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Article

Improving Cognitive and Chemosensory Function in Caenorhabditis elegans Through Polyphenol-Rich Sugarcane Extract

by
Deniz Heydarian
1,
Matthew Flavel
1,2,
Mihiri Munasinghe
1,3,
Markandeya Jois
1 and
Jency Thomas
1,*
1
Department of Microbiology, Anatomy, Physiology & Pharmacology, School of Agriculture, Biomedicine and Environment, La Trobe University, Melbourne, VIC 3086, Australia
2
TPM Bioactive Division, The Product Makers Pty. Ltd., Melbourne, VIC 3173, Australia
3
Department of Food Science and Technology, Faculty of Applied Sciences, University of Sri Jayewardenepura, Gangodawila, Colombo 10250, Sri Lanka
*
Author to whom correspondence should be addressed.
Stresses 2024, 4(4), 816-826; https://doi.org/10.3390/stresses4040054
Submission received: 4 November 2024 / Revised: 15 November 2024 / Accepted: 29 November 2024 / Published: 2 December 2024
(This article belongs to the Collection Feature Papers in Human and Animal Stresses)
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Abstract

:
Polyphenols are recognized for their potential benefits in enhancing lifespan and stress resistance. This study investigates the impact of Polyphenol-Rich Sugarcane Extract (PRSE) from Saccharum officinarum on the chemosensory behavior, learning, and memory in Caenorhabditis elegans (C. elegans). The C. elegans worms were administered PRSE at 5 mg/mL from the first larval stage. Chemotaxis assays, positive butanone learning, and short-term associative memory assays were conducted at days four, eight, and twelve to evaluate chemosensory response, learning index, and short-term memory loss index. PRSE significantly improved the naïve chemotaxis index by 28.8% on day four, 30% on day eight, and 35.3% on day twelve compared to controls. The learning index increased by 14.5% on day four, 21% on day eight, and 31.9% on day twelve. Additionally, PRSE reduced the short-term memory loss index by 46.4% one hour after conditioning on day four and by 48.6% two hours after conditioning on day four, with similar reductions observed on days eight and twelve. These findings indicate that PRSE has the potential to enhance chemosensory behavior, learning, and memory in C. elegans, suggesting the need for further research to explore its applicability in addressing age-related chemosensory and cognitive decline.

1. Introduction

The role of polyphenols in aging and chemosensation has been extensively studied, revealing their potential benefits in mitigating age-related decline and diseases. The study by Akter et al. [1] highlights the prophylactic effects of polyphenolic compounds in preventing neurodegenerative diseases by reducing oxidative stress and inflammation, which are key factors in aging-related neuropathologies. Menaa et al. [2] discuss the benefits of polyphenols in skin aging, emphasizing their antioxidant and chemopreventive properties that protect against environmental stressors. The molecular processes through which polyphenols influence aging characteristics, such as genomic instability and cellular senescence, highlighting their extensive anti-aging potential, have been investigated [3]. Polyphenols influence aging through gene regulation and epigenetic mechanisms, indicating their role in lifespan extension across species [4]. Evidence has been provided of polyphenols’ protective effects against age-associated diseases, particularly cancer, by highlighting their roles in preventing oxidative damage and chronic inflammation [5]. Polyphenol compounds are known to activate distinct human bitter taste receptors, contributing significantly to the bitterness of various foods and beverages [6]. Polyphenol-rich extract from grape and blueberry improves age-related memory decline in individuals with the highest cognitive impairments [7].
In this study, the aim was to investigate the effects of a novel bioactive compound, Polyphenol Rich Sugarcane Extract (PRSE), extracted from the sugarcane plant Saccharum officinarum, on the aging process and chemosensation in the model organism Caenorhabditis elegans (C. elegans). The chemosensory system of C. elegans, including the AWC neurons, is well-mapped and understood. These neurons are critical for detecting environmental cues and can be genetically manipulated to observe the effects on sensory behaviour [8,9]. Butanone is one of the many odorants that C. elegans can detect, and the ability to sense and move towards butanone (chemotaxis) is mediated primarily by the Amphid Wing C (AWC) neurons. AWC-mediated chemotaxis to butanone can be modified by experience, indicating a form of olfactory learning and memory that involves AWC neurons [10]. Caenorhabditis elegans serves as a valuable model for aging studies due to its conserved insulin/IGF-1 signaling pathway, which regulates longevity across species, including humans [11,12]. Additionally, C. elegans models with altered oxidative stress responses, such as daf-2 mutants, provide valuable insights into the relationship between oxidative stress and lifespan (Braeckman & Dhondt [13]), suggesting their relevance for human aging studies.
C. elegans’ chemotaxis assay, positive butanone learning, and short-term associative memory assay have been utilized to evaluate age-related changes in neuronal function and to assess the impact of PRSE supplementation. The naïve chemotaxis index represents the natural response of C. elegans to butanone, while the learning index measures the degree to which the worms learn to associate butanone with food. Additionally, short-term memory loss, which assesses the decline in memory within two hours after conditioning, was evaluated.

