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Determination of the antifungal properties of polyphenols extracted from the common grape
Determination of the antifungal properties of polyphenols extracted from the common grape (Vitis vinifera)
Owen Ng
Barker College
Purpose: This research aims to determine whether polyphenolic compounds extracted from grapeseeds (Vitis vinifera) are able to inhibit the growth of common grape fungi (Botrytis sp) and thus to consider their suitability as an organic fungicide in grape production. Design/methodology/approach: A method for extracting polyphenols in a secondary school laboratory was developed. The extracted compounds were tested via well diffusion on agar plates lawned with Botrytis sp against a control of potassium bicarbonate, a compound used widely in viticulture enterprises as an antifungal. Findings: A positive linear association was found between concentration of polyphenols and inhibition. However, it was also observed that polyphenols are less effective than that of the potassium bicarbonate control. Research Limitations/implications: This research was primarily focused on the polyphenols present in seeds, which may not reflect the composition and antifungal nature of other parts of the grape such as the skin. A larger sample size and more sophisticated laboratory analysis could improve the reliability of this research. Practical/social implications: If successful, this may allow for the development of new, ecologically friendly organic fungicides and sustainable horticultural practices in the future to meet the growing demand for mass produced food in a growing global food crisis. Originality/value: Rather than predict potential antifungal properties with radical scavenging molecules, this study uses live samples of Botrytis cinerea to determine antifungal properties in a practical manner. In addition, through a rigorous process of trial and error, this research outlines a reliable method for extracting polyphenols in a secondary school laboratory. This is not a trivial contribution since it could be used to develop interesting, engaging laboratory learning experiences for secondary chemistry students. Keywords: Botrytis cinerea, Polyphenols, Inhibition Paper Type: Research paper
Literature Review
Botrytis cinerea is an airborne, necrotrophic fungus frequently observed on soft fruits such as grapes and can be found in virtually all environments (Kan, Shaw & Grant‐Downton 2014). Since 2015, B. cinerea has cost the global wine industry between USD 10-100 billion per year (Brito et al. 2021), which is up to 23.8% its total market size (Huron, Behm & Helmus 2021). Able to infect over 1000 crop species (Fillinger 2016), it can destroy up to 80% of any given harvest, making it one of the most prolific plant fungal pathogens globally (Petrasch et al. 2019).
According to Williamson et al. (2007), plants infected by B. cinerea will begin to exhibit necrosis, as parenchyma tissues collapse and fill with water, presenting brown lesions up to 15cm long and soft rotting of aerial plant parts. The compromised plant is now more susceptible to opportunistic pathogens such as Aspergillus niger or Penicillium expansum that can further damage the crop. Prolific grey conidiophores become present post necrosis and can infect a neighbouring plant within 12 hours, or live on its surface for 3 months (Reignault et al. 2000).
Figure 1: Inhibition of fludioxonil, iprodione and other antifungals on B. cinerea conidial germination. (Source: Kim et al. 2016).
Currently, the most common treatment for B. cinerea is the use of anti-Botrytis fungicides known as botryticides, which currently represent 10% of the
The broth was sampled onto a microscope slide and covered with a cover slip. The slide was then observed for morphological traits such as hyaline conidia and rounded, apical cells to confirm that B. cinerea was grown. A malt agar plate was cultured from the broth to check that the sample was not contaminated.
Once positive, 5-10mm of infected skin was removed from the grape under the Bunsen and placed in a nutrient broth. This was left for 3 days at 25°C to produce a pure culture of B. cinerea.
Extraction of polyphenols Extraction
1kg of V. vinifera grapes were deseeded using a scalpel and the seeds left to dry in a dehydrator at 40°C for 2 days. The dry seeds were then removed and grinded into a fine powder with a mortar and pestle.
According to Nilüfer et al. (2018), the following conditions optimised the yield of polyphenols by ultrasound assisted extraction. In a beaker, 61.75mL of pure ethanol was mixed with 38.2mL of distilled water. This was repeated once to produce 200mL of 61.75% ethanol solution.
1.6g of grapeseed power was added to 50mL of ethanol solution in a 200mL conical flask. The flask was then fixed with tape in a 2L ultrasonic bath, filled with 1.3L of distilled water, heated to 50°C and a frequency of 28Hz. After 20 minutes, the flask was removed and cooled in room temperature water where it was filtered through Whatnam 40. filter paper.
