CHRISTIAN HANBALI 19010025
Dr. Robert D. Hancock
Marsia Gustiananda, S.Si., Ph.D.
INDONESIA INTERNATIONAL INSTITUTE FOR LIFE SCIENCES (i3L)
Comparative Analysis of Phytonutrients from Organically and Conventionally
Produced Horticultural Crops
PREFACE
This internship report was made to fulfill the requirements for the credited internship program. The title of this project is “Comparative analysis of Phytonutrients from Organically and Conventionally Produced Horticultural Crops.” This research period up to the writing of this report was from July to August 2022 in Dundee, Scotland.
I would like to express my deepest gratitude toward The British Council which funded this program, and the University of Dundee faculties: Professor Kim Dale, Dr. Paul Davies, and Dr. Sheriar Hormuzdi. Without them, this overseas internship would not be possible. I also would like to extend my gratitude towards my field supervisor Dr. Robert D. Hancock who showed me the ropes during my time at James Hutton Institute and is still helping me write this report. To coworkers in JHI: Raul Huertas, Simon Pont, Ceri Austin, and also Richard who were so kind and warm, and helped me through my time in JHI. To my friends Enrica Salim and Aurellia Ramara who supported each other during our time in Dundee. And to my friend Lowis, Patrice, and Tamung for being the support system while writing this report.
Jakarta, Indonesia, 6 October 2022
Christian Hanbali
ACKNOWLEDGEMENT
Comparative analysis of Phytonutrients from Organically and Conventionally Produced Horticultural Crops
This internship was done at Indonesia International Institute for Life-Sciences (i3L)
AUTHOR:
Christian Hanbali 19010025 Biomedicine
ACKNOWLEDGE BY:
SUPERVISOR AT i3L: FIELD SUPERVISOR:
Marsia Gustiananda, S.Si., Ph.D. Dr Robert D. Hancock
HEAD OF DEPARTMENT AT i3L:
Elizabeth Sidhartha, B.Sc., M.Sc.
TABLE OF CONTENTS
Preface...ii
Acknowledgements... iii
Table of Contents...iv
List of Figures and Tables...v
List of Abbreviations...vi
Summary...vii
Chapter 1: Introduction... 1
Chapter 2: Project Description... 3
Chapter 3: Findings...7
Chapter 4: Conclusion and Recommendations... 12
Chapter 5: Self Reflection... 13
Appendices... 14
Reference... 18
LIST OF FIGURES AND TABLES
List of Figures:
Figure 1. AsA content of various products with concentration below 200 ppm Figure 2. AsA content of various products with concentration above 200 ppm
Figure 3.AsA content of courgettes with various types of cultivar and grown organically and conventionally.
Figure 4.AsA content of potatoes grown with various natural fertilizers against conventional potatoes.
Figure 5.Total Antioxidant content of various products with concentration below 200 ppm.
Figure 6.Total Antioxidant content of various products with concentration above200 ppm.
Figure 7.Total Antioxidant content of courgettes with various types of cultivar and grown organically and conventionally.
Figure 8.Total Antioxidant content of potatoes grown with various natural fertilizers against conventional potato.
LIST OF ABBREVIATIONS
AsA : Ascorbic Acid
DHA : Dehydroascorbic acid SMs : Secondary Metabolite FD : Freeze dry
MPA : Meta-phosphoric Acid
TCEP : Tris(2-carboxyethyl)phosphine MetOH : Methanol
HPLC : High Performance Liquid Chromatography
SUMMARY
Organic farming is a method of farming that limits or even forbids the use of artificial fertilizers and pesticides. Organically grown crops are frequently associated with healthier foods than conventionally grown crops. Kinkell Farm is a family-run organic farm based in Lennoxtown, Scotland.
The key objective of the farm is to produce healthy organic products. Hence, the study was done to compare the Kinkell Farm organically grown products with conventional products. The plant extract was observed for its phytonutrient content, which was quantified using HPLC and a spectrophotometer. The result shows that products grown by Kinkell Farm on average have increased amounts of vitamin C or ascorbic acid. But different species and cultivars have a lesser increase or even decrease in vitamin C content. Some organic crops also have higher total antioxidant content, even triple that of conventional products. Although the increase of phytonutrients is not the case for all plant species, Other factors such as cultivars and ambient environmental conditions seem to play a bigger role in determining the nutritional content of the crops than the method of farming alone.
