component from Kufri Jyoti (Rf : 0.61) potato varieties in combination exhibited synergistic antioxidant interactions in combination. Spectrometric analysis (UV/Vis, FT-IR and HR-LCMS/MS) revealed that the active component with Rf : 0.57 from Kufri Chipsona-3 is prodelphinidin trimer and the other component with Rf : 0.61 from Kufri Jyoti is 5-hydroxy-3’,4’,7-trimethoxyflavone. In a root growth inhibition test (LC50 > 200 µg/ml), on Allium cepa, these two compounds showed no cytotoxicity. The results provide evidence that these two phenolic compounds, prodelphinidin trimer and 5-hydroxy-3’,4’,7-trimethoxyflavone in combination may serve as a stronger and efficacious novel natural antioxidant blend in pharmaceutical and food industries.

<h5>Introduction</h5>
Lipid oxidation is considered to be one of the main problems with the preservation of fatty acids in food <a href="#reference_list_0">[1]</a>. Since it has been established that omega-3 fatty acids exhibit significant health benefits, meals are now supplemented with them to boost the nutritional value of foods and lower the risk of acquiring chronic diseases. The two primary components of omega-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are highly susceptible to autoxidation because of the high levels of unsaturation in their molecular structures <a href="#reference_list_1">[2]</a>. This makes it challenging to produce stable functional foods fortified with omega-3 fatty acids. Synthetic antioxidants are frequently employed in the food sector to address these issues. However, there is a strong evidence to suggest that they can also be hazardous and in some cases nitrite and nitrate food preservatives may lead to stomach cancer <a href="#reference_list_2">[3]</a>. Therefore, new, safer and effective natural antioxidants should continue to be sought as preservatives, especially those derived from plants, to protect foods from oxidative deterioration, particularly omega-3 fatty acid-fortified functional foods.  
Due to their powerful antioxidant effectiveness, plant polyphenols may provide a possible source of such molecules [4, 5]. Given that these leftovers are one of the major sources of polyphenols, using fruit and vegetable waste for research has gained popularity recently. As a result, effective recycling and use of phenolic-rich fruit and vegetable waste could lead to financial gains for the food industry, contribute to addressing food nutritional issues, have a positive impact on health and reduce environmental pollution [6, 7]. Phenolic chemicals are abundant in potato peels. It has been discovered that potato peels have a higher phenol concentration than potato tubers. In the potato processing industry, large amounts of phenolic-rich potato peels are produced as a waste product. As a result, the industry in question faces a major challenge in terms of waste disposal <a href="#reference_list_7">[8]</a>. Potato peels are another waste product that is produced in large quantities by families. However, due to their significant antioxidant activity, these potato peels with high phenolic content can be employed as a source of natural antioxidants for preserving foods <a href="#reference_list_8">[9, 10]</a>. Researchers are now conducting a systematic search for these beneficial chemicals from used potato peels in response to the growing need to replace synthetic antioxidants with natural ones.
Only the peel extract of the potato varieties Kufri Chipsona-3 and Kufri Jyoti showed synergistic antioxidant interactions when combined, according to our recent investigation on the peel extracts of five different potato varieties <a href="#reference_list_10">[11]</a>. Therefore, an attempt has been made in the current work to isolate, characterize and identify the bioactive compounds from peel extracts of these two potato varieties (Kufri Chipsona-3 and Kufri Jyoti). They were found to be responsible for synergistic antioxidant interactions when combined with each other using a TLC-bioautography led isolation technique, which was followed by isobologram analysis and spectrometric characterization.
<h5>Materials and methods</h5>
Collection and processing plant materials 
These two potato varieties, Kufri Chipsona-3 and Kufri Jyoti, were collected from the vegetable farm of Bidhan Chandra Krishi Viswavidyalaya (BCKV), West Bengal, India, and were recognised by a botanist. The harvest period for them lasts for 90-100 days on average. They were used in the current investigation. These different kinds of potatoes were carefully rinsed in tap water before being manually peeled to a depth of around 1 mm.  The peels were then air dried for 5-6 days until they had reached a constant weight and were then grounded into a coarse powder using a manual grinder.