As ethanol is an antimicrobial, it needed to be removed by heat as both solutions are soluble in water. In a separate beaker, 200mL of distilled water was heated and maintained at 90°C on a heating plate. The filtrate was measured in a measuring cylinder before being transferred to a smaller beaker. This was partially submerged in the hot water until its contents reached 85°C and left to evaporate for 10 minutes. The water temperature was continually monitored to ensure it was below 90°C, preventing the denaturation of any polyphenols present. The beaker was then removed and left to cool at room temperature. The contents were transferred again to the same measuring cylinder, which confirmed the expected volume of ethanol had evaporated.
Test for presence
5 drops of the treated solution and ethanol solution were placed in two separate test tubes. 3 drops of FeCl3 was added of each test tube. Polyphenols present in the treated solution underwent a complexation reaction
The iron phenol complex caused an immediate colour change to a dark green to be observed, confirming the presence of polyphenols. The control test tube of ethanol solution remained a very feint yellow.
Testing for a zone of inhibition
The polyphenolic solution was added in 0.5mL increments to 9 test tubes from 1-4 mL in volume. An 9th test tube was left empty as a control. Each test tube was then made up to 5mL in volume with distilled water. 9 small circles of filter paper, 10mm in diameter, were cut outand one was placed in each test tube in contact with the solution. This was repeated 3 times.
The agar plate was divided into 3 sections with a permanent marker. Pure B. cinerea culture was then applied evenly on the plate with a spreader. A KHCO3 disk (an industry standard antifungal) and a filter paper disk of polyphenol solution was placed in each section of the plate, while the third was left blank. This was repeated for the remaining 26 agar plates. The plates were left in a dark area at room temperature for 5 days and results were tabulated.
Scientific Research Question
Do polyphenolic compounds extracted from grape seeds have anti-fungal properties?
Scientific Hypothesis
That the polyphenolic compounds extracted from grape seeds will exhibit a zone of inhibition around a well on a malt agar plate.
Results
Concentration and inhibition have a positive linear association
The raw data of the area of inhibition together with the independent variable is shown in Table 1. This
was illustrated as a scatter plot and shown in Figure 2a. To examine the association between inhibition diameter in millimetres and concentration, the Pearson’s correlation coefficient was first calculated (r = 0.93) between inhibition and concentration. Figure 2a illustrates this relationship as a scatter plot where a strong positive correlation between concentration and inhibition zone was observed including a clear upward trend. Next, a linear regression model was fitted to estimate the least square regression line and the fitted line is expressed as
with a corresponding ��������2 of 0.87. Even though the ��������2 is high, the residuals vs fit plot (Figure 2b) showed a non-random curvature pattern which indicates that it may be better to fit a non-linear relationship between concentration and inhibition.
Table 1: Raw data
Concentration (%)
Zone of inhibitioncontrol (mm)
Zone of inhibition –polyphenols (mm)
100
87.5
75
62.5
50
37.5
25 35
35
35
35
35
35
35 1.3
0.8
0.46
0.33
0.03
0
0
Piece-wise linear association to model the relationship between concentration and inhibition
Two approaches were attempted to capture the nonlinear relationship between concentration and inhibition. The first involves a log-linear model and the second involves a piece wise regression where a regression line was fitted to the non-zero inhibition data. For the log-linear model, a log transformation to the variable inhibition was performed and the fitted log-linear regression model with the corresponding scatter plot shown in Figure 3a. However, residuals vs fit plot (Figure 3b) still showed a non-random curvature pattern which is likely due to the zero values in the lower concentration data. Thus, the second approach fitted a linear model to the non-zero inhibition data and the piece-wise linear model is expressed as
with the corresponding scatter plot shown in Figure 3c. The goodness of fit statistic for part of the linear regression ��������2 is 0.92 and no pattern was shown in the residual vs fit plot (Figure 3d).
Figure 2: (a) A scatter plot showing the level of inhibition (y-axis) against concentration (x-axis). The actual values are shown as black dots and the line of best fit (least square regression line) is shown in blue. The shaded regions show the confidence bound associated with the line of best fit. (b) A diagnostics plot showing that the residuals between the fitted and real values of concentration and inhibition were non-random.