2. Results

2.1. PRSE Supplementation Improves Chemosensation

Administering PRSE at a concentration of 5 mg/mL significantly improved the naïve chemotaxis index towards butanone at multiple time points during the experiment. Specifically, there were notable increases on day four (28.8%), day eight (30%), and day twelve (35.3%) compared to the control group on the same days (p < 0.05, Figure 1).

2.2. PRSE Supplementation Enhances Learning

Despite a general decline in the learning index (LI) with age, PRSE supplementation significantly improved the LI at all observed time points. The LI increased by 14.5% on day four, 21% on day eight, and 31.9% on day twelve compared to untreated worms (p < 0.05, Figure 2).

2.3. PRSE Supplementation Might Reduce Memory Loss

PRSE supplementation at a dose of 5 mg/mL significantly reduced the short-term associative memory loss following conditioning. Specifically, the memory loss index was reduced by 46.4% one hour after conditioning on day four and by 48.6% two hours after conditioning on day four. Furthermore, reductions of 48.2% on day eight and 33% on day twelve after two hours were observed (p < 0.05, Figure 3).

3. Discussion

Leow et al. [14] found that individuals with Parkinson’s disease (PD) have reduced chemosensitivity and mechanosensitivity at the base of the tongue, leading to complications like aspiration pneumonia. Environmental factors can accelerate aging and affect chemosensory functions [15]. Diminished olfactory function is also common in Alzheimer’s disease, worsening food intake and disease progression [16]. C. elegans is a valuable model for studying chemosensory disorders and age-related sensory decline, with its well-mapped chemosensory system, including AWC neurons [17].
At a concentration of 5 mg/mL, PRSE significantly improved the naïve chemotaxis index towards butanone at various stages: 28.8% on young day four, 30% on middle-aged day eight, and 35.3% on elderly day twelve worms, compared to the controls. Despite an age-related decline in the learning index (LI), PRSE supplementation increased the LI by 14.5% on day four, 21% on day eight, and 31.9% on day twelve compared to untreated worms. PRSE also notably reduced the memory loss index after conditioning, with reductions of 46.4% one and 48.6% two hours after conditioning on day four, 48.2% on day eight, and 33% on day twelve. Previously, Munsinghe et al. [18] found significant learning improvement at middle-age day eight with 5 mg/mL cocoa supplementation. Polyphenols from traditional Chinese medicine and the Mediterranean diet have been shown to decrease amyloid-β aggregation and enhance chemotaxis in C. elegans, suggesting improved neuronal function [19]. However, no prior studies have investigated PRSE’s effect on age-related cognitive decline in C. elegans.
Sugarcane extract enhances longevity and reduces heat stress by improving antioxidant activity, contributing to overall health improvement [20]. PRSE inhibits monoamine oxidase (MAO) and acetylcholinesterase (AChE), crucial enzymes for neuronal health, thereby reducing memory loss and preserving cognitive functions in aging neurons [21]. PRSE may play a role in reducing oxidative stress and inflammation, promoting neurogenesis, and inhibiting neurodegenerative enzymes, thereby supporting neuronal health and functionality while enhancing cognitive resilience [18,22,23]. The neuroprotective benefits of PRSE are particularly evident in its ability to improve chemosensory performance and cognitive resilience, primarily attributed to its antioxidant properties [22,23,24]. Continued research using diverse animal models and relevant assays is vital to advance studies involving rodents and other models. Positive results from these investigations could pave the way for human clinical trials. Key factors that warrant investigation include the skin penetration of PRSE, its digestion and absorption levels, its stability following first-pass metabolism in the liver, and the dose response in various animal models. Addressing these parameters will enhance our understanding of PRSE’s potential in human health and is strongly recommended for future research. Successful findings in these areas could ultimately lead to clinical applications in humans.
In summary, PRSE shows promising potential in improving health parameters and mitigating aging and stress effects through enhanced antioxidant activity, inhibition of neurodegenerative enzymes, reduction of oxidative stress and inflammation, and overall neuroprotective benefits. By reducing oxidative stress and inflammation, promoting neurogenesis, and inhibiting neurodegenerative enzymes, PRSE supports the health and functionality of AWC neurons. These combined effects enhance chemosensory performance and cognitive resilience in C. elegans, validating our results [21,24].
Studies on phenolic compounds in Polyphenol-rich sugarcane extract (PRSE), indicate considerable cognitive benefits, such as improved chemosensation, learning, and short-term associative memory. In mouse models, these protective effects are mediated through the inhibition of glycogen synthase kinase-3 (GSK-3) and modulation of the transient receptor potential canonical 6 (TRPC6) pathway [25].