Extraction of plant materials
The phenolic extract of the potato peels of Kufri Chipsona-3 and Kufri Jyoti was made respectively, according to the procedure described by Abu-Reidah et al. <a href="#reference_list_11">[12]</a>. 25 g of coarsely ground potato peels were macerated at 25 ± 1º C in the dark for 24 hours with 250 ml of a solution of water and ethanol at a ratio of  20:80 v/v ratio, while being occasionally agitated. The mixture was then filtered using Whatman filter paper No. 1. Then the filtrates were blended after the second and third extraction of the residue. 100 ml of the pooled filtrate and 4 ml of Carrez reagent A were mixed and vortexed for two minutes before being left for one minute. The mixture was then combined with 4 ml of Carrez reagent B, then vortexed for 20 seconds. These two chemicals were utilized to separate the proteins and polysaccharides, respectively. The mixture was then centrifuged at 2000 g for 20 minutes. Next, the supernatant was removed, centrifuged at 2000 g for a further 10 minutes and then filtered.  Anhydrous sodium sulphate was used as a dehydrating agent, and the filtrate was subsequently dried by evaporation in a rotary evaporator at a bath temperature of 40°C. Most of the dried potato peel extracts were stored at 20 °C until needed [yield: Kufri Chipsona-3: 3.92%; and Kufri Jyoti: 3.72%].
Isolation, identification and chemical characterization of bioactive compounds
The phenolic extract from the peels of Kufri Chipsona-3 and Kufri Jyoti were chemically analyzed by TLC-bioautography guided detection and isolation, followed by isobologram analysis and spectrometric characterization as follows. This was done for isolating, identifying, and chemically characterizing the bioactive compounds responsible for antioxidant efficacy.   
TLC bioautography guided detection and isolation of antioxidant components from potato peel extract
Analytical TLC
In accordance with the methodology by Gu et al. (2009) <a href="#reference_list_12">[13]</a>, the Rf values of separated components of active extracts from the peels of the potato varieties Kufri Chipsona-3 and Kufri Jyoti (which showed synergistic antioxidant and antimicrobial interactions in combination) were determined using analytical TLC separation technique. The analytical TLC plates (5 × 10 cm, 0.25 mm thickness, Silica gel G 60 F254, Merck, Darmstadt, Germany) were baked in aluminium.  These plates were preconditioned by heating for an hour at 120 ℃. 5μl (10 mg/ml) of the active extracts from the peels of  Kufri Chipsona-3 and Kufri Jyoti potato varieties were applied using a capillary pipette. The plate was then developed for 3-4 hours at room temperature using a solvent solution made up of butanol, ethyl acetate, and formic acid [5:4:1 (v/v)]. After developing the TLC plate, the absorbent layer was air dried to eliminate all traces of the solvent mixture. For visualisation, the p-anisaldehyde-sulphuric acid reagent was sprayed on the sample and heated at 110°C for 5 minutes. The spots on the plate that contained separated active components of peel extract were measured for their Rf values using the equation below <a href="#reference_list_13">[14]</a>.   
Rf = Distance traveled by solute/Distance traveled by solvent
In order to prevent potential interference with the visualisation reagent, which could affect the antioxidant activity of test samples in bioautograms, TLC plates for the detection of antioxidant compounds in TLC bioautography were prepared simultaneously using the same procedure as described above without spraying the visualisation reagent <a href="#reference_list_14">[15]</a>.
TLC bioautography guided detection of antioxidant components
To confirm the presence of antioxidant components, the developed air-dried plate from analytical TLC that had been prepared for the detection of antioxidant compounds was then placed in a sterile Petri dish and sprayed with a 0.02% w/v DPPH solution in methanol. This was then heated at 110°C for 5 minutes. The Rf values of the antioxidant spots on the analytical TLC plate were calculated from the corresponding Rf values of the component’s spots.
Preparative TLC for isolation of antioxidant components
To separate the antioxidant components of the active extracts of Kufri chipsona-3 and Kufri Jyoti potato varieties, a streak of active test peel extracts was manually applied to a preparative TLC glass plate (20×20 cm, 1 mm thickness) (Sigma-Aldrich, USA) and air dried. After air drying, the plate was developed in a pre-saturated glass chamber with the same solvent mixture used for analytical TLC. On the produced bioautograms, a 0.02% (w/v) DPPH solution in methanol was sprayed on the entire plate. Antioxidant spots with Rf values that showed antioxidant activity in the TLC bioautography were scraped out of the processing. The bands with antioxidant activity were carefully removed from the silica gel and dissolved in 80% methanol. Then the methanol-based antioxidant components were centrifuged at around 10,000 g for an additional 15 minutes. The supernatants and silica gel were collected. The supernatants were then dried in vacuum after being filtered through a 0.22 m membrane filter. The extracted antioxidants, which had been dried at various Rf values, were then stored in a refrigerator for later use.
Following the isobologram analysis, the extracted antioxidant components from the peel extracts of Kufri Chipsona-3 and Kufri Jyoti potato varieties were submitted to a combined antioxidant efficacy research.
