Utilizing Environmental DNA for the Detection of Invasive Goldfish (Carassius Auratus) and Koi (Cyprinus rubrofuscus) in Southeastern Massachusetts Water System 

By: Mia Oliviera

I am happy to report that this semester’s research has been a huge, fun, learning opportunity, and I am very appreciative of the OUR’s grant to assist me in my work this Spring. In March, I spent my spring break taking water samples and negative controls at all ten sites across Southeastern Massachusetts. These samples were then brought to the UMD campus to be filtered over the following few weeks within Dr. Drew’s genetics lab. Samples were filtered using a vacuum pump setup under a sterile hood, and bleach was used to ensure no cross-contamination between samples.

Starting in early April. I utilized the DNeasy Blood and Tissue Kit and the Powersoil PowerLyzer kit to optimize an eDNA extraction method that best suits my extraction needs. My two positive control samples (goldfish and koi ponds) were extracted with both kits to determine which would produce the best product. It was found that the Powersoil PowerLyzer kit allowed the filter and cells to break down best and produce a clearly visible PCR product. I determined the best extraction method was to utilize the DNEasy Powersoil PowerLyzer kit for the remainder of my samples, with a few modifications to the listed protocol.

Once all samples and negative controls were extracted, I ran a PCR utilizing Dr. Drew’s genetics lab resources and using universal fish DNA primers that amplify all fish species. I chose to use fish primers instead of my species-specific primers to ensure the filtering, extraction, and PCR methods captured fish eDNA. The PCR products were run in an electrophoresis gel and imaged. The first PCR I ran produced a lot of DNA bands but showed signs of potential contamination within the PCR step. I chose to do a second PCR while under a sterile hood, which produced a cleaner product and a few samples containing DNA, but did show some amplification of fish DNA within my negative control samples. Dr. Drew and I worked through troubleshooting methods and discussed ways we could optimize protocols for the Fall semester and ways to use different primers, as well as my species-specific primers. I decided to do more research on eDNA sampling, extraction, and PCR methods over the summer to find the best protocols for my research in the Fall.

I have learned so much about the world of eDNA over the last few months. I find the troubleshooting aspect not to be a roadblock, but a huge opportunity to problem-solve and optimize my own protocols. eDNA research is still in its early stages, but it has so much potential in its application. I am very excited to continue my research on this in the Fall, and once again thank the OUR for this funding opportunity.

(Sampling at Snows Pond, Rochester, MA)

Chemical Profiling and Evaluation of Antioxidant and Neurodegenerative Enzyme Inhibitory Potential of Flavonol and Flavonol Glycoside Fractions of Cranberry Pomace and Fruit Extracts

By: Ryley Thatcher

Abstract

The large cranberry (Vaccinium macrocarpon) is widely known for its antioxidant potential due to its potent polyphenolic chemical constituents, such as anthocyanins, flavonols, phenolic acids, and proanthocyanidins. The composition of polyphenolic compounds within a cranberry sample varies with different factors such as growth stage, growth region, and the cultivar of the berry, which successively affect antioxidant capacity. This study aimed to analyze and compare the phytochemical composition and antioxidant activity of five new 2024 cranberry cultivars (Ruby Star, Mullica Queen, Pilgrim King, Welkers, and Haines) grown by the Cranberry Station in Wareham, MA. A small-scale extraction method was optimized, and antioxidant activity was determined by colorimetric assays, including the DPPH and FRAP assays. Results from all cultivars showed a dose-dependent response for both assays. Welkers had the best DPPH scavenging activity (IC50 =104 μg/mL ∓ 17), and Pilgrim King had the best reducing power based on FRAP (IC50 =184 μg/mL ∓ 11). The total phenolic content and proanthocyanidin (PAC) content of samples were measured by the Folin-Ciocalteu and DMAC assays, respectively. Haines had the highest total phenolic content, and Mullica Queen had the highest PAC content. HPLC-DAD analysis will be conducted to identify and quantify various polyphenolic compounds in the samples. Results will be shared with the Cranberry Station and could potentially inform future agricultural practices and dietary recommendations.

Introduction

Cranberries consist of various bioactive compounds known for their health benefits and have been used in traditional medicine for their antioxidant, anti-inflammatory, and antimicrobial properties. The large cranberry (Vaccinium macrocarpon), also known as the American Cranberry, is native to North America. In the United States, the large cranberry is most commonly grown in New Jersey, Massachusetts, Oregon, and Wisconsin. Beyond North America, V. macrocarpon is also extensively cultivated globally in locations such as Canada, Europe, and South America [1]. While other species of cranberry also contain significant amounts of nutritional phytochemicals, in the modern marketplace, the large cranberry is considered an important success story of the functional foods industry due to its notably high concentration of antioxidant compounds [2].

A specific constituent of cranberries largely associated with their high antioxidant activity is a class of compounds known as polyphenols. The polyphenolic contributors in cranberries are primarily flavonoids, including flavonols, proanthocyanidins, anthocyanins, phenolic acids, and benzoates [3]. The phenolic compounds in cranberries achieve antioxidant effects by scavenging the free radicals of harmful ROS (reactive oxygen species) that, when accumulated in the human body, can be linked to illnesses such as cancers and neurodegenerative diseases [1].

Figure 1. Cranberry Polyphenols: anthocyanins, phenolic acids, flavonols, and phenolic acids

The phytochemical composition of these polyphenols is influenced by multiple factors: cultivar, genotype, growing season, ripening, processing, and storage of cranberry fruit [4]. A cultivar’s influence on the phytochemical composition in cranberries is particularly substantial, given that each cultivar is a specific variety of cranberry, bred for its desirable traits. It is crucial to understand the impact specific cultivars have on the content of these phenolic compounds to maximize the gain of nutritional benefits from cranberries and for any potential medicinal applications in the future. This research will aim to compare the phytochemical composition and antioxidant activity of five Massachusetts Cranberry Cultivars collected on October 2nd, 2024, from the Cranberry Station in Wareham, MA. Results from this project will be shared with the cranberry station to influence future growing strategies and dietary recommendations.

Figure 2. Cultivar Breeding Map (not pictured: Ruby Star: Hy red x Bergman hybridPilgrim King: Pilgrim x GH2 hybrid)

MATERIALS AND METHODS

Sample Collection

Several cultivars of cranberry fruit were collected from the cranberry bogs at the Cranberry Station in Wareham, MA, in October 2024. After collection, samples were flash-frozen with liquid nitrogen and stored at -20°C until further use.

Sample Preparation and Extraction

Flash-frozen cranberry fruit samples from each cultivar were freeze-dried and ground into a fine powder. One gram of cranberry powder per cultivar was dissolved in 10 mL of a 40:40:19.9:0.1 acetone:methanol: formic acid: DI water v/v solvent. The resultant solution was mixed by a stir plate for one hour, followed by ultrasonication for 30 minutes. After mixing, the solutions were stored in the fridge for 24 hours. The next day, suction filtration was performed twice; the first time suction filtration was performed, the residue was re-dissolved in 5 mL of extraction solvent. For each filtration, the filtrate was kept as the liquid extract. Extracts were stored in the freezer until further antioxidant analysis, and the extraction process was performed in triplicate for each cultivar.

Folin-Ciocalteu Assay

The total phenolic content of each cultivar was measured by the Folin-Ciocalteu Assay according to Ainsworth et. al [5] with modification. 20 mg of cranberry powder from each cultivar was dissolved in 2 mL of ice-cold 95% v/v methanol, resulting in a 10 mg/mL sample concentration. Samples were mixed by vortexing and ultrasonication for 30 minutes, and then left in the fridge overnight. The next day, 10% v/v Folin-Ciocalteu reagent was prepared and kept in the dark until plating. Samples from the previous day were centrifuged at 13,000 g for 5 minutes, and the supernatant was used for analysis. 40 μl of each sample was transferred to an Eppendorf tube, and 95% v/v methanol was used as the blank. 800 μl of Folin reagent was transferred to each tube, and samples were left to stand for 5 minutes. 800 μl of 700 mM Na2CO3 was added to each tube and mixed by vortexing. Lastly, 360 μl of distilled water was added to each tube, reaching a final volume of 2 mL. Samples were incubated in the dark for 2 hours at room temperature. After incubation, 200 μl of each sample was plated in triplicate on a clear 96-well microplate. Once samples were plated, absorbance was read at 760 nm using a UV-Spectramax plate reader. Total phenolic content was determined as gallic acid equivalents by comparison to a gallic acid standard curve.

DMAC Assay

The total proanthocyanidin (PAC) content of each cultivar was determined by the DMAC (P-Dimethylaminocinnamaldehyde) assay as previously described by Feliciano et. al [8] with modifications. 50 milligrams of samples from each cultivar were dissolved in 1 mL of a PAC (75%/24.5%/0.5% acetone/water/acetic acid) extraction solution in triplicate. To mix the samples, they were sonicated for five minutes and placed on a Line-lab Orbital Shaker for one hour at 250 rpm. Once the samples were mixed, they were kept in the freezer overnight. The next day, samples were centrifuged at 3,000 rpm for five minutes, and the supernatant was kept for plating. DMAC reagent was prepared by dissolving 0.05 grams of DMAC in 50 mL of a 75%, 12.5%, 12.5% v/v/v ethanol/hydrochloric acid/DI water solution. 5 μl aliquots of each cultivar sample were plated in triplicate on a clear 96-well microplate, followed by the addition of 65 μl PAC extraction solution to each well. 70 μl of PAC extraction solution was plated in triplicate as the blank.= 100 μl of DMAC reagent was added to each well to reach a final volume of 170 μl, and the plate was incubated for 30 minutes. Absorbance was read at 640 nm on the UV Spectramax plate reader. Absorbances were compared to a PAC standard curve to express results as mg PAC per g DW.

