Biology

Effect of differential growth conditions and environmental stress factors in reactive oxygen species (ROS) generation and polyphenol biosynthesis in Salvia Rosmarinus

Geethika Reddy Biddala‡* Amador Valley High School , Neha Mandava‡* Santa Clara High School , Aryan Makhija*Irvington High School , Edison Liu*Mission San Jose High School, Soumya Suresh

1 Author affiliation

KEYWORDS (Word Style “BG_Keywords”). Polyphenols, ROS, Salvia Rosmarinus, Growth Conditions, Environmental Stress

 

ABSTRACT

Polyphenols are organic compounds that are naturally produced by plants to combat stressors, as well as the regulation of plant processes. Many medicinal plants such as herbs, fruits, vegetables, and even oils contain large amounts of these molecules. In addition to aiding plants in their defense, polyphenols have been found to lower the deficits caused by Alzheimer’s disease by weakening the oxidative stress caused by the accumulation of beta-amyloid in the brain. In order to increase the polyphenol production in plants to work effectively against AD, we altered the environmental factors of 4 polyphenol producing plants by affecting them with saline solution, UV irradiation, and hydrogen peroxide, respectively and adding a control. In this experiment, the herb rosemary was used as it is not only rich in polyphenols, but also contains over four different types of these molecules and has a low germination period. To identify the effects of these stress factors in the rosemary plants, we conducted a FOX assay to detect the amount of hydrogen peroxide in the plants and quantified the number of polyphenols produced through the Folin-Ciocalteu (FC) assay. Later, the rosemary extracts from each growth condition were screened for potential anti-amyloidogenic activity against wild type human amyloid-beta 42.

INTRODUCTION / BACKGROUND

Consumption of polyphenols has been proven to lower risks of encountering chronic illnesses such as Alzheimer’s Disease. AD occurs when extracellular amyloid-beta plaque builds up in the body until it reaches the brain and results in neurological deficits and immense oxidative stress. Fruits, vegetables, tea, and wine are all high in polyphenols which attenuate oxidative stress in the body by correcting ROS imbalance and preventing them from causing damage to imperative molecules such as DNA, proteins, and lipids in cells. Certain polyphenols, such as flavonoids, have shown to inhibit and clear out almost all amyloid-beta aggregation in patients with AD. Rosemary, as aforementioned, contains a variety of polyphenols that are shown to inhibit AD and is especially abundant in 4 different types of polyphenols. A regular diet of Polyphenols can aid in cutting down the amyloid-beta aggregation in the brain.

INSPIRATION

We drew our inspiration for altering environmental stress factors in rosemary plants to induce increased polyphenolic production from taxol. Taxol is a cytotoxic cancer chemotherapy drug derived from the Pacific Yew tree. Because of this compound’s scarcity and high ecological cost, alternative sources for taxol have been developed, including a chemically synthesized precursor of taxol from a relative of this tree. In addition to this, scientists were able to alter the environmental conditions of the Pacific yew tree, such as the pH, water activity, and elicitors of different concentrations to successfully enhance the production of taxol, similar to the experiment we conducted.

GROWTH CONDITIONS

In order to discern which environmental stress factor positively influenced polyphenol production the most, we stressed out three rosemary plants using saline solution, UV irradiation, and hydrogen peroxide along with a DI water control plant. The UV plant was placed under an ultraviolet lamp and watered with 400 mL of deionized water daily. The salt stressed rosemary and hydrogen peroxide plant were watered with a saline and H2O2 solution respectively in increasing concentration from 25mM to 300mM over the course of eight days. All four plants were kept under a growth light to stimulate the sun. After the stress period, plant material was gathered from all four plants and frozen. We noticed a pronounced change in all of the plants over the course of the week; the saline stressed plant grew away from the light and was studded with salt crystals, the UV plant was nearly translucent, and the hydrogen peroxide plant grew the most in length, substantially noticeable in its leaves, in contrast to the other plants.

