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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