DPPH Free Radical Scavenging Antioxidant Activity Assay

The free radical scavenging antioxidant activity of each cranberry sample was evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay as previously described by Liu et al [6]. A 100 μg/mL DPPH solution was prepared using methanol. 75 μl of cranberry extracts were diluted with 4.925 mL of methanol to achieve a target stock concentration of 1000 μg/mL. A two-factor serial dilution was performed for each cultivar extract from 1000 μg/mL to 31.25 μg/mL. For this assay, different 96-well plates were used for the different cultivar samples, resulting in the use of five different microplates. 100 μl of each concentration was plated in triplicate, followed by the addition of 25 μl of DPPH reagent. 125 μl of methanol was plated in triplicate as the blank. Once all samples were plated, they were incubated for 30 minutes at room temperature in the dark, and absorbance was read at 517 nm by the Spectramax plate reader. Percent inhibition was calculated using the following equation:

% Inhibition= A₀-A₁/A₀ *100%

Where A₀ was the absorbance of the control solution with no radical, and A₁ was the absorbance
of the sample at 517 nm. Percent Inhibition results were converted to IC50 values by OriginLab Pro software.

FRAP Ferric Reducing Antioxidant Power Assay

A method to examine the ferric reducing antioxidant power of each cranberry sample was developed by previous FRAP procedures with modifications [9][10]. A 10:1:1 acetate buffer, tripyridyltriazine (TPTZ), FeCl3 FRAP working solution was prepared as follows: 50 mL of 300 mM pH 3.6 acetate buffer, 5 mL of 10 mM TPTZ solution in 40 mM HCl, and 5 mL of 20 mM FeCl3 were added to a 250 mL Erlenmeyer flask. The FRAP working solution must be used within three hours of preparation for accurate measurement. Sample preparation was the same as performed for the DPPH assay, obtaining a stock target concentration of 1000 μg/mL to perform a two-factor serial dilution down to 31.25 μg/mL. Similar to the DPPH assay procedure, five separate 96-well microplates were used for each cultivar. 20 μl of each concentration was pipetted into the microplate in triplicate, and methanol was used for the blank. 180 μl of FRAP solution was transferred to each well and mixed by pipette. 200 μl of methanol was plated in triplicate as the blank. Samples were incubated in the dark at room temperature for 30 minutes, and the absorbance was read at 593 nm by the Spectramax microplate reader. The percent reducing power of each sample was calculated by the following equation:

FRAP% = Sample Absorbance – Blank Absorbance/ Sample Absorbance x 100

Percent reducing power results were converted to IC₅₀ values by the Origin Lab Pro software.

RESULTS + DISCUSSION 

Total Phenolic Content 

The objective of this project was to analyze and compare the antioxidant activity of five new 2024 cranberry cultivars, Mullia Queen, Ruby Star, Welkers, Haines, and Pilgrim King, grown by the Cranberry Station in Wareham, MA. The first parameter examined was the total phenolic content by the Folin-Ciocalteu assay. The Folin assay measures the ability of polyphenols present in the cranberry fruit samples to reduce the Folin Reagent, which is an oxidant that consists of a combination of phosphotungstic and phosphomolybdic acids. A color change from yellow to blue occurs at 760 nm, signifying the completion of the reduction reaction. Results showed that cultivar Haines had the highest total phenolic content (32.5土2.5 mg gallic acid/g dry powder), while Mullica Queen (MQ) had the lowest total phenolic content of the five cultivars (25.7 土4.4 mg gallic acid/g dry powder). Cultivars Welkers (30.4 土2.2 mg gallic acid/ g dry powder), Ruby Star (29.1 土2.1 mg gallic acid/ g dry powder), and Pilgrim King (27.8 土 0.51 mg gallic acid/ g dry powder) fell in the middle. Error bars indicate overlapping standard deviations between some cultivars, indicating that the total phenolic content between these cultivars was not completely significantly different.

Figure 3. Comparison of Total Phenolic Content by Folin Assay

Total Proanthocyanidin Content

Proanthocyanidins are widely regarded in the industry for their urinary tract health benefits. PACS are specifically known for their potential in the prevention and treatment of UTIs due to their anti-adhesion capabilities that can prevent E. coli bacteria from sticking to the walls of the bladder and urinary tract. The DMAC assay was used to quantify total soluble PAC content in each cranberry cultivar sample. The DMAC reagent reacts with PACS through a condensation mechanism between the aldehyde of the DMAC and the C8 of the PAC’s terminal unit, which is read at an absorbance of 640 nm. The DMAC assay is a more accurate colorimetric detection of PACs due to the specificity of the reaction that minimizes interference from other polyphenolic compounds that may be present in the samples.

Figure 4. Comparison of Total PAC Content

Mullica Queen was found to have the highest PAC content (78.7 土 2.7 mg/ g dry powder) of the five cultivars analyzed, and Haines was found to have the lowest PAC content (39.5 土 2.7 mg/ g dry powder). Pilgrim King, Ruby Star, and Welkers were in the middle of the range with a PAC content of 64.7 土 4.3 mg/ g dry powder, 62.2 土 1.7 mg/g dry powder, and 43.6 土 2.6 mg/g dry powder, respectively. Error bars are relatively small, indicating consistent results across replicates. Interestingly, even though Haines was the highest in total phenolic content, the trend shows Haines to have the lowest PAC content, which could suggest the Haines cultivar is higher in other polyphenolic constituents like anthocyanins, phenolic acids, and flavonols. MQ was the lowest in total phenolic content but highest in PAC content, indicating that it has a high PAC: phenolic compound ratio. Since one of the goals of cultivating Mullica Queen was to produce a cultivar with high PAC content, MQ being high in PAC content was expected.

Free Radical Scavenging Antioxidant Activity

The DPPH assay was used to determine the free radical scavenging ability of the cranberry fruit samples. The assay measures the antioxidant activity by measuring the cranberry polyphenols’ ability to scavenge the DPPH radical reagent. The neutralization of the radical is signified by a visible color change from purple to yellow at 517 nm.

Cultivar	IC50 (μg/mL)
Welkers	105 土 17
Ruby Star	144 土 24
Pilgrim King	172 土 42
Haines	227 土 32
Mullica Queen	330 土 57

Table 1. Comparison of Free Radical Scavenging by IC₅₀ Value

Free radical scavenging results were reported as IC50 values, which measure the concentration of the sample that would be required to inhibit 50% of the DPPH radicals. IC50 is inversely related to antioxidant activity, implying that a lower IC50 specifies a stronger antioxidant ability. Based on the data presented in Table 1 and Figure 5, Welkers had the highest free radical scavenging capacity (IC50= 105 μg/mL 土 17) followed by Ruby Star (144 μg/mL土 24), Pilgrim King (172 μg/mL 土 42), and Haines (330 μg/mL 土 57). Mullica Queen had the weakest free radical scavenging ability (IC50=330 μg/mL 土 57).

Figure 5. Free Radical Scavenging Capacity Measured by DPPH Assay

The results show that even though Mullica Queen had the highest total PAC content, it had the lowest free radical scavenging capacity. Welkers had the best free radical scavenging activity, and it performed moderately in both total phenolic and total PAC assays. This could suggest that some types of polyphenols contribute more to free radical scavenging capacity than others. For example, the MQ results propose that PACS may not have a significant effect on the antioxidant activity for this reaction. Figure 6 shows an example of how, for all samples, antioxidant activity decreased with concentration.

Figure 6. Example of Dose Response DPPH Results

Ferric Reducing Antioxidant Power

Another mechanism of antioxidant activity was measured by the FRAP assay, which measured the ferric reducing antioxidant power of each cultivar. The FRAP assay quantifies the ability of antioxidants in a sample to reduce Fe³⁺ to Fe²⁺ in an acidic environment. The reaction produces a blue-colored complex, which has a wavelength of 593 nm. The intensity of the consequent blue color correlates with the reducing power of the sample.

Cultivar	IC50 (μg/mL)
Pilgrim King	184 土 11
Mullica Queen	234 土 6
Welkers	273 土 25
Ruby Star	303 土 21
Haines	377 土 37

Table 2. Comparison of Ferric Reducing Power Measured by FRAP Assay

Similarly to the DPPH results, ferric reducing power was expressed as IC₅₀ values. As seen in Table 2 and Figure 7, Pilgrim King had the lowest IC₅₀ value (184 μg/mL 土 11), indicating it had the highest ferric reducing power out of the five cultivars. Mullica Queen and Welkers had relatively similar IC₅₀ values (234 μg/mL 土 6 and 273 μg/mL 土 25, respectively), followed by Ruby Star (303 μg/mL 土 21). Haines had the lowest ferric reducing power of the samples with an IC50 of 377 土 37 μg/mL).

Figure 7. Ferric Reducing Power Measured by FRAP Assay

Interestingly, some of the FRAP trends disagree with the DPPH results. For example, Mullica Queen had the weakest free radical scavenging activity in the DPPH assay, but had the second-best ferric reducing power as seen in Figure 7. These discrepancies between the DPPH and FRAP results highlight that the antioxidant activity performance of samples depends on the reaction mechanism being measured, and different cultivars may be better or worse depending on the parameter being examined. Figure 8 shows an example of the dose-dependent response data that was obtained for each cultivar, demonstrating how, as concentration decreases, reducing power decreases.