METHODS & RESULTS

The colorimetric FOX assay was used to measure the concentration of hydrogen peroxide(ROS) in the plants and quantify the oxidative stress. To begin the FOX assay we froze 10, 0.2g samples of each plant and then used a mortar and pestle to crush the plants along with the extraction solution (To 200 mM perchloric acid (PA): Add 1.7 mL of HClO4 (M=12) to 98.3 mL of deionized water). Next, each sample mass was measured again using a scale, in order to preserve the same mass. The samples were then centrifuged and the supernatant was collected and pipetted into eppendorf tubes. To every 1 mL of supernatant, 1 mL of working reagent (100 mL of working reagent containing 500 μM ammonium ferrous sulfate [19.6 mg, MW=392], 50 mM H2SO4 [0.28 mL, M=18 M], 200 μM xylenol orange [tetrasodium salt, 14.3 mg, MW=716.6], and 200 mM sorbitol [3.64 g, MW=182]). was added. If the resulting solution was below 0.5 mL the solution was diluted by two fold or even three fold depending on the volume, and later multiplied by the absorbance. The absorption rate of all 40 samples were then measured at 560 nanometers using a UV vis spectrophotometer and the concentration of hydrogen peroxide was later calculated with the extinction coefficient (2.24×105M-1cm-1and expressed as µmol/g FW) to determine the amount of stress.

In order to accurately quantify the phenolic compounds in rosemary, we used the Folin Ciocalteu, or FC, assay. This assay allowed us to identify which stress factor in rosemary produced the largest quantity of polyphenols. To start off the FC assay we extracted the polyphenols by blending 30g of each rosemary plant and 100 mL of ethyl acetate together and subsequently added acetone. Dimethyl sulfoxide was then pipetted into the compound and the resulting solution was left on a stir plate overnight. Later, the extractions were placed in a buchner flask and vacuumed, isolating the insoluble solids from solvent. The solutions were added to a round bottomed flask and rotovapped until only a small amount of solvent remained. After the extraction process, each extraction of polyphenol material was added to a cuvette with isopropanol, FC reagent, and water. These cuvettes sat for 30 minutes, and in the meanwhile a blank was created as a control to help identify the absorbance rates. The spectrophotometer was then used to figure out the absorbances, as well as quantify the value of polyphenols. After this, we utilized the Beer’s Law plot of gallic acid as a standard to find the total polyphenolic content in each stress factor. We noticed that the control contained the highest total polyphenol concentration, while the most oxidatively stressed plant, the saline stressed, contained the lowest total polyphenol concentration. This was not anticipated, as polyphenols are generally produced to counteract stressors, but the control had not been stress-induced. We initially thought that the polyphenolic production would positively correlate with the oxidative stress, but it was the contrary.

IN-VITRO SCREENING

To determine which of the four plants had a substantially negative effect on the aggregation of the amyloid-beta protein, we used in-vitro screening. We started off by adding a 20uL buffer in well A of the microplate and 20uL of buffer in C1, C2, C3. Then the Alzheimer’s peptide, or AB-42, was induced with our own polyphenolic extractions, into well A, B, and C1, C2, C3。The microwell plate was scanned in a microwell plate spectrophotometer that contained fluorescent lights. Subsequently, the plate was placed into an incubator which was set to the ideal temperature at which the protein could cultivate. The results were measured with the spectrophotometer every 5 minutes. The microwell plate results revealed which polyphenols from the oxidative stressed plants reduced the aggravated cells in the Alzheimer’s peptide. This indicated that the saline stressed plant had a profound negative effect on the aggravated protein, while the UV and the control plant initially slowed down, but then increased the aggregation of the amyloid-beta.

ANALYSIS

After calculating the hydrogen peroxide and the amount of oxidative stress in each plant, we observed that according to our results, stress and polyphenolic content vary indirectly with each other. An explanation of this interesting phenomenon could be that the short stress time shuts down the plants’ biosynthetic capabilities. Upon further research, we found that several stressors like salt, inhibit metabolic processes until they are relieved. Regarding the screening of beta-amyloid along with rosemary, we noticed that namely the saline and H2O2 stressed extractions had a negative effect on the aggregation of the protein, which would be beneficial in people with AD.