Keywords:Organic Agriculture, Phytonutrient, Ascorbic acid, Antioxidant
CHAPTER 1: INTRODUCTION
1.1. Host Institution / Company
The internship was done at the James Hutton Institute, which is located in Dundee, Scotland.
The University of Dundee (UoD), as the host institute, invited three i3L students to participate in their summer internship program and served as the liaison between i3L and the James Hutton Institute.
Summer lab-based research traineeships at the University of Dundee were open to students from all over the UK and affiliated universities around the world who wanted to hone their skills and knowledge outside of theater classes and practical courses. The James Hutton Institute, which focuses on botanical biosciences, was also one of the institutes and labs affiliated with the UoD School of Life Sciences.
1.1.1 Description of the company
The James Hutton Institute, named after one of Scotland's most famous geologists, James Hutton, is an interdisciplinary scientific research institute in Scotland that was founded in 2011.
The Institute has two sites in Scotland; one in Aberdeen and one in Dundee. The Dundee site also hosts the Plant Sciences department of the University of Dundee. The institute has the vision to be at the forefront of innovative and transformative science for sustainable management of land, crops, and natural resources that supports thriving communities. The actualization of their vision can be observed in the mission of their work: to conduct excellent science and engage in new ways of working across disciplines, with business, policy, and society, that guide contemporary thought and challenge conventional wisdom, ensure trust, and deliver the best outcomes for all.
1.1.2 Description of Department
The Cell and Molecular Sciences (CMS) department is based in Dundee and is home to more than 100 plant scientists with expertise in cell and molecular biology, genomics, genetics, pathology, and physiology. CMS's primary focus is crop improvement, with a portfolio that includes genetic improvement of cereals, potatoes, and soft fruit crops in terms of yield and quality, resource use efficiency, and pest and disease resistance. CMS investigates processes from the gene and molecular level to the field scale, providing knowledge to address food security issues and develop sustainable production systems in the face of environmental change.
1.1.3 Product of the Host Institution / Company
The James Hutton Institute is mainly a research institute that collaborates with the government or the private sector in fundamental and applied research to optimize their crop
growing conditions and the crop product itself. The James Hutton Institute also provides sequencing and microarray services, plant transformation, containment glasshouses, temperature-controlled growth rooms, and an imaging suite using state-of-the-art microscopy and imaging equipment.
CHAPTER 2: PROJECT DESCRIPTION
2.1. Internship Project
2.1.1. Project Background
Pharmacognosy is the study of plants and other natural substances as possible sources of drugs. Plants have always been used as medicine by mankind to treat health-threatening diseases and it is still popular to obtain new drug candidates as it is the oldest medical practice for humans (Orhan, 2017). For example, Madagascar periwinkle was used in folk medicine to treat diabetes. However, after the researchers were able to isolate the active compound, the chemical compound was used to treat cancer instead after various experiments showed its capability to reduce actively proliferating cells. Vinblastine and vincristine are chemotherapy drugs used to treat many kinds of cancer, in which the drugs are made from the aforementioned Madagascar periwinkle extract (Ravina, 2011).
Balanced diet of nutrients from whole foods is also known to have a role in maintaining optimal health conditions. Many nutrients are essential to humans only obtainable from eating green products, such as vitamin C. While many lesser essential nutrients such as antioxidants and other plant based micronutrients consumption are also associated with optimal health. As cited by Hancock, R.D. in 2007, there are protective effects of vegetable consumption against cardiovascular diseases (Ness and Powles, 1997) and various types of cancers (Cooke et al, 2005).
And as cited by Barański, M., et al in 2014, numerous human dietary intervention studies have reported that increased dietary intake of antioxidant or polyphenolic-rich foods do gives protection against various chronic diseases, including CVD, certain cancers (e.g. prostate cancer) and neurodegenerative diseases (Del Rio et al., 2013)
Phytochemicals or phytonutrients are bioactive compounds that are generated from secondary plant metabolism in response to environmental changes. (Monjotin, Amiot, Fleurentin, Morel & Raynal, 2022). Plants produce 2 kinds of metabolites: primary and secondary metabolites (SMs). Plants have two primary metabolites: sugars, amino acids, organic acids, and fats, which are found in all plants tissue most of the time and are crucial for their growth and survival; and secondary metabolites, which are composed of diverse chemicals including carotenoids, phytosterols, polyphenols, and saponins. The content of the secondary metabolites is sometimes specific to some types of plants and may have specific functions for the plant as a form of adaptation toward external stimuli (Meskin, 2002).