PRSE is composed of various components, with polyphenols comprising 23% of its content as determined by the Folin’s assay. Our study emphasizes the overall bioactivity of PRSE, which extends beyond its polyphenol content. The synergistic effects of different components, including minor polyphenols and other bioactive compounds, are thought to contribute to the observed cognitive and chemosensory improvements. The literature supports the insight that complex mixtures of bioactive compounds can produce effects not solely attributable to their individual components [23]. The combined neuroprotective effects of polyphenols including promoting neurogenesis, inhibiting neurodegenerative enzymes, and reducing oxidative stress and inflammation, enhance chemosensory performance and cognitive resilience, particularly in models like C. elegans [20].
In summary, phenolic compounds in PRSE support cognitive health by reducing oxidative stress and inflammation, promoting neurogenesis, and inhibiting neurodegenerative enzymes, thereby enhancing learning, memory, and chemosensory performance. Research highlights the impact of phenolic compounds on AWC neurons in Caenorhabditis elegans, which are crucial for olfactory responses and chemotaxis. These neurons are responsive to chemical stimuli and demonstrate mechanosensitivity, with calcium signaling events playing a pivotal role in their functionality [26,27]. Furthermore, genetic studies have identified genes such as odr-2 and lim-4 as essential for defining the functional roles and behavioral outcomes of olfactory neurons. Mutations in these genes have been shown to significantly impair the function of AWC neurons, highlighting their critical contribution to olfactory signaling and behavior. [28].
Overall, PRSE might modulate AWC neuron sensitivity, potentially affecting the olfactory responses and chemotactic behavior of C. elegans by influencing chemosensation and neural circuits. Although quercetin is not present in PRSE, it is used as a comparative standard due to its well-documented effects. This is not to suggest its presence in PRSE. Polyphenols from Blumea laciniata and PRSE extend lifespan and enhance stress resistance in C. elegans via the insulin signaling pathway, improving cognitive functions and stress resilience [29,30].
Research highlights the effects of polyphenol-rich sugarcane extract (PRSE) on inhibiting GLUT2 and its potential implications for chemosensation, learning, and cognition. Apigenin, a phenolic compound in PRSE, is among the strongest inhibitors of both GLUT2 and GLUT5. These findings suggest that PRSE can inhibit glucose transporters like GLUT2, potentially benefiting chemosensory responses and cognitive functions through various biochemical pathways [29,31]. Polyphenols from sugarcane and other sources have demonstrated inhibitory effects on glucose uptake and transport in human intestinal cells, supporting their roles in regulating GLUT2 activity [32].
Polyphenols in sugarcane extract can modulate signaling pathways, influencing learning and cognition. For instance, quercetin enhances the Nrf2/ARE pathway, associated with neuroprotection and cognitive function improvement [33]. Under normal conditions, nuclear erythroid-2-like factor-2 (Nrf2) is marked for degradation by ubiquitination via Kelch-like ECH-associated protein1 (Keap1) and broken down in the proteasome. During oxidative stress, Keap1 becomes inactive, allowing Nrf2 to be phosphorylated. Phosphorylated Nrf2 (p-Nrf2) accumulates in the nucleus, binds to antioxidant response element (ARE) sites, and activates genes responsible for producing antioxidants, detoxifying enzymes, and transporting proteins [34]. The Nrf2/HO-1 pathway plays a key role in antioxidant defense, as Nrf2 activation triggers the transcription of genes responsible for antioxidant production and detoxification, helping protect cells from oxidative damage. PRSE may affect this pathway by increasing nuclear Nrf2 levels, enabling it to bind to antioxidant response elements (ARE) and activate protective genes. Polyphenols in PRSE might further support this activation by disrupting Keap1, a protein that ordinarily binds and inhibits Nrf2, allowing Nrf2 to stabilize and move into the nucleus [35,36]. Studies indicate that polyphenols can suppress the NFκB pathway, which helps lower inflammatory responses. For instance, compounds like epigallocatechin-3-gallate (EGCG) from green tea and polyphenols from pomegranate peel have been observed to inhibit NF-κB activation, resulting in decreased levels of pro-inflammatory cytokines like IL-6 and TNF-α [37,38]. Moreover, polyphenols from coffee cherry husks and extra-virgin olive oil have been shown to regulate inflammatory mediators by inhibiting NF-κB, offering protective benefits against chronic inflammation [39,40]. These findings highlight the potential of polyphenols to reduce inflammation via NF-κB pathway inhibition, which may be advantageous for managing aging-related inflammatory conditions, including those studied in C. elegans. Using C. elegans strains with skn-1 mutations (the Nrf2 homolog) would help assess whether PRSE’s antioxidant effects involve this pathway, as skn-1 regulates oxidative stress response and longevity genes. Additionally, strains with mutations or reporters for genes like gst-4 and hsp-16.2 could offer further insights, while fluorescent reporters for skn-1 or ARE-linked genes would enable real-time visualization of PRSE’s effects on antioxidant defenses. These options could therefore be examined in future studies. Future studies are still needed to confirm whether PRSE can influence the Nrf2/HO-1 and NF-κB pathways in the C. elegans model.