Figure 8. Example of Dose-Dependent FRAP Data

CONCLUSIONS

This project highlights that the cranberry cultivar has a substantial impact on polyphenolic composition and antioxidant activity between cranberry fruit samples. A variety of colorimetric assays were utilized to measure parameters of each cultivar sample, such as total phenolic content, total proanthocyanidin content, free radical scavenging capability, and ferric reducing power. Cultivar Haines was found to have the highest total phenolic content, while Mullica Queen was found to have the lowest phenolic content of the five cultivars. Oppositely, MQ was found to have the highest PAC content, and Haines was found to have the lowest PAC content. DPPH IC50 data show Welkers to have the overall strongest radical scavenging antioxidant capacity, and Mullica Queen to have the least free radical scavenging capacity. FRAP IC50 values demonstrated Pilgrim King to have the highest percent ferric reducing power, and cultivar Haines had the weakest ferric reducing power. Inconsistencies in trends between assays indicate that antioxidant activity differs based on the assay or mechanism that is being measured, so different cultivars may be desired for different purposes. As the present work provides an initial idea on the different cultivars’ polyphenolic composition and antioxidant activity, future work for this project would be to quantify and compare the polyphenolic composition of anthocyanins, phenolic acids, flavonols, and PACS present in the samples by HPLC-DAD analysis.

ACKNOWLEDGMENTS

The authors would like to acknowledge the support of the UMass Cranberry Health Research Center, Giverson Mupambi at the UMass Cranberry Station, and the UMD Office of Undergrad Research for this research project.

REFERENCES

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Chemical Profiling and Evaluation of Antioxidant and Neurodegenerative Enzyme Inhibitory Potential of Flavonol and Flavonol Glycoside Fractions of Cranberry Pomace and Fruit Extracts

By: Elena De Pra

Abstract

The imbalance between reactive oxygen species and antioxidant defense is often implicated in neuronal damage associated with neurodegenerative diseases like Alzheimer’s and Parkinson’s. Quercetin, a naturally occurring flavonoid, has been extensively studied for its promising neuroprotective potential due to its ability to effectively combat oxidative stress as a potent antioxidant. Typically found in the standard diet as quercetin glycosides in foods such as cranberries, quercetin is known for its free radical scavenging abilities, though its poor bioavailability limits its therapeutic effectiveness. This research aimed to investigate the antioxidant and enzymatic inhibitory potential of two fractions isolated from cranberry pomace and cranberry fruit extract containing quercetin and quercetin-3-galactoside, respectively. The extracts were profiled for polyphenolic compounds, and the desired flavonoids were isolated and confirmed using high-performance liquid chromatography (HPLC). Antioxidant activity was assessed using DPPH, ABTS free radical scavenging, and FRAP reducing power assays. Results demonstrate that the aglycone quercetin fraction exhibited higher antioxidant capacity than the quercetin-3-galactoside fraction. The quercetin fraction also demonstrated strong inhibitory activity against acetylcholinesterase. Future research will evaluate the inhibitory effect of these fractions to inhibit other enzymes associated with neurodegenerative diseases, including butyrylcholinesterase and monoamine oxidase A/B. Results from this study aim to shed light on how glycosylation may influence therapeutic potential with respect to inhibitory effects, to link antioxidant activity to neuroprotection.

Introduction 

Neurodegenerative disorders represent a critical public health crisis affecting millions of individuals regardless of age, sex, education, or income. The World Health Organization (WHO) estimates 6.8 million people die per year of neurological disorders¹. The significance of this issue grows as human life spans extend, making elderly populations increasingly vulnerable to neurological diseases like Alzheimer’s and Parkinson’s. This topic is sensitive not only to directly affected individuals, but also to their families, caregivers, and society at large, who fear the uncertainties of aging. Modern therapeutics focus on symptom management, as no treatments currently exist that reverse neuronal death or effectively delay progression.

Natural products have been used for thousands of years in traditional medicine for their biological and pharmacological properties. Natural product-derived compounds show promising potential in drug development for their role in treating neurodegenerative disorders, particularly attributed to their polyphenolic content². Polyphenolic compounds are proven to have neuroprotective effects, notably through their capacity to neutralize reactive oxygen species, characteristic of their antioxidant activity². Consuming these naturally occurring compounds, rich in various fruits and vegetables, is a preferred alternative over pharmaceutical drugs as it has both restorative and preventative potential without adverse effects (Fig. 1).

Fig. 1. Schematic Illustration of Neuroprotective Flavonoids Against Oxidative Stress-induced Neurodegeneration

Flavonoids, a class of polyphenolic compounds, exhibit significant cognitive potential, partly through their ability to enhance antioxidant defenses and protect against oxidative stress³. Quercetin, a naturally occurring flavonoid, has been extensively researched for its neuroprotective and anti-inflammatory properties and promising tool in treating neurodegenerative diseases such as Alzheimer’s and Parkinson’s⁴. Quercetin-3-galactoside is the predominant glycosidic form of quercetin present in cranberries⁵. The main structural difference lies in the sugar moiety attached in place of the 3’ OH group connected by a glycosidic bond on the central-C-ring of the core quercetin structure (Fig. 2).

Fig. 2. Quercetin and Quercetin-3-galactoside Chemical Structure

Quercetin glycosides are more commonly found in derivatives than free form quercetin⁶. Glycosides are more water-soluble than their respective non-sugared aglycones and have generally been reported to possess greater bioavailability in vivo⁷. Although glycosides often have reduced antioxidant activity, their structural differences can enhance compound stability and absorption into the bloodstream, highlighting their potential as a preferred alternative in targeting specific drug properties^7.

Although quercetin has been investigated for its inhibitory effects on neurodegenerative enzymes, there is limited research investigating quercetin-3-galactoside’s potential as an inhibitor of neurodegenerative-related enzymes. Acetylcholinesterase (AChE) is an enzyme responsible for hydrolyzing acetylcholine, a critical neurotransmitter that plays a role in memory, learning, and attention⁸. Butyrylcholinesterase (BChE) is a nonspecific enzyme that catalyzes the hydrolysis of choline and non-choline esters, including acetylcholine⁹. Low levels of acetylcholine are characteristic of individuals affected by Alzheimer’s disease⁸. Monoamine oxidase (MAO) exists as two isoforms: MAO-A, which oxidizes serotonin, norepinephrine, and epinephrine, and MAO-B responsible for oxidizing dopamine¹⁰. Monoamine oxidases are relevant to Parkinson’s disease, a neurological disease impacted by reduced levels of dopamine, norepinephrine, and serotonin¹¹. Targeting these enzymes and discovering these compounds’ inhibitory potential could highlight their therapeutic relevance.

Comparing isolated fractions of quercetin and its glycosides’ antioxidant capacity and neuroprotective potential is relevant to the development of modern therapeutics that can be used in combination with other potential natural compounds to address root mechanisms of disease. It is important to explore the relationship between flavonoids and their glycosides and how structural differences can impact antioxidant and neuroprotective properties to optimize treatment. In addition to its potential in modern therapeutics, findings from this study, if successful, could influence dietary recommendations to reduce the risk of onset of neurodegenerative diseases¹².

Experimental 

Preparation of Crude Extracts 

The Mullica Queen (MQ) cranberry cultivar was collected from cranberry bogs at the Cranberry Station in Wareham, MA, in September 2023. Cranberry pomace samples, consisting of a mixed variety of cultivars, were obtained from Ocean Spray. All samples were flash-frozen in liquid nitrogen and stored at -20°C. Samples were ground, lyophilized, and further ground into a fine powder. Dried fruit powder (40 g) was dissolved (400 mL) in an extraction solvent composed of acetone/methanol/distilled water/formic acid (40:40:19:1 v/v). The mixture was stirred for 1 hour, sonicated for 30 minutes, and refrigerated overnight. The following day, the sample was vacuum filtrated, and the solid residue was re-extracted using half the original solvent volume (200 mL). The same process was repeated the following day with half the original solvent amount for a total of three extractions. All filtrates were combined, rotary evaporated, frozen, and
lyophilized for further analysis.

Isolation of Concentrated Fruit Extracts 

A Diaion HP-20 chromatography column was prepared for the purification of polyphenolic compounds from the Mullica Queen (MQ) extract. A glass chromatography column was packed with clean sand and glass wool. The resin was pretreated by activating with methanol for 15 minutes and washing with distilled water prior to being transferred into the column, allowing the resin to settle. Crude extract (20 g) was dissolved in a minimal volume of methanol/formic acid (99.9:0.1v/v) and was loaded onto the column and allowed to absorb into the packing. After 15 minutes had elapsed, the elution of free sugars was initiated using distilled water until the eluate appeared colorless; colorless eluates were discarded. Colored bands containing polyphenols and terpenoids were eluted using methanol/formic acid (99.9: 0.1 v/v). Acetone was used to elute the residual yellow that appeared in the packing. All eluates were collected, combined, and rotary evaporated to remove solvent. Samples were freeze-dried to obtain a concentrated powder and stored at 0°C.

Isolation of Flavonol Fractions

Sephadex LH-20 (3.0 x 22.0 cm), a hydroxypropylated, cross-linked dextran resin with an exclusion limit of 4-5 kDa and flow rate capacity of < 60 cm/hr, was used to isolate flavonoid derivatives. A glass column is packed with glass wool and sand. The Sephadex resin (40 g) was pretreated by swelling in 70% MeOH for 3 hours at room temperature. Particles were decanted, resuspended, and poured into the column, which was allowed to stand overnight in 70% MeOH before separation. Concentrated fruit extract (1 g) was dissolved in a minimal volume of formic acid/water/methanol (1:50:48.9 v/v/v) and loaded onto the column. After 15 minutes of absorption, phenolic compounds were eluted using 70% MeOH, and colored bands were collected separately. Brown bands containing proanthocyanidins were eluted with 70% acetone, followed by 100% acetone to elute any remaining proanthocyanidins. All collected fractions were rotary evaporated, frozen, lyophilized, and stored for further analysis.