These SMs are not only beneficial for the plant; they are often also used by humans to make medicine since the dawn of time. Kittakoop, Mahidol & Ruchirawat, in their 2013 research
stated that alkaloids which are a class of plant secondary metabolites have numerous chemicals synthesized from it that have been used for medicinal purposes. To name a few: Cocaine, Berberine, Vincristine, Galantamine, Atropine, Quinidine, and Quinine. The function of each drug varies from anesthetic to anticancer drug and their usage is already well recorded. This diversity of function shows the potency of using plant SMs as various therapeutic agents.
Despite the usage of phytochemicals already dating back to the early civilization. There is no one sure way to consistently produce crops with high phytochemical value. There are many factors that change the nutrient content of a plant, even when two of the same cultivar are planted. Various genetic, ontogenetic, morphogenetic, and environmental factors can influence the biosynthesis and accumulation of SMs. SM accumulation is strongly dependent on a variety of environmental factors such as light, temperature, soil water, soil fertility, and salinity, and for most plants, a change in an individual factor may alter the content of SMs even if other factors remain constant (Yang et al., 2018).
Organic farming is an agricultural system that uses fertilizers of organic origin, such as compost manure or green manure, and avoids using synthetic fertilizers or pesticides. Kinkell Farm is a family-run organic and microgreen farm based in Lennoxtown, East Dunbartonshire. A key objective of the farm is to produce healthy organic produce and hence the objective of the current project was to measure key nutritional parameters in Kinkell farm produce compared to conventionally produced products bought from a local supermarket.
2.1.2. Scope of the Project
The scope of the project covers preparations of horticultural products for analysis, extraction using various techniques and solvents, and phytochemical assay which includes AsA content assay and total antioxidant assay.
2.1.3. Objectives
The aim of this project is to compare various nutritional qualities of products grown in organic farm conditions with conventionally grown products extracted using MPA/ TCEP and Methanol solution; and measured using HPLC and spectrophotometer.
2.1.4 Problem formulation and proposed solutions
The hypothesis to be tested was that production of fruit and vegetables using organic cultivation techniques resulted in produce with higher nutritional quality. Hence significant difference in nutritional quality can be observed by performing various phytochemical quantification assays.
Freeze-drying of a sample
Freshly obtained vegetables are cleaned from any debris or dirt and then cut into matchstick-like shapes. The vegetable cuttings are then put into a 50mL falcon tube and then snap frozen by immersing the tubes into a liquid nitrogen vat. After all the samples are frozen, the sample is then brought to the freeze-dry machine. The falcon tube caps were replaced with poked aluminum foil and then the tubes were loaded into the machine to freeze dry for days.
Freeze Dried Sample Milling
The freeze-dried samples were checked for their consistency. If the sample snaps when bent, then the sample is fully dried. Fully dried samples were then weighed and then grinded using a coffee grinder into a very fine powder. The freeze-dried powders are then kept in a -20 °C freezer until needed.
Ascorbic Acid Extraction
20 mg of freeze dried (FD) powder was put into a 2mL centrifuge tube, and then filled with 1 mL of cold 5% MPA/TCEP solution. The chemical reaction can be seen as inAppendix 3. in which cold MPA will prevent ascorbic acid oxidation and precipitate enzymes; TCEP acts as electron donor for DHA to revert back into AsA. The tubes were then put into a rolling mixer machine and then left to run at 40 RPM for 30 minutes in a 4 °C cold room. Using a refrigerated centrifuge, the tubes were spun down at maximum RPM for at least 15 minutes at 4°C to separate the sediment from the supernatant.