Determination of antioxidant activity of active components alone and in combination by DPPH radical scavenging method
According to Wang et al. (1998) <a href="#reference_list_15">[16]</a>, the ability of antioxidant elements from the potato peel extracts of Kufri Chipsona-3 and Kufri Jyoti to scavenge DPPH (2,2-diphenyl-1-picrylhydrazyl) free radicals was investigated both separately and in combination. In brief, test tubes were added with 100 µl of antioxidant compounds alone and in combination (1:1) at different concentrations (3.12 µg/ml to 100 µg/ml), and then 3.9 ml of a 0.1 mM solution of DPPH in methanol was added. The test tubes were then shaken vigorously. The tubes were then left at room temperature in the dark for 30 minutes. The test substances described above were used to establish the control. The zero value was changed by using methanol. The absorbance of the samples was measured at 517 nm. The percentage radical scavenging activity of antioxidant components alone and in combination was evaluated as follows:   
（%）Free radical scavenging = （Ablank - Asample)/Ablank × 100
Where, Asample is the absorbance of DPPH solution after reacting with a given concentration  of antioxidant component and Ablank is the absorbance of DPPH solution with methanol blank instead of antioxidant component. The IC50 values (the concentration required to scavenge 50% of the DPPH radical) of antioxidant components alone and in combination were determined from dose-response curve.
Isobologram analysis for determination of combination index (CI) based on IC50 values
The antioxidant combination index (CI) of combined isolated antioxidant components was evaluated using isobologram analysis based on IC50 values. The data were analyzed using the traditional calculation by isobologram combination index (CI) <a href="#reference_list_16">[17]</a>.
CI = D1/Dx1+D2/Dx2                            
where (D1) and (D2) are the doses (IC50 values) of two different test antioxidant components in combination; (Dx1) and (Dx2) are the doses (IC50 values) of two individual test antioxidant components that are combined <a href="#reference_list_16">[17]</a>.
Determination of type of antioxidant interactions based on combination index (CI)
Based on the CI values, the type of antioxidant interactions in combination were interpreted     as follows: CI < 1: synergistic; CI = 1: additive; CI > 1: antagonistic <a href="#reference_list_17">[18]</a>.
Spectrometric analysis
In order to characterize the chemical composition of the antioxidant compounds, the spectrometric (UV/Vis, FTIR, HR-LCMS/MS) analyses of the peel extract of the potato varieties Kufri Chipsona-3 and Kufri Jyoti were performed on the antioxidant components,  which demonstrated synergistic antioxidant interactions in combination in isobologram analysis.
Following spectrometric investigation for chemical identification, only one component from peel extract of Kufri Chipsona-3 (Rf: 0.57) and Kufri Jyoti (Rf: 0.61) in combination was found to show synergistic antioxidant interactions.
UV/Vis absorption spectra
A Perkin Elmer Lambda 950 UV-Vis spectrometer was used to record the UV/Vis absorption spectra of antioxidant components from peel extract of these two selected potato varieties, which together demonstrated synergistic antioxidant interactions. The spectral range was 200-2500 nm. The calibration curve was prepared. Samples were subjected to UV/Vis absorption spectral analysis.
FT-IR analysis
The FT-IR spectral analysis of test antioxidant components from Kufri Chipsona-3 and Kufri Jyoti was conducted by using the Fourier-transform infrared instrument (make and model:3000 Hyperion Microscope with Vertex 80 FTIR System, Burker, Germany) in the scan range 450-4000 cm−1.
HR-LCMS/MS analysis
The HR-LCMS/MS spectrometric analysis of test antioxidant compounds was done by using HR-LCMS instrument ( make and model : Agilent Technologies, USA, 1290 Infinity UHPLC System,1260 infinity Nano HPLC with Chipcube, 6550 iFunnel Q-TOFs) in the mass range 50-32200 amu and resolution 40000 FWHM. The instrument was equipped with (1) UHPLC & Nano HPLC for small molecules as well as for large molecules; (2) Direct Infusion Mass with ESI & APCI (Positive & Negative mode ionization; (3) UHPLC PDA Detector -Mass spectrometer and (4) Nano HPLC with Chipcube (Microfluidic column) -Mass spectrometer. Agilent software was used for data analysis.