Identification of Compounds by HPLC

Chromatographic Conditions

High-performance liquid chromatography (HPLC) analysis was performed using an Atlantis T3 C18 column (4.6 x 150 mm, 3 µm) on a Waters HPLC system equipped with a pump (Empower e2695), a photodiode array (PDA) detector (Waters 2998), an online degasser, and an autosampler. Mobile phase A consisted of water/phosphoric acid (99.5:0.5, v/v), and mobile phase B contained water/acetonitrile/glacial acetic acid/phosphoric acid (50:48.5:1.0:0.5, v/v/v/v). A reverse-phase gradient program was used, developed by Liang Xue 2021 and modified by Maureen Otieno (2023). The injection volume was 20 µL, with a flow rate of 0.900 mL/min and a total run time of 31 minutes. Chromatographs were recorded at 520, 355, 310, 280 nm to measure anthocyanidins, flavonols, phenolic acids, and proanthocyanidins, respectively.

Standard Preparation 

A stock solution of the standard was prepared by dissolving 2 mg of each standard in 2 mL of HPLC-grade methanol to obtain a concentration of 1000 ppm. Serial dilutions were performed to obtain concentrations of 100, 50, 25, 12.5, 6.25, and 3.13 ppm. Solutions were sonicated and filtered through a 0.45 mm syringe filter before injecting 20 µL into the HPLC system.

Sample Preparation

Extract samples (2 mg/5 mg/10 mg) were dissolved in 1 mL of methanol to obtain the desired concentration. Solutions were sonicated and filtered through a 0.45 mm PTFE syringe filter and 20 µL of each sample was injected into the HPLC system.

Folin-Ciocalteau Assay

The quantification of total polyphenolic content in both the crude fruit and pomace extracts was measured using a modified protocol of the Folin-Ciocalteau assay as described by Ainsworth et al.¹³. 20 mg of cranberry/cranberry pomace powder samples were weighed in triplicate and dissolved in 2 mL of ice-cold 95% v/v methanol to obtain a concentration of 10 mg/mL. Samples were ultrasonicated for 30 minutes and refrigerated overnight. The following day, samples were centrifuged for 5 minutes at 10,250 rpm. 40 µL of the supernatant was collected into duplicate Eppendorf tubes and reacted with 800 µL of 10% v/v F-C reagent. The solution was vortexed and allowed to stand for five minutes. 800 µL of sodium carbonate was then pipetted, followed by 360 µL of distilled water. A blank sample was prepared by substituting the stock solution with 40 µL of 95% v/v methanol. Samples were incubated in the dark at room temperature for two hours before measuring absorbance. 200 µL of respective solutions were pipetted in triplicate onto a 96-well microplate. Absorbance was measured at 760 nm using a SpectraMax 190 microplate reader. Total phenolic content was calculated as gallic acid equivalents (GAE) using the regression equation between gallic acid standards at A760 nm, using the following calculation:

T = C/C¹ X MW

T = total phenolic content in mg/g, in GAE (gallic acid equivalents)

C = concentration of gallic acid established from the calibration curve in mg/m

C¹ = concentration of the extract in mg/mL

MW = the molecular weight of gallic acid

DPPH Radical Scavenging Assay

The DPPH radical scavenging activity of both quercetin and quercetin-3-galactoside fractions was evaluated according to a modified method of Baliyan et al.¹⁴. 5 mg of each respective fraction was dissolved in 20 mL of methanol, vortexed briefly, and subjected to ultrasonication to ensure samples were fully dissolved and obtain a concentration of 250 µg/mL. From this stock solution, a serial dilution was performed to obtain the following range of concentrations: 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95 µg/mL. A DPPH solution (300 µM)was prepared by dissolving 5 mg of 2,2-diphenyl-1-picrylhydrazyl in 50 mL of methanol. 100 µL of each respective concentration was pipetted in triplicate onto a 96-well microplate. 25 µL of DPPH solution was added and mixed by pipetting. A control solution was prepared by pipetting 100 µL of methanol with 25 µL of DPPH. The blank solution contained 125 µL of methanol. The plate was incubated in the dark at room temperature for 30 minutes, and absorbance was measured at 517 nm using a SpectraMax 190 microplate reader. The following formula was used to compute percent inhibition:

DPPH % Inhibition = A₀ – (A₁ -A_b)/A₀ x 100%

A₀: Control absorbance with no radical scavenger (DPPH + MeOH)

A₁: Sample absorbance (DPPH + scavenger)

A_b: Blank absorbance (MeOH)

Ferric Reducing Antioxidant Power

The ferric reducing power of quercetin and quercetin-3-galactoside fractions was measured using a modified version of the method described by Gashaye et al.¹⁵. Samples were prepared by dissolving 5 mg of extract in 20 mL of methanol, briefly vortexing, followed by ultrasonicating to obtain a concentration of 250 µg/mL. From this stock solution, a serial dilution was performed to obtain the following range of concentrations: 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95 µg/mL. A FRAP working solution was prepared containing 25 mL of acetate buffer (300 mM, pH 3.6), 2.5 mL of 10 mM TPTZ in 340 mM HCl, and 2.5 mL of 20 mM FeCl3 • 6H2O solution (10:1:1 v/v/v). This working solution was designated for usage within three hours of preparation. 20 µL of the respective sample was pipetted in triplicate into a 96–well microplate, followed by the addition of 180 µL of FRAP working solution and thoroughly mixed by pipetting. As a control, 20 µL of methanol and 180 µL of FRAP working solution were plated in triplicate. A blank solution consisting of 200 µL of methanol was plated in triplicate to account for background absorbance. The plate was incubated at room temperature in the dark for 30 minutes. Absorbance was measured at 593 nm using a SpectraMax 190 microplate reader. Results were calculated as percent reducing power using the following formula:

FRAP % = A₁ -A_b/A₁ x 100%

A₁: Sample absorbance (FRAP + reducer)

A_b: Blank absorbance (MeOH)

ABTS Radical Scavenging Assay

The relative ability of the flavonol fractions to scavenge ABTS was determined using a colorimetric assay as described by Tomasina et al¹⁶. A 7 mM ABTS stock solution was prepared by dissolving 0.1921 g of ABTS in 50 mL phosphate-buffered saline (PBS). A 2.45 mM potassium persulfate solution was prepared by dissolving 0.0331 g of potassium persulfate in 50 mL PBS. Equal aliquots (50 mL) of 7 mM ABTS and 50 mL 2.45 mM potassium persulfate solution were mixed and allowed to stand in the dark at room temperature for 16 hours to generate the ABTS•+ radical cation. The resulting ABTS•+ solution was diluted with 0.01 M PBTS to an absorbance of 0.70 ± 0.02 at 734 nm. Once adjusted, the solution was left to stabilize for 30 minutes and monitored for significant changes in absorbance. The reaction was initiated by reacting 190 µL of diluted ABTS•+ reagent with 10 µL of extract in triplicate wells of a 96-well microplate. A control was prepared using 10 µL of methanol in addition to 190 µL of ABTS•+, with 200 µL of methanol serving as the blank. Absorbance was read at 734 nm using a SpectraMax190 microplate reader. The following formula was used to compute percent inhibition:

% Inhibition = A₀ – (A₁ -A_b)/A₀ x 100%

A₀: Control absorbance with no radical scavenger (ABTS•+ + MeOH)

A₁: Sample absorbance (ABTS•+ + scavenger)

A_b: Blank absorbance (MeOH)

Acetylcholinesterase In Vitro Enzyme Assay

The AChE inhibitory activities of the isolated fractions were examined using Ellman’s method¹⁸ in conjunction with the AmpliteTM Colorimetric Acetylcholinesterase Assay Kit (AAT Bioquest, Inc.), and optimized based on the procedure described by Koseki et al17. One milligram of extract was dissolved in 1 mL of DI water with 0.4% DMSO. A series dilution was performed to obtain the following concentrations: 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95 µg/mL. 10 µL of the sample solutions were pipetted into triplicate onto a 96-well microplate, followed by 30 µL of assay buffer and 10 µL of acetylthiocholinesterase (200 mU/mL in assay buffer). Galantamine (0.1 mg/mL), a selective inhibitor of AChE, was used as a positive control. Samples were incubated in the dark at room temperature for 20 minutes to facilitate binding. The reaction was initiated by adding the working reagent containing 5,5’dithiobis(2-nitrobenzoic acid) and acetylthiocholine iodide, which, upon hydrolysis, forms thiocholine. Thiocoline reacts with the reagent to form a yellow 5-thio-2-nitrobenzoate anion. After 30 minutes of incubation, absorbance was measured at 405 nm using the SpectraMax 190 microplate. The AChE inhibitory activity was calculated as follows¹⁹:

Percent Inhibition = E x S/E x 100%

E: Activity of enzyme without the inhibitor

S- Sample Absorbance

*E and S were each subtracted by their respective blank

Results and Discussions 

HPLC Quantification of Polyphenolic Compounds in MQ & CP

Polyphenolic constituents were characterized using reversed-phase high-performance liquid chromatography (HPLC) to compare both Mullica Queen (MQ) and cranberry pomace (CP) extracts for their polyphenolic content at various wavelengths. Anthocyanins. flavonols, phenolic acids, and proanthocyanidin chromatograms were extracted at 520, 355, 310, and 280 nm, respectively, and compared to standard compounds for identification. The detection of these polyphenolic compounds is relevant to their bioactive potential and provides insight into their active antioxidant components. Compounds marked in orange were identified using a published reference²⁰ or require standard spiking. Relevant peak wavelengths are detailed below the chromatograms.