Ascorbic Acid Quantitative Analysis
200 μL of each sample supernatant from the ascorbic acid extraction process was transferred into HPLC vials. The standard was made using solid crystalline L-ascorbic acid powder dissolved in the 5% MPA/TCEP, making a 10 mg/mL AsA solution, which will be diluted into a series of solutions: 500 μg/mL, 300 μg/mL, 200 μg/mL, 150 μg/mL, 100 μg/mL, 80 μg/mL, 60 μg/mL, 40 μg/mL, 20 μg/mL. The sample order was randomized to prevent drifting bias. The sample and standards were then loaded into the HPLC machine according to the randomized order. The HPLC column was a Coregel64H cation interaction column (4 X 300 mm), the mobile phase was 4 mM H2SO4 and AsA was detected by UV/vis absorbance at 245 nm. HPLC was then run according to the preset setting for ascorbic acid, and peak analysis was also done to detect and measure the AsA content in the solution.
Total Antioxidant Extraction
50 mg of FD powder was put into a 2mL centrifuge tube, and then filled with 1 mL of cold 50% MetOH + 1% Formic solution. The tubes were then vortexed at the highest power. Using a refrigerated centrifuge, the tubes were spun down at maximum RPM for at least 15 minutes at 4°C to separate the sediment from the supernatant.
Total Antioxidant Content: Quantitative Analysis
Standards were made by using a 10 mg/mL trolox solution diluted in pure methanol, the stock solution is then diluted into a series of solutions with lower concentration using distilled water. The chemical reaction can be seen as inAppendix 4.in which the trolox or antioxidants will reduce the folin reagent. 50 μL of sample and trolox standard were loaded into a 96-well plate. To each well, 50 μL of 1x Folin-Ciocalteu’s reagent was added, followed by 100 μL of 13%
sodium carbonate solution. The plate is then shaken and allowed to incubate in the dark for an hour. Using a plate reader, measure the absorbance at 750 nm with DW as a blank.
CHAPTER 3: FINDINGS
3.1. Results
3.1.1 Ascorbic Acid Quantitative Analysis
Ascorbic acid content was measured in five batches with HPLC. Each batch was measured along with a set of standards. On average, the R2value was 0.99, which shows all batches have reliable linear regression as shown inAppendix 5. Overall comparative results can be observed in Figure 1. and Figure 2. While Figure 3. and Figure 4. show the in-depth comparison of conventional courgette against various courgette cultivars grown organically; and conventional potato against organic potato grown using various natural fertilizers respectively. The results are then checked for its statistical significance using 2 way ANOVA (for overall comparative figure) and 1 way ANOVA (for In-depth comparison of courgette and potato).
Figure 1.AsA content of various products with concentration below 200 ppm.
Figure 2.AsA content of various products with concentration above 200 ppm.
Figure 3.AsA content of courgettes with various types of cultivar and grown organically and conventionally.
Figure 4.AsA content of potatoes grown with various natural fertilizers against conventional potato.
3.1.2 Total Antioxidant Content: Quantitative Analysis
Total antioxidant content was measured in five batches using plate reader. Each batch was measured along with a set of standards. On average, the R2value was 0.981, most of the batches have near perfect linear regression with the lowest value at 0.957 as shown in Appendix 6.
Overall comparative results can be observed inFigure 5.andFigure 6. whileFigures 7.andFigure 8.show the in-depth comparison of conventional courgette against various courgette cultivars grown organically; and conventional potato against organic potato grown using various natural fertilizers respectively. The results are then checked for its statistical significance using 2 way ANOVA (for overall comparative figure) and 1 way ANOVA (for In-depth comparison of courgette and potato).
Figure 5.Total Antioxidant content of various products with concentration below 200 ppm.
Figure 6.Total Antioxidant content of various products with concentration above 200 ppm.
Organic crops that notably have higher amounts of antioxidants compared to their conventional counterparts are: carrots, peas, spring onion, peppers, and broccoli. The rest of the organic crops do not have significantly higher antioxidant content levels. Carrots, peas, and
cabbage notably have 2 to 3 times the antioxidants compared with their conventional crop counterparts. The p-value for carrot and peas were calculated independently from its cohort due to their lower concentration of antioxidants and much less standard deviation.
Figure 7.Total Antioxidant content of courgettes with various types of cultivar and grown organically and conventionally.
Figure 8.Total Antioxidant content of potatoes grown with various natural fertilizers against conventional potato.
3.2 Discussion
Ascorbic Acid Quantitative Analysis
Quantification of AsA was done with HPLC since HPLC also removes interfering impurities from the sample which provide more accurate results when water soluble chemical is measured. The overall AsA concentration graph was separated into 2 graphs to accommodate crops with lower AsA concentration to be compared along cohorts with similar range of AsA. Courgette and potato result was separated due to presence of sub variable: cultivar and fertilizer content respectively. There are some organic crops that have higher AsA content than its conventional counterpart: beet tops and
cabbage. But overall, there are not many organic crops that have significantly higher amounts of AsA compared to the conventional crops.