CONCLUSION

As aforementioned, the control contained the most abundant amount of phenolic content, which is unforeseen because we believed that plants that had induced stress would contain the highest phenolic content. In order to produce the most accurate results, the stress time could have been prolonged and there could’ve been more plants of each stress type. Through understanding the role of polyphenols in cellular functions like stress and defense, we can help further studies of other areas and come closer to finding a potential cure to neurodegenerative diseases like Alzheimer’s.

FIGURES

Figure 1: Day one of stress inducing the four rosemary plants (from left to right: Saline, Hydrogen peroxide, UV, Control)

Figure 2: Day eight of stress inducing the four rosemary plants (from left to right: Hydrogen peroxide, Control, UV, Saline)

 

Figure 3: Taxus Brevifolia is chemically synthesized to to Pacitaxel (taxol), a anticancer drug that was FDA approved 1993

CHARTS

Chart 1. Effect of Growth Conditions on ROS production

Chart

We observed that the saline-stressed plant had the most ROS species; hence, it was the most stressed.

Chart 2. Effect Of Growth Conditions on Polyphenol production

Chart

Contrary to our belief, the control contained the most phenolic content.

Chart 3. Total Polyphenol Content vs. Oxidative stress

Chart

Polyphenolic production had a negative correlation with the amount of oxidative stress induced

Chart 4. Change in Absorbance vs. Time

The Saline stressed plant had the most profound negative effect on Amyloid beta protein aggregation Chart

TABLES

Table 1. FOX Assay Absorption Rates (Au)

Control

UV

Salt

Hydrogen Peroxide

0.49

0.5

1.14

0.82

0.4

0.86

1.9

0.39

0.66

1.52

1.42

0.86

0.25

0.82

2

0.72

0.31

1.52

1.96

1.98

0.22

1.58

3.4

2.43

0.32

1.16

1.82

1.74

0.5

0.96

3.4

2.4

0.37

1.47

3

1.7

0.33

1.64

2.97

2.13

Table 2. FOX Assay Hydrogen Peroxide Concentration (μmol/g)

Control

UV

Salt

Hydrogen Peroxide

0.00208

0.02126

0.00485

0.00349

0.00170

0.00366

0.00808

0.00166

0.00281

0.00646

0.00604

0.00366

0.00106

0.00349

0.00850

0.00306

0.00132

0.00646

0.00833

0.00842

0.00094

0.00672

0.01446

0.01033

0.00136

0.00493

0.00774

0.00740

0.00213

0.00408

0.01446

0.01020

0.00157

0.00625

0.01276

0.00723

0.00140

0.00697

0.01263

0.00906

Table 3. FOX Assay Hydrogen Peroxide Concentration Averages (μmol/g)

Control: 0.001637

UV: 0.007674

Salt: 0.00979

H2O2: 0.00821

Table 4. FC Assay Absorption Rates (Au)

Control

UV

Salt

Hydrogen Peroxide

0.713

0.408

0.192

0.342

AUTHOR INFORMATION

Corresponding Author

* Geethika Reddy Biddala

* Neha Mandava

Author Contributions

The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript. / ‡These authors contributed equally.

Funding Sources

ASDRP and Olive Children Foundation

ACKNOWLEDGMENT

We thank Ms. Soumya Suresh greatly for allowing us this opportunity to be a part of this project and guiding us in this experiment. We thank Mr Edward Njoo for providing scientific guidance and advice throughout the lab work. Thank you to our parents for their continuous support. Finally, last but not least, thank you to the Olive Children Foundation and ASDRP for creating this program allowing us to experience and be a part of this project.

ABBREVIATIONS

AD, Alzheimer’s Disease; FC, Folin-Ciocalteu; UV, Ultra-Violet; AB, Amyloid-Beta; Au, Absorbance Units

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ABOUT THE AUTHOR

Neha Mandava is currently a rising junior at Santa Clara High school and is extremely passionate about the scientific and medical field. In her free time, she tutor students in science and volunteer throughout the community, as well as dance and participate in robotics tournaments. She is also a part of a directed research program and is working on the synthesis of phenols and NNRTIs.

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