Fig. 3. HPLC Chromatogram of Mullica Queen (MQ) Fruit Extract (Run 3, 5 mg/mL) at 520 nm

Fig. 4. HPLC Chromatogram of Cranberry Pomace (CP) Extract (Run 3, 10 mg/mL) at 520 nm

HPLC chromatograms were extracted at 355 nm to reveal flavonoids present in both MQ and CP extracts, with several peaks corresponding to known flavonol standards. Peaks observed at retention times 11.79 min and 13.16 min in the MQ fruit extract were identified as myricetin-3-O-galactoside and quercetin-3-O-galactoside, respectively (Fig. 5). The aglycones of these compounds, myricetin and quercetin, were confirmed to be present in the CP extract at later elution times of approximately 18.07 min and 22.21 min, respectively (Fig. 6).

Fig. 5. HPLC Chromatogram of Mullica Queen (MQ) Fruit Extract (Run 3, 5 mg/mL) at 355 nm

Although the cranberry pomace was run at a higher concentration, the MQ extract demonstrates a broader distribution and abundance of flavonols. The difference in composition contributes to their antioxidant performance. The profiling of the whole extracts confirms the presence of the target quercetin aglycone and its glycoside, but also provides information as to other phytochemicals that may contribute to their observed antioxidant effect.

Fig. 6. HPLC Chromatogram of Cranberry Pomace (CP) Extract (Run 3, 10 mg/mL) at 355 nm

Fig. 7. HPLC Chromatogram of Mullica Queen (MQ) Fruit Extract (Run 3, 5 mg/mL) at 310 nm

Fig. 8. HPLC Chromatogram of Cranberry Pomace (CP) Extract (Run 3, 10 mg/mL) at 310 nm

Fig. 9. HPLC Chromatogram of Mullica Queen (MQ) Fruit Extract (Run 3, 5 mg/mL) at 280 nm

Fig. 10. HPLC Chromatogram of Cranberry Pomace (CP) Extract (Run 3, 10 mg/mL) at 280 nm

Quantification of Polyphenolic Compounds in MQ Extract

Quantitative analysis of the MQ extract reveals that quercetin-3-O-galactoside was the most abundant polyphenolic compound with an average concentration of 13.9 ± 0.19 mg/g of desugared extract. Quercetin glycosides constituted much of the polyphenolic content in this extract. Although glycoside derivatives are present in high concentrations in this sample, trace amounts of free quercetin were also detected, though present at significantly lower concentrations. CP extract likely contains higher levels of quercetin aglycones, potentially due to processing conditions during juice extraction, where these water-insoluble aglycones may be concentrated in the pomace.

Table 1. Mullica Queen Polyphenolic Content Quantification

Compound	Average Concentration (mg analyte/g of Desugared Extract)
FLAVONOLS
Quercetin	0.33 ± 0.00
Quercetin-3-O-Galactoside	13.9 ± 0.19
Myricetin-3-O-Galactoside	12.2 ± 0.09
Isorhamnetin	0.11 ± 0.01
PHENOLIC ACIDS
p-Coumaric Acid	2.55 ± 0.02
Ideain Chloride	0.51 ± 0.0
Peonoidin-3-O-Galactoside Chloride	6.65 ± 0.04
ANTHOCYANINS
Cyanidin-3-O-Galactoside	0.51 ± 0.0
Peonidin-3-O-Galactoside	0.60 ± 0.0
PROANTHOCYANINS
PACs A	7.39 ± 0.02
PACs B	3.5 ± 0.35

 Isolation and Confirmation of Quercetin & Quercetin-3-galactoside in Isolated Fractions

Fractionation of the fruit and pomace extracts using Sephadex gel filtration chromatography was proven successful in the isolation of both quercetin and its glycoside (Table 2). Sephadex fractionation relies on size exclusion principles, where larger molecules elute first and smaller molecules are retained longer in the porous matrix. A peach-colored eluate corresponding to 70%methanol Eluate IV was the final fraction eluted with this solvent and confirmed to contain quercetin-3-galactoside in this MQ fraction. The compound’s strong retention onto the column indicates strong interactions with the Sephadex matrix. HPLC spectral data were used for identification as the compound strongly absorbed at 355.3 nm, corresponding to standard data and retention times (Fig. 11).

Table 2. Mullica Queen Sephadex LH-20 Fractionation Output

 

Fig. 11. MQ 70% Methanol Eluate IV Fraction Chromatogram (Run 3, 2 mg/mL) at 355 nm

A yellow-colored eluate corresponding to 70% Acetone Eluate III was confirmed to contain the aglycone quercetin as the main active component in this CP fraction. Spectral data showed a strong absorbance at 363.6 nm corresponding to the standard quercetin wavelength and retention times (Fig. 12). Solubility differences between quercetin and its more polar glycoside impact their interaction with the nonpolar stationary phase. Polar mobile phase solvent, 70% methanol, facilitates the elution of the more polar glycoside, whereas 70% acetone is strong enough and less polar to elute the aglycone with reduced polarity.

INSERT FIGURE 12 HERE

Fig. 12. CP 70% Acetone Eluate III Fraction Chromatogram (Run 3, 10 mg/mL) at 355 nm

Determination of Total Phenolic Content 

Phenolic compounds are critical for antioxidant defense to neutralize free radicals. The Folin-Ciocalteau (F-C) assay is a widely used method to measure total phenolic content (TPC). Reaction of phenolic compounds reveals their antioxidant power based on the reduction of yellow phosphotungstate-phosphomolybdate complex by antioxidants that reduce the complex to a blue chromagen measured at 760 nm21. Reducing capacities of the powdered CP and MQ extracts were measured to compare the broader polyphenolic composition of the extracts the quercetin and its glycosides were isolated. The total phenolic content was measured in milligrams of gallic acid per gram of dry powder. The MQ fruit extract exhibited a higher total phenolic content compared to the CP extract, which is consistent with the presence of more diverse polyphenolic compounds, suggesting the fruit may serve as a richer source of flavonol glycosides. The TPC of the MQ powder was significantly higher (192.1 ± 1.53 mg/g ) than CP (137.3 ± 2.28).

Fig. 13. Total Phenolic Content in CP & MQ

DPPH Free Radical Scavenging Antioxidant Activity

Another commonly used bioanalytical method to measure antioxidant activity is the 1,1-diphenyl-2 picrylhydrazyl (DPPH) assay. This method evaluates antioxidant capacity based on spectrophotometric measurements of antioxidants’ ability to scavenge DPPH free radicals. The underlying mechanism involves the reaction of DPPH· radicals with hydrogen-donating antioxidants to form a reduced hydrazine compound (DPPH-H). Upon the formation of hydrazine, a neutralized, yellow-colored solution appears, indicating the radical neutralization from its original dark purple complex, spectrophotometrically observed at 517 nm22. This assay was employed to compare the antioxidant capacity of de-sugared CP and MQ extracts, as well as their respective quercetin and quercetin glycoside fractions.

Fig. 14. (a) Percent Inhibition of DPPH radical by CP and MQ de-sugared extracts. (b) Percent inhibition of DPPH radical by CP Quercetin, and MQ Quercetin-3-Galactoside fractions

Results indicate that the MQ extract exhibited a higher percentage of DPPH radical inhibition than the CP extract, though data did not significantly vary (Fig. 14a). Both extracts displayed dose-dependent behavior with increasing concentrations resulting in greater inhibition of DPPH radicals. Analysis of the CP quercetin and MQ quercetin-3-galactoside fractions revealed significant differences in antioxidant activity at lower concentrations, as higher concentrations were omitted to demonstrate a more pronounced difference. CP-derived quercetin demonstrated higher antioxidant activity with respect to its glycoside fraction, despite originating from the CP extract with overall lower antioxidant activity and TPC. At 31.3 μg/mL, CP quercetin exhibited 81.0 ± 12.1% inhibition, outperforming its glycoside, which only reached 73.7 ± 3.79 % at the same concentration (Fig. 14b).

Ferric Reducing Antioxidant Power

Further antioxidant analysis was performed using the Ferric Reducing Antioxidant Power assay(FRAP), which measures antioxidant reduction potency based on the reduction of a colorless Fe3+- TPTZ complex into an intense blue Fe²⁺-TPTZ in the presence of antioxidants. This reduction in acidic medium is measured spectrophotometrically at 593 nm. Notably, the ferricreducing antioxidant power between the desugared CP and MQ extracts demonstrates differing results in respect to the DPPH assay.

Fig. 15. (a) Ferric reducing antioxidant power by CP and MQ de-sugared extracts (b) Ferric-reducing antioxidant power by CP Quercetin and MQ Quercetin-3-Galactoside fractions

Results show CP extract exhibited greater ferric reducing antioxidant power at varying concentrations (250 – 3.91 μg/mL). Although it’s important to note that the dose-dependent response is not as significant for CP extracts as previously noted, considering lower concentrations still exhibited significant reduction potential with respect to MQ (Fig. 15a). Analysis of the fractions reveals CP quercetin demonstrates significantly higher ferric reducing antioxidant power with respect to its MQ quercetin-3-galactoside fraction. At 31.3 μg/mL, CP quercetin exhibited 57.4 ± 4.29% reducing power, outperforming its glycoside, which only reached 43.5 ±1.28 % at the same concentration (Fig. 15b).

ABTS Radical Scavenging Activity

To further validate the antioxidant potential of the isolated fractions, the 2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS)assay was utilized. The principle of this method relies on the electron transfer of antioxidants in reducing ABTS radical cation (ABTS•+) to its colorless, neutralized form with a decreased absorbance measured spectrophotometrically at 734 nm. Both CP quercetin and MQ quercetin-3-galactoside fractions demonstrated strong free radical scavenging at 250 μg/mL, with ABTS inhibition values of 98.2 ± 0.96 % and 95.9 ±4.88 %, respectively. CP quercetin maintained relatively higher activity, exhibiting 26.3 ±3.51% inhibition, outperforming its glycoside fraction, which showed lower inhibition at 24.9 ±2.39%. Findings from the ABTS assay, along with FRAP and DPPH results, support the conclusion that CP-derived quercetin exhibits stronger antioxidant activity than its glycosylated counterpart.