Evaluation of cytotoxic potential of antioxidant compounds that showed synergistic antioxidant interactions in combination  
Allium cepa root growth inhibition assay
When evaluating the cytotoxic potential of antioxidant compounds in the peel extract of the potato varieties Kufri Chipsona-3 and Kufri Jyoti, which exhibited synergistic antioxidant interactions in combination, Fiskesjo's method from 1985 was used <a href="#reference_list_18">[19]</a>. Briefly, we purchased 20 to 25 g of robust common onion bulbs from a neighboring market in Bura Bazar, Kolkata, India. Before the experiment, the bulbs were allowed to dry and the outer scales were removed, which were made simpler by stopping the destruction of the root primordial. The remainders of the pesticides and herbicides were then carefully removed from the bulbs by washing them under running water. After that, bulbs were kept in tap water at room temperature for 3-4 days without light to germinate, at this time the new roots were 3-4 cm long. Nine groups of bulbs, with six for each group, were collected after the development of new roots. Each glass tube contained 10 ml of tap water. The antioxidant compounds of Kufri Chipsona-3 and Kufri Jyoti were then added into tubes containing seven groups of onion bulbs at seven different concentrations (3.125, 6.25, 12.5, 25, 50, 100, and 200 µg/ml), which demonstrated synergistic antioxidant interactions in combination in the DPPH method and isobologram analysis. Positive control group received sodium azide (10 µg/ml), while the negative control group did not receive any medication. Following that, both control and antioxidant component-treated groups were stored at room temperature in the dark for 96 hours. The top 10 roots of Allium cepa bulbs in each group were measured at the conclusion of the exposure period (96h), and the mean root length (cm) was calculated. Onion bulbs in the negative control group were considered as a standard, and measurements of the average root length in the treatment groups that received antioxidant chemicals and the positive control group were made in comparison. At the conclusion of the exposure period, whether the roots have any bulges, hooks, or twists, as well as other clear morphological transformations, including root consistency and color changes, were also carefully studied.
Statistical analysis
The statistical analysis of data was performed using the SPSS software: version 18.0. A one-way ANOVA (analysis of variance) followed by a Tukey range test was used to analyse the data with the significance level set at P < 0.05.
<h5>Results</h5>
Separation and detection of antioxidant components by analytical TLC and TLC bioautography analysis
Figure 1(a) shows the Rf values of separated components of peel extract of Kufri Chipsona-3 obtained by analytical TLC analysis, and Figure 1(b) shows the Rf values of antioxidant components of peel extract of Kufri Chipsona-3 obtained by TLC- bioautography. The analytical TLC separation of peel extract of Kufri Chipsona-3 (Figure 1(a)) revealed the presence of four components (Rf: 0.33, 0.47, 0.52 and 0.57) in Kufri Chipsona-3 potato peel extract. Of these four separated components, only three components with Rf : 0.33, 0.52 and 0.57 were found to have antioxidant activity in TLC bioautography guided detection technique as yellow spots against a purple background (Figure 1(b)).
 
<img src="https://file.luminescience.cn/6606856ddbf7e1711703405" alt="" width="350" height="274" />
 Figure 1. (a) Rf values of separated components of Kufri Chipsona-3 by analytical TLC； (b) Rf values of antioxidant components of Kufri Chipsona-3 by TLC-bioautography
                        
Figure 2(a) and Figure 2(b) respectively represent the chromatograms of analytical TLC and TLC bioautography analyses of peel extract of Kufri Jyoti potato variety. It was observed from Figure 2 (a) that the peel extract of Kufri Jyoti potato variety contains five components ( Rf : 0.14, 0.22, 0.36, 0.61, and 0.73) and of them only four components (Rf : 0.14, 0.22, 0.36, and 0.61) demonstrated antioxidant activity in TLC bioautography analysis (Figure 2 (b)).
<img src="https://file.luminescience.cn/660685bcdb12b1711703484" alt="" width="400" height="255" />
Figure 2. (a) Rf values of separated components of Kufri Jyoti by analytical TLC； (b) Rf values of antioxidant components of KufriJyoti by TLC-bioautography
Results of isobologram analysis for determination of combination index (CI) based on IC50 values
Table 1 shows the Rf values of seven antioxidant components of peel extracts of Kufri Chipsona-3 (Rf: 0.33, 0.52, 0.57) and Kufri Jyoti (Rf: 0.14, 0.22, 0.36, 0.61) potato varieties obtained by TLC bioautography analysis and their antioxidant potential (IC50) alone and in combination determined by DPPH radical scavenging method as well as their combination index (CI) values based on IC50 values using isobologram analysis. It was observed from Table 1 that among the possible combinations tested, only one combination between one component (Rf : 0.57) of Kufri Chipsona- 3 and one component (Rf :0.61) of Kufri Jyoti peel extract exhibited synergistic antioxidant interactions (CI < 1), whereas other possible tested combinations showed additive antioxidant effect (CI=1).