4. Materials and Methods

4.1. Strain, Culture Conditions and Harvesting Synchronous Cultures

Escherichia coli OP50 (E. coli OP50) and the wild-type N2 (Bristol) C. elegans strain were obtained from the Caenorhabditis Genetics Center in Minneapolis, MN, USA. Concentrated E. coli OP50 was prepared by diluting 1 g of the cultured E. coli OP50 pellet in 12 mL of M9 buffer. To achieve age-synchronized L1 wild-type N2 (Bristol) nematodes, gravid adult nematodes were treated with a mixture of 1 mL bleach and 0.5 mL of 5N NaOH [41]. The eggs were then incubated in 3 mL of M9 buffer for 48 h. All cultured nematodes on NGM plates were maintained at 20 °C throughout their entire lifespan.

4.2. Polyphenol-Rich Sugarcane Extract and Treatment

PRSE from the Saccharum officinarum plant was utilized in this study. This patented product is developed by The Product Makers in Keysborough, Victoria, Australia, who provide its specifications and information about the manufacturing process [22,23,29]. PRSE has a total polyphenol content of 22.1%, equivalent to 221 mg/g Gallic Acid Equivalency (GAE). The primary phenolic compounds identified in the composition include Apigenin, Luteolin, Tricin, Diosmin, Syringic acid, and Chlorogenic acid listed in Table 1 along with antioxidant activity, as derived from Ji et al. [23] and Deso et al. [22].
A single sample of PRSE was used in this study. The PRSE was prepared with M9 buffer at a concentration of 5 mg/mL, as this concentration was found to be the most effective among those in the 1–5 mg/mL range for its impact on longevity and heat stress during aging [29]. In a previous study, a dose–response experiment tested concentrations ranging from 1 to 5 mg/mL, finding that 5 mg/mL provided the most notable improvements in lifespan and heat stress resistance and was the minimum effective dose for the C. elegans model. Consequently, its effectiveness on various aging parameters was investigated, including chemosensation and short-term associative cognitive decline [29]. In similar studies, this PRSE suspension was then applied to a layer of E. coli OP50 bacteria that had been cultured overnight on a 60 × 10 mm NGM (nematode growth medium) agar plate containing 5 mL of NGM agar. The application was performed at a 2:1 volume ratio, with 400 μL of the PRSE suspension mixed with 200 μL of E. coli OP50. As a result, the final concentration of the diluted PRSE was 0.1 mg/mL. The PRSE suspension was introduced from the first larval stage onwards in triplicate and maintained at 20 °C.