According to statistical analysis of the results, it can be seen that the organic crop and conventional crops do not have significant differences in AsA content (P-value above 0.05). This can be interpreted that most organically grown crops do not have higher AsA content compared to the conventional crop counterparts.
This finding aligns with the study of Duarte, A., et al. in 2010 in which they stated that organic farming methods do not consistently produce citrus with higher vitamin content, despite the fact that citrus fruit with the highest AsA content was found in the organically grown citrus group. They stated that the main factors that contribute to the AsA content would be the crop species and cultivars.
Plants also have been shown to change secondary metabolite composition in response to external environmental changes (light, humidity, and temperature), which could interfere with the results.
Despite the insignificance of increasing the crops' AsA content, organic farming methods provide some benefits, such as more soluble solids and a lower maturation index (Duarte, A., et al., 2010).
Another study that also backs up these experiment results is a study by Worthington, V. in 2001. He compiled various paper or journal articles and showed that there is a 27% mean increase in AsA content in organic crops when compared to conventional crops of the same species. The result, as shown inAppendix 1., is that when put into average, there are around 19% more AsA in organic crops. But the same paper also stated that the difference in AsA content could vary from -100% to +507%. Appendix 1. shows that there are various species of crops that show an increase in AsA content when grown organically, and yet there are also a few species that have lower AsA content when grown organically.
Total Antioxidant Content: Quantitative Analysis
Overall comparative results shown inFigure 5.andFigure 6.show that few organic crops do have a slight increase in antioxidant content. This finding aligns with the meta-analysis of Baraski et al. in 2014, in which their analysis found that there are statistically significant and meaningful differences in phytochemicals between organic and non-organic crops, especially in their antioxidant content as seen in Appendix 2. Although looking at the graph, we can also see that not all organic crops show a difference in antioxidant content. Hence, we can assume that plant species play an important role in determining whether organic farming methods will affect the crops antioxidant content.
Organic courgettes, on the other hand, show a decrease in antioxidant capacity as seen in Figure 7. All courgette cultivars that were grown organically have slightly fewer antioxidants than those of conventional courgettes. This demonstrates that organic farming methods do not always
result in crops with higher antioxidant content; rather, the courgette's cultivars play a larger role in determining the plant's antioxidant content.
When looking at the data on organic potato antioxidant capacity or content inFigure 8., it aligns with the findings of Moreno-Reséndez et al. in 2016, who discovered that plants (especially peppers) grown with organic manure have higher antioxidant capacity in general. The statistical test using one-way ANOVA showed that the difference in antioxidant content is significant, boasting a p-value of 0.0001. Potatoes grown organically using horse manure as their fertilizer are shown to have the highest antioxidant content of all samples, with almost 4 times the amount of conventional potato antioxidant content. Other organic potatoes grown with green waste and mushroom compost have antioxidant content that is around two times that of conventional potatoes.
Executive summary:
Although most organic crops do not have significant increases in essential nutrients such as vitamin C, some species have positive increased profiles of antioxidant levels when they are grown organically. Meanwhile some species and cultivars have lower amounts of vitamin C and total antioxidants than its conventional product counterpart. The change of phytonutrient of those crops can be attributed to the change of soil composition and ambient growing environment which was introduced by the organic farming method.
In hindsight, organic farming methods do not consistently produce crops with higher nutritional value; especially because plant species and cultivars have a bigger role in determining the plants nutrition value. Hence we can conclude that organic production can’t be viewed as a universal and sure method for improving food quality.
But there are still various health benefits from consuming organic products compared to consuming conventional/ mass produced products. Baraski et al. in 2014 found that organic products have 75% lower amounts of pesticides residues and toxic metals such as cadmium that are associated with pesticide usage. Also stated by Walter J. Crinnion in his article written in 2010: organic food contains less trace amount of pesticides and artificial fertilizers. The article also cited that decrease in toxic chemicals improves the health benefit in breastfeeding and lower the amounts of allergic manifestation and eczema in two years old babies.