Table 1. Results of combined antioxidant efficacy (CI) of active components from peel extracts of Kufri Chipsona-3 and Kufri Jyoti potato varieties based on IC50 values of antioxidant components alone and in combination
<img src="https://file.luminescience.cn/660a2d9c347481711943068" alt="" width="686" height="747" />
Results of spectrometric analysis
UV/Vis absorption spectra results
Figure 3(a) represents the UV/Vis spectra of Kufri Chipsona -3 peel extract component (Rf: 0.57). It was observed that the maximum absorbance (UVλmax) of Kufri Chipsona-3 peel extract component (Rf: 0.57) was 278 nm, which corresponds with the absorbance maxima of  proanthocyanidin compounds <a href="#reference_list_19">[20]</a>.
<img src="https://file.luminescience.cn/660a4d5fc4eeb1711951199" alt="" width="400" height="245" />
                                                                                                                (a)
 
<img src="https://file.luminescience.cn/660a4d6fbaca61711951215" alt="" width="400" height="255" />
                                                                                                                (b)
Figure 3. (a) UV-Vis absorption spectra of Kufri Chipsona-3 peel extract component (Rf : 0.57); (b) UV/Vis absorption spectra of Kufri Jyoti peel extract component (Rf : 0.61)
Figure 3(b) represents the UV/Vis absorption spectra of Kufri Jyoti peel extract component (Rf: 0.61). It was observed that the maximum absorbance (UVλmax) of Kufri Jyoti peel extract component (Rf: 0.61) was at 350 nm and 260 nm, which corresponds with the absorbance maxima of flavones <a href="#reference_list_20">[21]</a> .
FT-IR analysis results
Figure 4(a) represents the FT-IR spectrum of Kufri Chipsona -3 peel extract component (Rf: 0.57). From FT-IR curves (Figure 4(a)) the major peaks of peel extract component (Rf : 0.57) of  Kufri Chipsona-3 were found at 3429.61 (stretching vibrations of O-H groups); 2906.48 and 2851.53 (stretching vibration of C-H (CH2, CH3); 1575.36 (aromatic ring vibrations); 1417.19 (related to CH2, CH3, flavonoids and aromatic rings where the vibrations would be bending (δ) vibrations of C-H and stretching vibration of aromatics); 1120.80-1020.44 (stretching vibration of C-O-C). These bands represent the structure of proanthocyanidins <a href="#reference_list_19">[20]</a>.
<img src="https://file.luminescience.cn/660686ce26e241711703758" alt="" width="400" height="259" />
                                                                                                                (a)
<img src="https://file.luminescience.cn/660686dcf376b1711703772" alt="" width="400" height="235" />
                                                                                                               (b)
Figure 4. (a) FTIR spectra of Kufri Chipsona-3 peel extract component (Rf : 0.57); (b) FTIR spectra of Kufri Jyoti peel extract component (Rf : 0.61)
Figure 4(b) represents the FT-IR spectrum of Kufri Jyoti peel extract component (Rf : 0.61). Major peaks at FT-IR spectra of Kufri Jyoti peel extract component (Rf: 0.61) were found at 3442.28 (stretching vibration of O-H group); 2927.83-2821.82 (stretching vibration of C-H); 1594.57 (aromatic ring vibration); 1353.23 (stretching vibration of aromatic C=C); 1075.60 (C-O stretching). These bands represent the molecular structure of flavonoids <a href="#reference_list_21">[22]</a>.
HR-LCMS/MS analysis results
Figure 5(a) represents the HR-LCMS/MS analysis of Kufri Chipsona-3 peel extract component (Rf : 0.57). The retention time of this component was found to be 7.89 min and its molecular mass (m/z) was found to be 897.0.
<img src="https://file.luminescience.cn/660686eeb15da1711703790" alt="" width="400" height="247" />
                                                                                                           (a)
<img src="https://file.luminescience.cn/660686fa495b61711703802" alt="" width="400" height="255" />
                                                                                                           (b)
Figure 5. (a) HR-LCMS/MS spectra of Kufri Chipsona-3
peel extract component (Rf : 0.57); (b) HR-LCMS/MS spectra of Kufri Jyoti peel extract component (Rf : 0.61)
Figure 5(b) represents the HR-LCMS/MS analysis of Kufri Jyoti peel extract component (Rf: 0.61). From HR-LCMS/MS analysis curves, the retention time of Kufri Jyoti peel extract component (Rf : 0.61) was found to be 10.04 min and its molecular mass (m/z) was 327.5.