Fig. 16. Comparison of ABTS scavenging activity between MQ Quercetin-3-galactoside and CP Quercetin fractions

Inhibitory Effect on Acetylcholinesterase(AChE)

Acetylcholinesterase (AChE) is a neurodegenerative-associated enzyme that catalyzes the hydrolysis of acetylcholine, a neurotransmitter critical to memory, attention, and learning⁸. AChE inhibitors are used to treat Alzheimer’s disease by inhibiting the breakdown of acetylcholine. To assess their potential neuroprotective properties of cranberry and cranberry pomace-derived fractions, AChE-inhibitory activity was evaluated in vitro. This assay employed Ellman’s reagent to quantify thiocholine, a product of acetythiocholine hydrolysis by AChE. Thiocholine reacts with DTNB (5,5’-dithiobis (2-nitrobenzoic acid)) to produce a yellow complex measured at 405 nm.

Fig. 17. Inhibition of AChE activity by CP Quercetin

Using the AmpliteTM Colorimetric Acetylcholinesterase Assay Kit (AAT Bioquest, Inc.) with protocol adjustments based on a method described by Koseki et al¹⁸, CP-derived quercetin exhibited significant inhibitory activity. At a concentration of 250 μg/mL, this fraction achieved 59.0 ± 2.41% inhibition of AChE activity, while at a lower concentration, 31.3 μg/mL, the sample reached 8.85 ± 2.86% AchE inhibition. Galantamine, a clinically approved AChE inhibitor used in the treatment of dementia, served as the positive control. As subsequent trials were conducted, tests at higher concentrations with the CP quercetin extract and the evaluation of the inhibitory potential of the MQ quercetin-3-galactoside fraction demonstrated inconsistent results. A reduction in galantamine’s inhibitory effect was also observed during these trials, indicating partial degradation of the enzyme. Solubility issues also arose when dissolving the glycoside fraction, which required increased DMSO concentrations that could have impacted its inhibitory effects. Overall, while CP-derived quercetin shows promising inhibitory activity against AChE, this method requires further optimization to allow for accurate comparisons of fraction AChE inhibitory activity.

Conclusions 

The results of this study suggest that cranberry pomace-derived quercetin exhibits stronger antioxidant activity than its glycosylated counterpart, quercetin-3-galactoside, isolated from Mullica Queen (MQ) fruit extract. HPLC profiling and fractionation confirmed these compounds as the primary active constituents of their respective extracts. Despite the CP powder having lower TPC than MQ, CP-derived quercetin consistently outperformed its glycoside-rich fraction in antioxidant assays (DPPH, FRAP, ABTS) and demonstrated moderate acetylcholinesterase(AChE) inhibitory activity. It should be noted that the DPPH antioxidant activity of the CP and MQ concentrated extracts themselves is relatively statistically equivalent, with the CP extract exhibiting greater ferric reducing antioxidant power maintained throughout lower concentrations. ABTS results show relatively similar potential in reducing ABTS radical cation, although CPquercetin still demonstrated higher inhibition than its glycoside. Results highlight CPquercetin’s neuroprotective potential through its ability to scavenge reactive oxygen species(ROS). The differences in activity between the aglycone and its glycoside are likely due to the absence of the sugar moiety. Findings from this study support the conclusion that glycosylation may reduce bioactivity. Future studies will explore their effects on additional neurodegenerative-related enzymes such as monoamine oxidases (MAOs) and butyrylcholinesterase, as well as potentially conducting in vivo studies to evaluate how glycosylation impacts absorption and therapeutic potential.

Acknowledgements

This work was supported by funding from the Office of Undergraduate Research at the University of Massachusetts Dartmouth and the UMass Cranberry Health Research Center. The author also gratefully acknowledges the support of Dr. Neto, Maureen Otieno, and the rest of the Neto lab research group.

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Synthesis and Characterization of Gold Nanorods and Gold Nanorod Dimers  

By Kayli Vieira 

Introduction 

Gold nanoparticles have become a hot spot in recent research due to their unique electrochemical properties. When light hits the surface of a gold nanoparticle, a collective oscillation of electrons results in a strong electromagnetic field, enhancing absorption and scattering properties. This phenomenon, known as plasmon resonance, gives gold nanoparticles their uniqueness and versatility in different areas of research. For example, gold nanoparticles have been utilized for research in drug delivery, biosensing, spectroscopy, and catalysis. In Professor Wei-Shun Chang’s lab at UMass Dartmouth, gold nanoparticles are studied using spectroscopic techniques to understand their physical and chemical properties for application in catalysis, microscopy, and biosensing. For this project, gold nanorods will be used to study substrate–induced chirality through circular dichroism (CD) measurements on a hyperspectral microscope. Samples of both chiral and achiral gold nanorods are required for single particle measurement, which can be synthesized using simple wet-lab procedures.  

Results 

The first step of the project requires the synthesis of pure, homogenous, medium–sized gold nanorods (22×66 nm in size). To accomplish this, multiple syntheses were performed using the seed-mediated growth method first developed by the Murphy Group (see Figure 1 for procedure schematic). 

Figure 1. Schematic of Seed-Mediated Growth Method of Gold Nanorods (Adapted from the Murphy Group) 

After extensive background research and alterations of reagent concentrations, the procedure was used to synthesize medium–sized gold nanorods, optimized for single particle measurement. For characterization, samples were analyzed using UV-Vis spectroscopy, scanning electron microscopy, and hyperspectral microscopy.  

Figure 2. UV-Vis Absorption Graph of Medium Sized Gold Nanorods

As seen in Figure 2, the UV-Vis absorption graph yielded important information about the sample’s purity and general aspect ratio, evident by the transversal and longitudinal plasmon bands. To determine the average size of the nanorods produced, SEM (scanning electron microscope) images were taken and analyzed. After analyzing over 150 individual gold nanorods, the dimensions of the rods produced yielded 59.6 ∓ 7.0 nm X 20 ∓ 3.18 nm (image can be seen in Figure 3).  

Furthermore, after being spin cast on a glass substrate and imaged on a darkfield inverted hyperspectral microscope, individual spectra for a given nanorod were produced using analyses of Hyperspectral images through the MATLAB program (see Figures 4 and 5). 

Figures 3 and 4. SEM Image and Hyperspectral Image of Medium Gold Nanorod Dimers 

Figure 5. Individual Gold Nanorod Spectra 

After successful synthesis of singular medium–sized gold nanorods, synthesis of a chiral sample is required. Using the previously made gold nanorod samples and a method developed by Kar et. al, samples of gold nanorod dimers (two gold nanorods linked end-to-end) were assembled. Using the polyelectrolyte coating and thiol-linking procedure schematized in Figure 6, chiral samples of gold nanorod dimers were synthesized and stable for a very short time.

Figure 6. Schematic of Gold Nanorod Dimers developed by Kar et. al 

Unfortunately, results varied from those in the literature. After a successful first attempt, reproducing the same results was almost impossible. Countless experiments were done altering individual factors at every part of the procedure to determine the driving force of the reaction. The hydrochloric acid concentration was determined to be the main contributor as to whether dimers were produced, likely due to the decrease of surface charge on the rods resulting in the ability for linkage. Furthermore, it was discovered that the addition of DABCO resulted in the destabilization of dimers formation likely due to surface charge imbalances. Thus, successful samples produced were only stable for a few hours.  

Figure 7. UV-Vis Absorption Graph of Gold Nanorod Dimers Sample

Characterization of each gold nanorod dimer sample was preformed using the same methods mentioned above. In Figure 7, the UV-Vis absorption graph revealed important information as to whether or not dimers were formed in the reaction, evident by the shoulder created after the longitudinal plasmon band. Similar graphs can be seen in the resulting spectra generated using the hyperspectral microscope (see Figures 8 and 9). Furthermore, SEM images depicted in Figure 10 clearly showed the configuration of the rods as dimers, aligning with the results of the hyperspectral images and UV-Vis graph. 

Figures 8 and 9. SEM Image and Hyperspectral Image of Medium Gold Nanorod Dimers

Discussion and Future Work 

Homogeneous and pure medium-sized gold nanorods were successfully synthesized for future single-particle measurement. Additionally, gold nanorod dimers were assembled using the polyelectrolyte coating and thiol linking method previously developed in the literature. Hydrochloric acid was identified to be the key parameter of the dimerization reaction, with alterations needed for each concentration of rods used for assembly. Currently, the gold nanorod dimers synthesis is continuing to be optimized for maximum reproducibility, with future plans of single-particle measurement. Both products synthesized will be used for analysis in the future study of substrate-induced chirality.  

Works Cited

Synthesis of Solution-Stable End-to-End Linked Gold Nanorod Dimers via pH-Dependent Surface Reconfiguration Ashish Kar, Varsha Thambi, Diptiranjan Paital, Gayatri Joshi, and Saumyakanti Khatua Langmuir 2020 36 (33), 9894-9899 DOI: 10.1021/acs.langmuir.0c01516

Seeded High Yield Synthesis of Short Au Nanorods in Aqueous Solution Tapan K. Sau, and Catherine J. Murphy Langmuir 2004 20 (15), 6414-6420 DOI: 10.1021/la049463z

Phosphorus Removal Through Plant Assimilation in Floating Treatment Wetlands

By Mia Oliveira, Sara Sampieri Horvet, Micheline Labrie 

Affiliation: Coastal Systems Program, Department of Estuarine and Ocean Science, School for Marine Science and Technology 

Abstract 

Excess phosphorus (P) in freshwater systems contributes to the degradation of aquatic habitats through algal blooms and low dissolved oxygen levels. This study investigates the deployment and effectiveness of floating treatment wetlands (FTWs) as a method for phosphorus removal in Long Pond, located in Barnstable, MA. Preliminary results demonstrate species-specific differences in P assimilation and overall plant biomass increase during the first year of growth. 