4.3. Chemotaxis Behaviour, Learning and Short-Term Associative Memory

Naïve chemotaxis, learning, and short-term associative memory assays were adapted from the chemotaxis assay defined by Margie et al. [42] and the positive butanone learning assay discussed by Kauffman et al. [43]. The chemotaxis index (CI) was calculated using the formula:
CI = ( T 1 + T 2 ) − ( C 1 + C 2 ) ( Total   number   of   scored   worms )
where (T1 + T2) is number of worms paralysed on butanone quadrants and (C1 + C2) is the number of worms paralysed on ethanol quadrants. Worms that remained within a 1 cm diameter origin circle were considered unscored and were excluded from the analysis. The test plate was prepared with 1 µL of 10% butanone as the attractant solution, 1 µL of 95% ethanol as the control solution, and 0.5 µL of 0.5 M sodium azide (NaN3) as the paralysis solution, following the optimization scheme (Figure 4).
On each day (day 4, 8 and 12) three independent experiments were performed which led to triplicates on each day. Age-synchronized hermaphrodites (600–1000) were cultured on high-growth media plates (100 × 10 mm) with an E. coli OP50 lawn. This served as one replicate of the control treatment. For the test treatment, another replicate of worms received 5 mg/mL of PRSE suspended in M9 buffer. The ratio of PRSE suspension to E. coli OP50 was 2:1 (v/v) as previously mentioned. Specifically, 600 µL of PRSE suspension and 300 µL of E. coli OP50 were used as the treatment on each culture plate. PRSE supplementation began at the L1 stage and continued until the worms reached day 4, day 8, and day 12 for assay testing. Three replicates of both treated and untreated worms were tested for naïve chemotaxis, positive butanone learning, and short-term associative memory assays. For each assay, an average of 100 to 250 worms were used. The modified naïve chemotaxis, learning, and short-term associative memory assays are shown below (Figure 5).
The learning index (LI) at different time points was calculated as follows:
L I   =   C h e m o t a x i s   i n d e x t − C h e m o t a x i s   i n d e x n a ï v e
Short-term associative memory loss after 1 h and 2 h of associating with butanone and food was calculated as:
S h o r t - t e r m   a s s o c i a t i v e   m e m o r y   l o s s   i n d e x t = L e a r n i n g   i n d e x 0 − L e a r n i n g   i n d e x t

4.4. Statistical Analysis

All statistical analyses were performed using IBM SPSS® Statistics software (version 29.0.2.0), and the visual representations were generated with GraphPad Prism 9.1.0. Data are expressed as mean ± SEM (Standard Error of the Mean), unless stated otherwise. A General Linear Model (GLM) with a multivariate test was applied to assess differences in chemotaxis, learning, and memory loss indices between groups. In this study, the GLM-Multivariate test was utilized to investigate the effects of two fixed factors: the day (indicating the worm’s age) and the treatment (PRSE vs. NO PRSE). This model facilitates a thorough analysis by accounting for potential interactions and correlations between the dependent variables. The selection of this model is validated by its proven effectiveness in similar research studies [18].