Table 2 shows the spectrometric analyses data (UV/Vis, FT-IR, HR-LCMS/MS) of two antioxidant components, one from peel extract of Kufri Chipsona-3 (Rf : 0.57) and the other from peel extract of  Kufri Jyoti (Rf : 0.61) that showed synergistic antioxidant  interactions in combination. Comparing the obtained data with MS library data as well as available literature data [23, 24], the antioxidant component (Rf : 0.57) obtained from peel extract of Kufri Chipsona-3 was identified as prodelphinidin trimer and the antioxidant component (Rf : 0.61) obtained from peel extract of Kufri Jyoti was identified as 5-Hydroxy-3’,4’,7-trimethoxyflavone.
 
Table 2. Spectrometric analyses data of active compound from peel extract of Kufri Chipsona-3 (Rf : 0.57) and Kufri Jyoti (Rf : 0.61) potato varieties that showed synergistic antioxidant interactions
<table>
<tbody>
<tr>
<td width="201">
Spectrometric
parameters
</td>
<td width="273">
Active components from peel extract of Kufri Chipsona-3 potato
(Rf : 0.57)
</td>
<td width="260">
Active components from peel extract of Kufri Jyoti potato
(Rf : 0.61)
</td>
</tr>
<tr>
<td width="201">
 
UVλmax(MeOH)
</td>
<td width="273">
 
Only one band at 278nm
</td>
<td width="260">
Two bands. Band A at 350 nm and B at 260 nm
</td>
</tr>
<tr>
<td width="201">
HR-LCMS/MS(m/z)
[M-H]-
</td>
<td width="273">
897(calcd.molecular formula:C45H38O20;898.8)
</td>
<td width="260">
327.5  (calcd.molecular formula :C18H16O6;328.3)
</td>
</tr>
<tr>
<td width="201">
Retention Time(min)
</td>
<td width="273">
7.89
</td>
<td width="260">
10.04
</td>
</tr>
<tr>
<td width="201">
FT-IRKBr(cm-1)
</td>
<td width="273">
Major peaks are at 3429.61(stretching vibrations of OH groups); 2906.48 and 2851.53(stretching vibration of C-H (CH2,CH3);1575.36 (could be related to aromatic ring vibrations); 1417.19(related to CH2, CH3, flavonoids and aromatic rings where the vibrations would be bending(δ) vibrations of C-Hand stretching vibration of aromatics);1120.80-1020.44 (stretching vibration of C-O-C).
</td>
<td width="260">
Majorpeaksareat3442.28(stretching vibration of O-H group); 2927.83-2821.82
(stretching vibration of C-H);1594.57 (aromatic       ring
vibration); 1353.23 (stretching vibration of aromatic C=C);1075.60(C-O stretching).
</td>
</tr>
<tr>
<td width="201">
Proposed compound
</td>
<td width="273">
Prodelphinidin trimer
</td>
<td width="260">
5-Hydroxy-3’,4’,7-trimethoxy
flavone
</td>
</tr>
<tr>
<td width="201">
Molecular structure
</td>
<td width="273">
    <img src="https://file.luminescience.cn/6606874832a4f1711703880" alt="" width="238" height="112" />
       
</td>
<td width="260">
 <img src="https://file.luminescience.cn/66068752710911711703890" alt="" width="253" height="117" />
 
</td>
</tr>
<tr>
<td width="201">
References
</td>
<td width="273">
NIST MS search v.2.3
</td>
<td width="260">
NIST MS search v.2.3
</td>
</tr>
</tbody>
</table>
 
Allium cepa root growth inhibition assay results
The results of the inhibition assay on Allium cepa root growth were used to determine the cytotoxic potential of the bioactive compounds prodelphinidin trimer and 5-Hydroxy-3',4',7- trimethoxyflavone, which were isolated respectively from active peel extracts of the potato varieties Kufri Chipsona-3 and Kufri Jyoti. Cell viability of the bioactive compounds tested in combination did not differ significantly from the negative control up to a concentration of 200 µg/ml, but it did differ significantly (p < 0.05) from the positive control (Sodium azide), and the IC50 of the tested bioactive compounds in combination was discovered to be greater than 200 µg/ml (Figure 6).