Introduction 

Freshwater ponds and lakes in Massachusetts are degraded by excess inputs of phosphorus (P). Bioavailable dissolved forms of P, such as ortho-phosphate (PO43-), can stimulate phytoplankton blooms, potentially containing toxins. These blooms lead to low water column dissolved oxygen, resulting in poor habitat health. 

Long Pond (Barnstable, MA) is currently impaired by excessive P loading, primarily from wastewater sources (Eichner et al. 2022). Although sewering is expected to significantly reduce P inputs, it will not be implemented for ~25 years. The Town of Barnstable is exploring low-cost, non-infrastructural alternatives that can be implemented within 5 years. One such option is the deployment of floating treatment wetlands (FWTs). FTWs transform bioavailable P into plant biomass, which can be permanently removed upon harvest, effectively reducing P levels in the water (Lane et al. 2016). 

Using guidance from the Cape Cod Commission (Eichner et al. 2003), the target total P in the water column is 7.4 kg, consistent with levels measured in 2013 (7.8 kg) and 2011 (6.4 kg). This target contrasts sharply with the 2021 measurement of 16.2 kg, reflecting the need for additional water quality data collection and P reduction. 

Objective: Quantify in-pond P removal by FTW plant biomass via assimilation by seven aquatic plant species. Ultimately, success will be evaluated based on total P removal (kg/year), in situ P reduction from water quality measurements, and cost per kg P removal for full-scale implementation. 

Figure 1.

(Left) Aerial image of Long Pond, a relatively shallow, ~50-acre Great Pond. It is a public resource and subject to MA and federal regulations. The gray square represents the FTWs deployment location. Blue stars represent 2024 water quality sampling sites.

(Right) September 2024 Photo of the FTW rafts. 

Materials and Methods

Study Site

Ten FTW rafts were installed in the northern section of Long Pond in April 2024. Each raft measured 2.0 m × 2.1 m and consisted of a buoyant, high-density polyethylene matrix with designated planting holes for 36 (n=6) or 72 (n=4) plants per raft (Figure 2). The selection of plant species and planting configurations was directed by the Town of Barnstable, with 5 cm plugs sourced from New England Wetland Plants, Inc. 

Figure 2. Aquatic plant species and planting configuration determined by the town. The town obtained 5 cm plugs from New England Wetland Plants, Inc. 

Plant biomass measurements and tissue samples were collected in May (pre-planting) and September 2024 (end of Year 1 growing season). Sub-samples included whole plant wet weight, and wet and dry weights of above and below-water biomass. At the Year 1 season’s end, the ten rafts were surveyed to determine plant survival. 

Biomass was dried to a constant weight of 65°C and ground using a ball mill. Total P was measured via persulfate digestion and analyzed by the ascorbic acid-molybdenum blue method (Method 4500-PE) using a spectrophotometer set at 882 nm at the Coastal Systems Program Analytical Facility. 

Figure 3. Mia Oliveira conducting persulfate digestions at the Coastal Systems Program, SMAST-West. 

Results 

Plants deployed in rafts with 36 planting holes showed slightly higher survivability compared to rafts with 72 planting holes (Table 1). Preliminary data from the town indicated that most plant mortality occurred during July. Higher plant mortality coincided with greater incidence of weed growth. Species with higher survival rates contributed proportionally more to total P removal. 

Table 1. Summary of plant species remaining at the end of Year 1 for each raft. On average, rafts had 67% and 55% survival with 36 and 72 planting holes, respectively. 

Species	Raft #
	36 planting holes						72 planting holes
	1	2	3	4	5	6	7	8	9	10
C. lurida	9	5	9	2	8	17	9	0	7	14
J. effusus	6	8	8	10	8	3	9	21	13	11
P. cordata	0	0	0	0	0	0	0	0	0	0
A. americanus	3	2	2	4	13	3	5	10	11	10
A. incarnata	4	5	5	2	0	0	12	8	1	8
S. atrovirens	3	0	0	0	0	0	1	0	0	0
V. noveboracensis	0	0	1	2	1	1	2	0	3	4
Percent Survival	69%	56%	69%	56%	83%	67%	53%	54%	49%	65%

Initial P content varied across plant species (Figure 4). Acorus americanus displayed the highest P concentration in shoot biomass and significant root P content. Viburnum noveboracensis exhibited higher root P levels compared to shoots, though data was limited (n=1). Comparing initial and final phosphorus contents, phosphorus (umol/g dry plant biomass) decreased over the growing season. On average, root and shoot phosphorus content decreased by 58% and 52%, respectively. 

Whole plant biomass increased across all species from deployment to the end of Year 1, except for P. cordata, which showed no growth. C. lurida had the highest biomass increase, reaching 500 g wet weight. Sub-sampling in September 2024 identified a total of 80 C. lurida individuals, accounting for a 37 kg biomass increase. Similarly, J. effusus contributed 30 kg of biomass over the growing season. Overall, given the wet biomass increase (Figure 5) and the total number of plants remaining at the end of the growing seasons, the total wet biomass increase was approximately 100 kg. 

Figure 4. Total phosphorus content (umol/g; dry wt. basis) of plant roots and shoots subsampled at the time of deployment (a. Initial) or at the end of the Year 1 growing season (b. Final). 

Figure 5. Whole plant biomass (g wet wt.) measured at the time of deployment (May 2024) and at the end of the first year growing season (September 2024). 

Discussion 

The results highlight the different phosphorus uptake capacities among studied aquatic plant species. The growth and P assimilation data demonstrate the potential effectiveness of FTWs as a P mitigation strategy. However, the feasibility of FTWs will likely depend on the biomass increases observed during the second year growing season.

C. lurida and J. effusus showed promise for selection for future FTW implementations, given their biomass increase. It is likely that biomass increase, rather than phosphorus content, will control total P assimilation. The lower performance of P. cordata suggests that careful species selection is important for optimizing FTW efficiency. Factors such as planting density, environmental stressors (competition, predation), and species-specific nutrient uptake mechanisms likely influenced survival and growth outcomes.

Additionally, the cost-effectiveness of FTWs should be assessed relative to other mitigation strategies. Integrating FTWs with other methods, such as aluminum sulfate treatment, sediment dredging, or aeration systems, could provide a more comprehensive and reliable approach to P management in Long Pond. Future research should focus on long-term monitoring of FTWs, including multi-year survival rates, biomass increase potential, and the ecological impacts of large-scale deployments. Additional work is also needed to quantify in situ water column P reductions and evaluate potential secondary benefits, such as enhanced habitat for aquatic organisms and sedimentation. 

Acknowledgements 

We thank the Office of Undergraduate Research and the Town of Barnstable Department of Public Works for their invaluable support. Special thanks to the individuals and organizations dedicated to the protection and restoration of Barnstable’s aquatic ecosystems.  

References

Eichner, E.M., T.C. Cambareri, G. Belfit, D. McCaffery, S. Michaud, and B. Smith. (2003).

Cape Cod Pond and Lake Atlas. Cape Cod Commission. Barnstable, MA.

Eichner, E., Howes, B., & Schlezinger, D. (2022). Long Pond management plan and diagnostic assessment. Town of Barnstable, Massachusetts. Coastal Systems Program, School for Marine Science and Technology, University of Massachusetts Dartmouth. New Bedford, MA. 110 pp.

Lane, S., Sample, D., Lazur, A., Winston, R., Streb, C., Ferrier, D., Linker, L., & Brittingham, K. (2016). Recommendations of the expert panel to define removal rates for floating treatment wetlands in existing wet ponds. Final Report to the Urban Stormwater Work Group.

Standard Methods for the Examination of Water and Wastewater, 19th edition. Method 4500- PE.

Evolving Feminism in The Hunger Games Series

By Ellie Cook

My project analyzes the differences in feminine gender performance between Katniss Everdeen, the protagonist of the original The Hunger Games trilogy by Suzanne Collins, and Lucy Gray Baird, the protagonist of the prequel novel The Ballad of Songbirds and Snakes. It analyzes the two characters from an intersectional perspective and argues for the importance of cultural, economic, social, etc. contexts when accounting for differences in gender performance, as these factors are all intertwined with gender norms. From the basis of their contrasting gender performance, the paper then delves into agency as a theme and how each character’s relative agency impacts their feminine representation. These differences in gender performance and agency show a different archetype for a young adult feminine heroine over ten years after the original trilogy’s publication. 

From the end of May to mid-July, I completed the background reading to prepare for writing my paper. I read critical theory such as Judith Butler’s Gender Trouble, Undoing Gender, and Who’s Afraid of Gender? as well as Butler Matters: Judith Butler’s Impact on Feminist and Queer Studies by Warren J. Blumenfeld and Margaret Sönser Breen. I also read scholarship about The Hunger Games series that focused on gender and feminism. Some of these texts include: “You Love Me. Real’: Gender in the Hunger Games Trilogy” by Bienvenue Bray and “I Hunt. He bakes.’: Constructing and Deconstructing Gender Identity in Suzanne Collins’s The Hunger Games Trilogy” by Rakchuda Thibordee. I also read books that gave a general overview of the young adult dystopian genre, such as Female Rebellion in Young Adult Dystopian Fiction by Sarah K. Day, et al. and Contemporary Dystopian Fiction for Young Adults: Brave New Teenagers by Carrie Hintz, et al. Using these texts, I completed an annotated biography.