5. Conclusions

The research demonstrates that Polyphenol-Rich Sugarcane Extract (PRSE) at a 5 mg/mL concentration significantly enhances chemosensory abilities, learning, and memory in Caenorhabditis elegans, irrespective of age. PRSE notably improved naïve chemotaxis and learning indices, indicating heightened sensory responsiveness and learning capacity, with benefits observed across young, middle-aged, and older worms. Additionally, PRSE mitigated short-term memory decline, highlighting its potential to preserve cognitive function during aging. These findings suggest that PRSE could be an effective supplement for addressing age-related sensory and cognitive deterioration. Future studies could further investigate the mechanisms involved by examining different strains, including Alzheimer’s model strains expressing the pathogenic human amyloid-beta peptide (Aβ1–42).

Author Contributions

Conceptualization: D.H. and M.J.; methodology: D.H., M.J. and M.M.; formal analysis: D.H. and M.J.; resources: D.H. and M.F.; writing—original draft: D.H.; writing—review and editing: D.H., M.F., M.J., M.M. and J.T.; supervision: J.T.; project administration: D.H.; funding acquisition: D.H. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by La Trobe University and The Product Makers Pty. Ltd. Postgraduate Research Scholarship (LTUPRS) and La Trobe University Full Fee Research Scholarship (LTUFFRS). The authors declare that this study received partial funding from The Product Makers Pty. Ltd. via an industry-based PhD scholarship.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data supporting the findings of this study are available from the corresponding author upon reasonable request. Access to the data is subject to approvals by La Trobe University on a case-by-case basis.

Conflicts of Interest

Author Matthew Flavel was employed by the company The Product Makers Pty. Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The funder had the following involvement with the study: supplying their product and partially funding the La Trobe Post Graduate Research Scholarship (LTUPRS). The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication.