 <img src="https://file.luminescience.cn/66068763e16431711703907" alt="" width="500" height="256" />
 Figure 6. Results of cytotoxic potential of bioactive compounds in combination isolated from potato varieties Kufri Chipsona-3 (Rf : 0.57) and Kufri Jyoti (Rf : 0.61) in Allium cepa root growth inhibition assay. *No significant (P > 0.05) difference in root growth up to 200μg/ml concentration when compared with negative control but significantly (P < 0.05) different when compared with positive control Sodium azide
<h5>Discussion</h5>
It has been reported by several researchers that potato peels are rich in compounds, such as anthocyanins, glycoalkaloids, phenolic compounds and flavonoids <a href="#reference_list_24">[25]</a>. These bioactive compounds are associated with good health because they protect the body’s cells from oxidative processes and have antioxidative, anti-inflammatory, antitumor and anticancer properties. Due to their extensive application potential and beneficial properties, potato peels are well worth the attention of researchers and industry. Hence, new methods to discover these compounds and their applications to food matrices are being explored <a href="#reference_list_25">[26, 27]</a>. Take this into consideration, in our present investigation, an attempt has been made to isolate, identify and chemically characterize the bioactive compounds responsible for synergistic antioxidant interactions in combination from potato peel extracts of potato varieties Kufri Chipsona-3 and Kufri Jyoti with a aim to develop novel natural antioxidants for food and pharmaceutical industries.
To achieve the goal, at first, TLC bioautography guided detection and isolation of antioxidant components from peels extracts of potato varieties Kufri Chipsona-3 and Kufri Jyoti were carried out. TLC bioautography guided detection and isolation technique were used in the present investigation because  (1) this assay results in the direct and quick localization of bioactive compounds obtained from plant extracts; (2) it is a quick, low-cost and straightforward method for isolating compounds from complex plant extracts; and (3) it is discovered to be able to accurately identify chemical compounds in plant extracts; (4) It is crucial to avoid time consumption for inactive compound isolation, and this assay makes it easier to combine chromatographic separation with an in-situ activity measurement, which enables to locate and identify bioactive compounds and this technique in recent years has become  an important tool for detection and isolation of  bioactive compounds <a href="#reference_list_14">[15]</a>.
TLC bioautography analysis showed the presence of three antioxidant components in peel extract of Kufri Chipsona-3 that  have Rf values 0.33, 0.52 and 0.57, respectively (Figure 1(b)) and four antioxidant components with Rf 0.14, 0.22, 0.36 and  0.61 in peel extract of potato varieties Kufri Jyoti (Figure 2 (b)). These seven antioxidant components of different Rf values from peel extracts of two selected potato varieties were subjected to combined antioxidant efficacy study in DPPH radical scavenging method followed by isobologram analysis to observe their possible antioxidant interactions (synergistic, additive or antagonistic).  Results obtained from combined antioxidant efficacy study revealed that among the possible combinations tested, only one combination between components with Rf : 0.57 of Kufri Chipsona-3 peel extract and Rf : 0.61 of  Kufri Jyoti peel extract exhibited synergistic antioxidant interactions (CI < 1). Besides, most of the possible combinations showed an additive antioxidant effect (CI =1), whereas a few combinations showed antagonistic antioxidant efficacy (C>1) (Table 1).The possible reason behind the antagonistic  antioxidant interactions between a few tested combinations observed in the present investigation is not clear right now. According to Olszowy et al., it may be due to (1) antioxidant adduct formation that is competitive, (2) antioxidant regeneration by an antioxidant that is more effective and (3) antioxidant oxidation by the radicals of a less effective antioxidant; and antioxidant microenvironment modification by another antioxidant <a href="#reference_list_27">[28]</a>. In our previous study on antioxidant activity of potato peel extracts, we have used BHT as reference standard antioxidant <a href="#reference_list_28">[29]</a>. But,  in the present investigation , our main objective is to  identify and chemically characterize the bioactive components from  active  extracts of potato peels responsible for synergistic antioxidant interactions. That is why, in the present investigation we have not used any reference standard antioxidant like BHT.
Now, these two bioactive components from peel extract of potato varieties Kufri Chipsona-3 (Rf : 0.57) and Kufri Jyoti (Rf : 0.61) were subjected to spectrometric (UV/Vis, FT-IR, HR-LC MS/MS) analyses for chemical characterization of bioactive compounds responsible for synergistic antioxidant interactions. Collective spectrometric analyses data of these two antioxidant components of peel extracts of potato varieties Kufri Chipsona-3 and Kufri Jyoti are shown in Table 2. These data were compared with MS library data as well as relevant references <a href="#reference_list_22">[23, 24]</a> to chemically characterize the bioactive compounds. Comparing the obtained spectral data of bioactive components from peel extracts of  potato varieties Kufri Chipsona-3 and Kufri Jyoti (Table 2) with MS library data as well as relevant literature data, the antioxidant component from peel extract of Kufri Chipsona-3 with Rf : 0.57 and m/z: 327.5 was found to be prodelphinidin trimer and the antioxidant component from peel extract of Kufri Jyoti (Rf : 0.61; m/z 897) was found to be 5-Hydroxy-3’,4’,7-trimethoxyflavone.