However there are some limitations in this study. Kinkell farm organic farming method is not fully disclosed. Their techniques, ambient condition, and soil composition is not fully documented while these factors may contribute to the organic crops' varying nutrients. Some plant extracts also have strong coloration in which they will interfere with the plate reader measurement, namely beet roots and beet tops (chard). The kinkell farm sample cultivar is also not disclosed clearly. If true comparative study wished to be done, then these factors need to be put into the constant. PCR and sequencing can also be done in order to make sure plants of the same cultivar are being compared.
CHAPTER 4: CONCLUSION AND RECOMMENDATION
Conclusion
According to the results from this study, some species and cultivars of plants benefit from organic farming methods. These plants show a slightly higher amount of essential nutrients such as vitamin C and notable increase in antioxidants. Although better nutrition quality is not always the case with every species and cultivar of plants. Some crops even show lower amounts of vitamin C and antioxidants when planted organically. Hence organic farming is not a one for all solution or universal method for improving food quality.
Despite the inability to consistently increase the nutritional value of the crops, there are still benefits to consuming organic products. Organic Products have lower traces of toxic chemicals which are linked to many diseases such as cancer and cardiovascular diseases. Elevated levels of antioxidants and many nonessential nutrients also benefit consumer health in general. These benefits are already well documented by various authors and researchers through numerous studies around the world, hence it can be concluded that the benefits of organic crops are not pseudoscience.
Recommendations
Antioxidants are a class of chemicals that are made up of very diverse chemicals. More phytochemical screening and specific compound quantification is needed to map the change of chemistry on organically grown crops.
CHAPTER 5: SELF REFLECTION
From this internship, I found out the difference between eastern and western culture regarding work. We eastern scientists have a tendency to prioritize producing a large number of results in a short period of time, even if the results are not reliable. The providence of results seems to be our main selling point or value, and pumping out results means we are productive and reliable.
While being there, I was humbled. My working speed may match that of master or postgraduate individuals. But my rush to provide as many results as I can makes me exhausted and bored with repetitive tasks. In the end, due to my rushed nature, sometimes I need to redo some experiments.
Regarding my strength, I find my creative thinking can make me more productive since it tackles and eases the repetitive work that numbs the brain and muscles. My supervisor also praised me for my tenacity and ability to adapt, since even in a new environment and culture, I can still blend in and work. To add salt to the wound, during my work there, the laboratory was being demolished and relocated. Much equipment was removed and my work was hindered, but I still persisted and am able to deliver satisfying output.
From this internship, I learned about the difficulty of extracting various phytochemicals. I thought nutrient extraction was limited to maceration with solvent, but instead many factors need to be considered in order to keep the extraction and measurement valid. I also learned how to use various tools and equipment that i3L does not have or I did not have access to. I hope when the time comes when I need to extract some plant chemicals alone, the skills I got from this internship will help me. And during the writing of this report, it also came to my attention that I need to reduce my tendency to procrastinate and deal with my anxiety. Much of my time could be saved if I had consulted with my supervisor earlier. Instead, I am afraid to show my results despite mistakes being a common thing in the learning process.
Thanks to this internship opportunity, I have secured another 5-month long internship in Scotland. If the result of that internship is favorable and I have found a topic that interests me, I would like to apply for a PhD. Of course, in order to get to that, I need to keep my performance up despite the increasing workload of our final years. Or at least, if I have not found my passion or topic of interest, I would like to try to apply for an LPDP scholarship for my masters degree.
I would like to thank i3L for their robust curriculum and infrastructure/equipment. Despite the workload being massive, and the difficulty also being comparable to those of master's studies, surviving and being active in academia was very rewarding. My CV was adorned with various lab experiences that few can only dream of participating in. The i3L lab and curriculum were ahead of and above the rest of the life science universities in Indonesia, and can be compared with those of universities abroad.
APPENDICES
Appendix 1.Table of average AsA content in both organic and conventional crops.
Appendix 2.Table of average antioxidant content in both organic and conventional crops.
Appendix 3.Chemical reaction of TCEP converting DHA into AsA.
Appendix 4.Chemical reaction of antioxidants reduces folin ciocalteu, turning the folin reagent color into blue.
Appendix 5.Graphs of AsA standard linear regression analysis compilation.
Appendix 6.Graphs of Antioxidant standard linear regression analysis compilation.
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