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Figure 1. Naïve chemotaxis of control and Polyphenol-Rich Sugarcane Extract treated wild-type N2 Caenorhabditis elegans on day four, day eight and day twelve. Naïve chemotaxis assay was conducted at day four, day eight and day twelve to determine the naïve chemotaxis index. Although naïve chemotaxis index from day four to day twelve was a decreasing trend in both PRSE treated (0.26 to 0.19) and untreated worms (0.19 to 0.14), PRSE (5 mg/mL) significantly improved naïve chemotaxis index on each day compared to the control group (* p < 0.05). Data represent mean values ± SEM, based on three independent replicates.
Figure 1. Naïve chemotaxis of control and Polyphenol-Rich Sugarcane Extract treated wild-type N2 Caenorhabditis elegans on day four, day eight and day twelve. Naïve chemotaxis assay was conducted at day four, day eight and day twelve to determine the naïve chemotaxis index. Although naïve chemotaxis index from day four to day twelve was a decreasing trend in both PRSE treated (0.26 to 0.19) and untreated worms (0.19 to 0.14), PRSE (5 mg/mL) significantly improved naïve chemotaxis index on each day compared to the control group (* p < 0.05). Data represent mean values ± SEM, based on three independent replicates.
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Figure 2. The 1× learning indices of control and Polyphenol-Rich Sugarcane Extract treated wild-type N2 Caenorhabditis elegans on day four, day eight and day twelve. After one-hour starvation followed by one-hour conditioning with food and 10% butanone, learning assay was performed at day four, day eight and day twelve to calculate 1× learning index. As in the naïve chemotaxis assay, the learning index was gradually decreasing as part of aging from day four to day twelve; however, PRSE at dose of 5 mg/mL increased learning index compared to control group of each day significantly (* p < 0.05). Data represent mean values ± SEM, based on three independent replicates.
Figure 2. The 1× learning indices of control and Polyphenol-Rich Sugarcane Extract treated wild-type N2 Caenorhabditis elegans on day four, day eight and day twelve. After one-hour starvation followed by one-hour conditioning with food and 10% butanone, learning assay was performed at day four, day eight and day twelve to calculate 1× learning index. As in the naïve chemotaxis assay, the learning index was gradually decreasing as part of aging from day four to day twelve; however, PRSE at dose of 5 mg/mL increased learning index compared to control group of each day significantly (* p < 0.05). Data represent mean values ± SEM, based on three independent replicates.
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Figure 3. Short-term associative memory loss indices of control and Polyphenol-Rich Sugarcane Extract treated wild-type N2 Caenorhabditis elegans at day four, day eight and day twelve. Massed short-term associative memory loss was measured after 1 h and 2 h from conditioning at day four, day eight and day twelve. Differences between PRSE supplemented worms and control after one hour from conditioning at day four and day twelve were not statistically significant; however, PRSE significantly decreased memory loss index after 1 h from conditioning at day four (* p < 0.05). On the other hand, memory loss index significantly decreased after 2 h from conditioning at day four, day eight and day twelve (* p < 0.05). Data represent mean values ± SEM, based on three independent replicates.
Figure 3. Short-term associative memory loss indices of control and Polyphenol-Rich Sugarcane Extract treated wild-type N2 Caenorhabditis elegans at day four, day eight and day twelve. Massed short-term associative memory loss was measured after 1 h and 2 h from conditioning at day four, day eight and day twelve. Differences between PRSE supplemented worms and control after one hour from conditioning at day four and day twelve were not statistically significant; however, PRSE significantly decreased memory loss index after 1 h from conditioning at day four (* p < 0.05). On the other hand, memory loss index significantly decreased after 2 h from conditioning at day four, day eight and day twelve (* p < 0.05). Data represent mean values ± SEM, based on three independent replicates.
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Figure 4. Schematic diagram of the chemotaxis assay plate. The measurements of 10% butanone (T1 and T2) spots and 95% ethanol (C1 and C2) spots from the origin are shown on the chemotaxis assay plate (60 × 10 mm). The assay plates were divided into four quadrants: two quadrants for testing the chemoattractant (T1 and T2) and two quadrants for control (C1 and C2). A measurement of 0.5 M sodium azide was applied to all four marked spots to paralyze worms in the quadrants. Worms were released at the origin spot at the center of the plates, and after an hour, worms on each quadrant were counted for analysis. Worms inside the origin circle at the center were removed from analysis as unscored worms, and the scored worms were considered as the total number of worms outside the origin circle with a 1 cm diameter. The chemotaxis index calculation was based on the number of worms paralyzed on the C1 and C2 quadrants (neutral), subtracted from those on the T1 and T2 quadrants (chemoattractant), divided by the total number of scored worms.