Cytotoxicity studies of plant extracts or isolated bioactive compounds  are  essential because to be minimal nontoxicity is a crucial process that results in the successful development of a pharmaceutical or cosmetic product. In the present investigation, we therefore, evaluated the cytotoxic potential of two phenolic compounds prodelphinidin trimer and 5-hydroxy-3',4',7-trimethoxyflavone isolated respectively from potato peel extracts of Kufri Chipsona-3 and Kufri Jyoti  that showed synergistic antioxidant interactions in combination using Allium cepa root growth inhibition assay [30, 32]. The Allium cepa test was chosen because it is straightforward with a lower relative cost than other tests and is versatile, and only requires a minimal amount of laboratory equipment to be used <a href="#reference_list_30">[31]</a>. Due to the fact that root development inhibition can be seen as a manifestation of cell division arrest, it is considered to be the most sensitive measure [32, 33]. The IC50 of the tested bioactive components in combination in Allium cepa root growth inhibition assay was found to be >200µg/ml in this investigation. Furthermore, no bulges, hooks, or twists in the roots, as well as any discernible morphological alterations in root consistency and colour were seen and therefore the test compounds in combination  can generally be considered as safe <a href="#reference_list_33">[34, 35]</a>.
<h5>Conclusion</h5>
Thus, phenolic compounds prodelphinidine trimer and 5-hydroxy-3',4',7-trimethoxyflavone isolated  respectively from peel extracts of Kufri Chipsona-3 and and Kufri Jyoti were found to be responsible for synergistic antioxidant interactions in combination and can generally be considered as safe. In our previous study, peel extracts of Kufri Chipsona-3 and Kufr Jyoti showed synergistic antioxidant interactions both in various in-vitro models as well as against oxidation of omega-3 fatty acids enriched food supplements. Therefore, these two phenolic compounds isolated from Kufri Chipsona-3 and Kufr Jyoti potato peel extracts may serve as a potent antioxidant applied both in food and pharmaceutical industries. The findings may also help to convert waste potato peels from zero value waste to value added products. To the best of our knowledge this is the first report of synergistic antioxidant interactions of phenolic compounds isolated from potato peel extracts.
<h5>Acknowledgements</h5>
The corresponding author was given a research fellowship by the Dean of Studies at the Indian Statistical Institute in Kolkata, India, allowing the authors to continue their research. The Heads, SAIF, Indian Institute of Technology, Madras, India, and Indian Institute of Technology, Bombay, India, SAIF-CDRI Lucknow provided laboratory facilities to conduct spectrometric analysis of material, to whom the authors also acknowledge.  The authors also acknowledge the contribution of potatoes from Dr. Ashish Chakraborty of Bidhan Chandra Krishi Viswavidyalaya (BCKV), Kalyani, West Bengal, India.
<h5>Ethical approval</h5>
This article does not contain any experiment on human participants or animals performed by any of the authors.
<h5>Consent to participate</h5>
Informed consent was obtained from all individual authors included in the study.
<h5>Consent to publish</h5>
All authors consent for the publication of identifiable details, which can include data, tables, figures and details within the text to be published in the Journal of Food, Nutrition and Diet Science.
<h5>Authors' contribution</h5>
The study conception and design were done by Dr. Rabi Ranjan Chattopadhyay. Material preparation, data collection and analysis were performed by Dr. Abhishek Bhattacharya. The first draft of the manuscript was written by Dr. Rabi Ranjan Chattopadhyay and all authors commented on previous version of the manuscript. All authors read and approved the final version of this manuscript.
<h5>Funding  </h5>
This study has been funded by internally funded project of Indian Statistical Institute, Kolkata, India.
<h5>Conflict of interests</h5>
The authors have no relevant financial or non-financial interests to disclose.
<h5>Availability of data and materials</h5>
All data are included within the manuscript.
<h5>Declaration of generative AI and AI assisted technologies in the writing process</h5>
No AI or AI assisted technologies were used in writing process.

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Bhattacharya, A., & Chattopadhyay, R. R. (2024). Isolation, identification and chemical characterization of compounds from phenolic extracts of peels of Kufri Chipsona-3 and Kufri Jyoti potatoes having synergistic antioxidant interactions in combination. Journal of Food, Nutrition and Diet Science, 2(1), 29–40. https://doi.org/10.55976/fnds.22024123129-40

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Isolation, identification and chemical characterization of compounds from phenolic extracts of peels of Kufri Chipsona-3 and Kufri Jyoti potatoes having synergistic antioxidant interactions in combination

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Isolation, identification and chemical characterization of compounds from phenolic extracts of peels of Kufri Chipsona-3 and Kufri Jyoti potatoes having synergistic antioxidant interactions in combination

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