I met with my Apex mentor, Professor Caroline Gelmi, in mid-June, mid-July, and mid-August to check my progress. From mid-July to the end of August, I completed the first draft of my paper. I will continue to work on it with Professor Gelmi throughout the Fall 2024 semester and will finish my final draft at the end of the semester.  

I am grateful to Professor Gelmi for her feedback and mentorship and to OUR for supporting this project through a summer research grant.

Human-Induced Fear in Free-Living Raccoons 

By Ruby Sanger 

Abstract 

This past June and July, I spent time at Norcross Wildlife Sanctuary in Wales, Massachusetts. Norcross is a large intact forest of approximately 4200 ha with restricted human access and a small 17ha area near the visitor center, where human access is permitted for hiking along 4km of natural trails. 

I intended to study the effects of human-induced fear on the foraging effects of free-living white-tailed deer, both in the human-accessible area and the forest. This design included audio treatments (human talking and birdsong) and food treatments (plain corn and plain corn mixed with molasses) at eight different stations across the sanctuary. However, after a few weeks of experimentation, there was no deer activity at any of the stations. While thorough background research was conducted to decide the most appealing foods to bait deer with, and there was known to be a high density of deer at the sanctuary, it is possible that because the forest is so dense with natural browse and plants the deer eat, they simply not interested in what was being offered. As a result, Dr. Sherriff and I decided to adapt the experiment to study the foraging behaviors of raccoons, as there were reoccurring raccoon visits at select feeding stations on the public trails. 

None of the stations with molasses had traffic, nor did any of the stations in the deep woods, so we cut the molasses treatment while adding two audio treatments (human yelling and dog barking) for a total of four treatments. The experiment was limited to the public trails of the sanctuary. 

Introduction 

While predators can kill prey, they can also alter prey fitness through non–consumptive effects. The risk of predation can subsequently alter prey’s behavior, morphology, and physiology, which may all impact prey survival and reproduction. These effects may then influence prey population sizes (Sheriff et al., 2020). For example, a study by Cherry et al. (2016) showed that the presence of coyotes led to reduced lactation and ovulation in white-tailed deer, and an absence of coyotes related to an increase in feeding, lactation, and ovulation. These non-consumptive effects may reduce prey population size due to a lack of reproductive success and fecundity (Say-Sallaz et al., 2019). 

The term “landscape of fear” is defined as the spatial variation in prey perception of predation risk. These “landscapes” combine the elements of the physical environment that prey may inhabit or forage in, the predation risk and how it varies across locations, and a prey’s response to predation risk. There are generally two methods of prey response, one being avoiding areas perceived as higher risk, and the other being changes in behaviors while in the areas perceived as higher risk. The perception and fear of predation may be able to drive community-level changes within ecosystems, such as trophic cascades. (Gaynor et al., 2019). A common way to study the spatial variation resulting from risk response is by looking at giving-up densities (GUDs) and analyzing foraging behaviors within the context of risk. GUDs are used to provide insight into metabolic and predation costs of foraging by determining when an individual may stop foraging (Brown, 1987). 

In a study by Darimont et al. (2015), it has been shown that human predators kill far more prey than non-human predators, as well as killing carnivores nine times more than natural predators (Smith et al. 2017). While humans kill at an unparalleled rate, they more often affect prey behavior through disturbance (Frid, Dill 2002). Fear of humans as a “super predator” is also known to lead to behavioral changes in both predators and prey, and the subsequent effects on populations and communities may be larger than those resulting from non-human predators (Crawford et al. 2022). Experimental non-consumptive behaviors from humans have even led to a decrease in feeding times for pumas, an animal without any natural predators (Smith et al. 2017). A landscape of fear of the perception of humans can result in significant changes in wildlife behavior and community dynamics. Suraci et al. (2019) conducted studies with free-living mountain lions, bobcats, medium-sized carnivores (such as opossums and skunks) and deer mice in the Santa Cruz mountains. 

Human predation risk was simulated by using playbacks of human vocalization. The carnivore groups all experienced behavioral changes in response to perceived predation risk: avoiding areas, making temporal changes, and being less efficient in foraging. 

However, deer mice seemingly benefited from human presence; they increased space use as well as foraging intensity. The fear that the carnivores perceived affected lower trophic levels, influencing the surrounding wildlife system (Suraci et al., 2019).

Methods 

i. Feeding Station Set-ups 

Figure 1: Map of the 8 feeding stations, marked on the GAIA app. 

 

Figure 2: Camera and speaker set-ups. The cups were used as a shield for the exposed speakers from the rain. 

 

Eight feeding stations were chosen among the public trails of Norcross Wildlife Sanctuary in Wales, Massachusetts (Figure 1). Each station consisted of a painted feeding bin, a field camera, and a speaker (Figure 2). 

The speaker and camera were programmed to be used together, using Arduino, so that when the camera was triggered by motion, the speaker was triggered to play a programmed playback. Two speakers were programmed to play conversational human speaking, two to play dogs barking, two to play bird songs native to the area, and two to play humans yelling. Each speaker was programmed to play at about 65-70 decibels. The camera-speaker set-ups were programmed for the speaker to trigger 20 seconds after the camera was triggered. 

80 ounces of dried whole grain corn (5 lbs) were set into the feeding tubs. Each empty tub, when closed with the lid, weighed 70 ounces. 

ii. Daily Protocol 

The stations were filled with 80 ounces of corn, and the cameras and speakers were switched on June 30th. Every morning from July 1st to July 10th, the combined weight of the corn and the tub were taken at each of the eight locations by closing the tub and weighing it with a digital fishing scale. If the weight was below 120 ounces, the corn was later refilled to the base weight of 150 ounces (80 ounces of corn plus the weight of the tub). Additionally, the SD cards in the cameras were checked to see if there was any raccoon activity or other significant animal activity during the night. The battery levels of the cameras and speakers, as well as the SD card storage amounts, were also checked every morning to ensure proper performance for the following night. In the case of heavy rain, the tubs were covered to prevent the corn from being waterlogged, which could provide inaccurate weights. 

Table 1: Key of auditory playbacks per feeding station

Feeding Station	Treatment 6/30-7/05	Treatment 7/05-7/10
1	Talking	Birdsong
2	Yelling	Dog barking
3	Dog barking	Yelling
4	Birdsong	Talking
5	Talking	Birdsong
6	Yelling	Dog barking
7	Birdsong	Yelling
8	Dog barking	Talking

On July 5th, after 5 nights of data collection, the playback treatments were changed to different locations (Table 1). At the end of data collection, the SD cards were collected, and the feeding stations were broken down. 

iii. Data Analysis 

The footage captured from the feeding stations are currently being analyzed manually. I will be scoring for behaviors including fleeing (running/leaving quickly), leaving (walking away), looking up, head-up foraging, head-down foraging, playing, freezing, and leaving a group. There are many cases of corn being eaten by mice, chipmunks, squirrels, and occasionally deer. The footage of these animals will be used to separate their consumption from the raccoon’s consumption. Additionally, statistics of each night of the experimental run are being gathered. I am counting the total number of foraging events as well as the time spent eating each night. The footage is currently being analyzed. 

Conclusion 

While the results are still being analyzed, information regarding how raccoons react to auditory playbacks will provide useful insight into the effectiveness of using sound as a method of pest control, as well as how small mammals such as raccoons are affected by the presence of humans. I hope to continue in this line of study and resume with the original design for studying white-tailed deer in the future. 

Works Cited 

Sheriff MJ, Peacor SD, Hawlena D, Thaker M. (2020). “Non‐consumptive predator effects on prey population size: A dearth of evidence.” Journal of Animal Ecology vol 89. 

Cherry, M. J., K. E. Morgan, B. T. Rutledge, L. M. Conner, and R. J. Warren. (2016). “Can coyote predation risk induce reproduction suppression in white-tailed deer?” Ecosphere 7(10):01481. 

Say-Sallaz, E., Chamaille-Jammes, S., Fritz, H., Valeix, M. (2019). “Non-consumptive effects of predation in large terrestrial mammals: Mapping our knowledge and revealing the tip of the iceberg.” Biological Conservation vol.235: 46-52. 

Suraci, J.P, Clinchy, M., Zanette, L.Y., Wilmers, C.C. (2019). “Fear of humans as apex predators has landscape-scale impacts from mountain lions to mice.” Ecology letters vol. 22,10: 1578-1586. 

Gaynor, K.M., Brown, J.S., Middleton, A.D., Power, M.E., Brashares, J.S. (2019). “Landscapes of Fear: Spatial Patterns of Risk Perception and Response.” Trends in ecology & evolution vol. 34,4: 355-368. 

Darimont, C.T, Fox, C.H, Bryan, H.M, Reimchen, T.E. (2015). “HUMAN IMPACTS. The unique ecology of human predators.” Science vol. 349,6250: 858-60. 

Smith J.A., Suraci J.P, Clinchy M., Crawford A., Roberts D., Zanette L.Y., Wilmers C.C. (2017). “Fear of the human ‘super predator’ reduces feeding time in large carnivores.” Proceedings. Biological sciences vol. 284,1857: 20170433. 

Frid, Alejandro, and Dill, L. (2002). “Human-Caused Disturbance Stimuli as a Form of 

Predation Risk.” Conservation Ecology, vol. 6. 

Crawford, D.A., Conner, M.L, Clinchy, M., Zanette, L.Y., Cherry, M.J. (2022). “Prey tells, large herbivores fear the human ‘super predator’.” Oecologia vol. 198,1: 91-98. 

Gaynor K., Hojnowski C., Carter N., Brashares J. (2018). “The influence of human disturbance on wildlife nocturnality.” Science vol. 360,6394 (2018): 1232-1235.