Figure 4. Schematic diagram of the chemotaxis assay plate. The measurements of 10% butanone (T1 and T2) spots and 95% ethanol (C1 and C2) spots from the origin are shown on the chemotaxis assay plate (60 × 10 mm). The assay plates were divided into four quadrants: two quadrants for testing the chemoattractant (T1 and T2) and two quadrants for control (C1 and C2). A measurement of 0.5 M sodium azide was applied to all four marked spots to paralyze worms in the quadrants. Worms were released at the origin spot at the center of the plates, and after an hour, worms on each quadrant were counted for analysis. Worms inside the origin circle at the center were removed from analysis as unscored worms, and the scored worms were considered as the total number of worms outside the origin circle with a 1 cm diameter. The chemotaxis index calculation was based on the number of worms paralyzed on the C1 and C2 quadrants (neutral), subtracted from those on the T1 and T2 quadrants (chemoattractant), divided by the total number of scored worms.
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Figure 5. Illustration of modified naïve chemotaxis, 1× learning and short-term associative memory assays. Between 600 and 1000 worms were washed off the treatment or control plate and immediately 100–250 washed worms with M9 buffer collected in microtubes were released on the original spot of naïve chemotaxis assay plate. After wiping the excess M9 on the original spot, worms were moved to butanone or ethanol spots and paralyzed there. After an hour, the number of worms paralyzed on each quadrant was recorded for naïve chemotaxis analysis. The remaining worms were kept in a microtube, starved for one hour in M9 buffer, transferred to NGM plates (60 × 10 mm), and seeded with E. coli lawn and 2 µL of 10% butanone stretched over the plate’s lid for conditioning. After an hour of conditioning, the worms were washed off the plate and were divided into three equal parts. The first part was used directly after conditioning for 1× positive butanone associative learning assay (t = 0) and the other two parts transferred to two NGM plates seeded with E. coli without butanone for one and two hours respectively (t = 1 and t = 2). After one hour, worms kept on NGM plate (t = 1) were washed off and tested for short-term associative memory on assay plate for an hour. The last part of worms kept on NGM plate with seeded E. coli for two hours (t = 2) were washed off the plate and released on assay plate for one hour of short-term associative memory assay. At the end of each assay, the number of worms paralyzed on each quadrant were recorded for analysis.
Figure 5. Illustration of modified naïve chemotaxis, 1× learning and short-term associative memory assays. Between 600 and 1000 worms were washed off the treatment or control plate and immediately 100–250 washed worms with M9 buffer collected in microtubes were released on the original spot of naïve chemotaxis assay plate. After wiping the excess M9 on the original spot, worms were moved to butanone or ethanol spots and paralyzed there. After an hour, the number of worms paralyzed on each quadrant was recorded for naïve chemotaxis analysis. The remaining worms were kept in a microtube, starved for one hour in M9 buffer, transferred to NGM plates (60 × 10 mm), and seeded with E. coli lawn and 2 µL of 10% butanone stretched over the plate’s lid for conditioning. After an hour of conditioning, the worms were washed off the plate and were divided into three equal parts. The first part was used directly after conditioning for 1× positive butanone associative learning assay (t = 0) and the other two parts transferred to two NGM plates seeded with E. coli without butanone for one and two hours respectively (t = 1 and t = 2). After one hour, worms kept on NGM plate (t = 1) were washed off and tested for short-term associative memory on assay plate for an hour. The last part of worms kept on NGM plate with seeded E. coli for two hours (t = 2) were washed off the plate and released on assay plate for one hour of short-term associative memory assay. At the end of each assay, the number of worms paralyzed on each quadrant were recorded for analysis.
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Table 1. Polyphenol composition and antioxidant activity of Polyphenol-Rich Sugarcane Extract (PRSE).
Table 1. Polyphenol composition and antioxidant activity of Polyphenol-Rich Sugarcane Extract (PRSE).
Total Polyphenols221 mg/g as Gallic Acid Equivalency (GAE)
Key polyphenols
Apigenin1.89 μg/g
Luteolin5.30 μg/g
Tricin27.40 μg/g
Diosmin227.00 μg/g
Syringic acid107.57 μg/g
Chlorogenic acid74.29 μg/g
Total flavonoids53.8 mg/g Catechin Equivalency (CE)
Total antioxidant activity (Oxygen Radical Absorbance Capacity—ORAC 5.0)18,837 μmol Trolox Equivalency (TE) per gram
ORAC against hydroxyl radicals13,785 μmol TE per gram
ORAC against peroxyl radicals2336 μmol TE per gram
ORAC against peroxynitrite255 μmol TE per gram
ORAC against singlet oxygen2011 μmol TE per gram
ORAC against superoxide anion450 μmol TE per gram
Cellular antioxidant assay229.12 μmol Quercetin Equivalency (QE) per gram
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Heydarian, D.; Flavel, M.; Munasinghe, M.; Jois, M.; Thomas, J. Improving Cognitive and Chemosensory Function in Caenorhabditis elegans Through Polyphenol-Rich Sugarcane Extract. Stresses 2024, 4, 816-826. https://doi.org/10.3390/stresses4040054

AMA Style

Heydarian D, Flavel M, Munasinghe M, Jois M, Thomas J. Improving Cognitive and Chemosensory Function in Caenorhabditis elegans Through Polyphenol-Rich Sugarcane Extract. Stresses. 2024; 4(4):816-826. https://doi.org/10.3390/stresses4040054

Chicago/Turabian Style

Heydarian, Deniz, Matthew Flavel, Mihiri Munasinghe, Markandeya Jois, and Jency Thomas. 2024. "Improving Cognitive and Chemosensory Function in Caenorhabditis elegans Through Polyphenol-Rich Sugarcane Extract" Stresses 4, no. 4: 816-826. https://doi.org/10.3390/stresses4040054

APA Style

Heydarian, D., Flavel, M., Munasinghe, M., Jois, M., & Thomas, J. (2024). Improving Cognitive and Chemosensory Function in Caenorhabditis elegans Through Polyphenol-Rich Sugarcane Extract. Stresses, 4(4), 816-826. https://doi.org/10.3390/stresses4040054

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