Figure 3. (a) A scatter plot showing the log transformed level of inhibition (y-axis) against concentration (x-axis) with the line of best fit (blue) represented by equation �����������������������������������������ℎ�����������������������������������������������������������������������������������������=3.45+0.004����������������������������������������������������������������. (b) A diagnostics plot showing that the residuals versus the fitted values. (c) The piece-wise linear model with break point at concentration 51. (d) Diagnostic plot of the non-zero where ���������������������������������������������������������������� > 51
The inhibition of poly was similar to the control KHCO3 The hypothesis was tested by determining whether the slope of the concentration for ���������������������������������������������������������������� > 51 was significantly different from zero. The estimated coefficient of ���������������������������������������������������������������� in the linear model was 0.246
with a corresponding t-statistic of 9.304 and a p-value of 0.003. The QQ-plot (Figure 4a) shows an approximate normal distribution, indicating the assumption behind the t-statistic is valid. Despite a positive non-zero coefficient which indicates a significant positive relationship between concentration and inhibition, the level is still less than the inhibition of the positive control (��������������������������������3). Even at 100% concentration, the inhibition area of the polyphenols was 1.3mm at its maximum, while the positive control consistently inhibited 3.5mm see Figure 4b).
Discussion
In this study, the antifungal properties of polyphenols extracted from V. vinifera were examined by plating the polyphenols on a plate of B. cinerea. The results revealed that polyphenolic compounds extracted from V. vinifera did have an inhibitory effect on B. cinerea. However, the size of this effect was marginal compared to the positive antifungal control of ��������������������������������3, potentially limiting the use of polyphenols as an effective method to inhibit B. cinerea.
Figure 5: Chemical structure of resveratrol (Source: Gambini et al. 2015).
Figure 4: (a) A scatter plot showing the level of inhibition (y-axis) against concentration (x-axis) compared to the positive control ��������������������������������3. The actual values are shown as black dots and the piece-wise function is shown in black. The ��������������������������������3 is shown in red. (b) A diagnostics plot showing the similarity between residual values and a normal distribution to validate the t-test
Polyphenols contained in V. vinifera, such as resveratrol, have a unique structure allowing it to inhibit the growth of most fungi. This is consistent with the data which showed a positive correlation (r = 0.93) between polyphenol concentration and B. cinerea inhibition. This is likely due to the presence of resveratrol (a stilbene), which is a well-known phytoalexin and has already been used to successfully inhibit the growth of B. cinerea at concentrations between 60–140 μg/mL (Abedini et al. 2021). These properties can be attributed to its structure, as resveratrol (3, 5, 4’-trihydroxystilbene) is the product of substituting phenyl groups for hydroxyls at 3, 5 and 4’ (Perrone et al. 2017, Figure 5). These hydroxyl groups mediate the production of other reactive oxygen species in the cell such as peroxides and hydroxides, leading to the activation of caspase and the release of cytochrome c into the cytosol, triggering cell apoptosis in fungi and the destruction of the pathogen (Redza-Dutordoir et al. 2016; Lee et al. 2014).
While some polyphenolic compounds such as resveratrol have exhibited anti-fungal properties, other polyphenols such as Flavonoids are ineffective as anti-fungal agents. Thus, if the proportion of ineffective polyphenols are high, polyphenols will be less effective at inhibiting B. cinerea. This can partially explain that while polyphenols from grapes inhibit B. cinerea growth, the most concentrated polyphenols mixture that was generated had a lower inhibition zone (1.3mm) than the positive control KHCO3 (3.5mm). One possible explanation for this trend is that fungal cell walls, comprised of multiple layers of polysaccharides and lipids, are difficult to penetrate (Garcia-Rubio et al. 2020). Thus, for a polyphenolic compound such as Flavonoids, which rely on the exploitation of weak cell walls to destroy pathogens, are likely to be ineffective against B. cinerea (Al Aboody et al. 2020; Mohammad et al. 2020). Specifically, B. cinerea’s cell wall undergoes extensive covalent cross-linkage during growth between polysaccharides (Cantu et al. 2009) and it secretes a complex layer of triacylglycerol as part of its extra cellular matrix (Doss 1999), making it resistant to certain types of antifungal agents such as Flavonoids (Figure 7) (Nawaz et al. 2006; Singh et al. 2015).
Figure 6: Chemical structure of Flavonoids (Nishiumi et al. 2011). The polar ketone in the second carbon group interacts with porin protein on the cell membrane of B. cinerea to inhibit the transport of glucose, preventing further growth of both gram positive (e.g. Staphylococcus aureus, Xie et al. 2014) and gram negative bacteria (e.g. Escherichia coli,) (Source: Xie et al. 2017).
Liang and collegues (Liang et al. 2014) have shown that Flavonoids are the predominant polyphenolic compound in V. vinifera and are found in concentrations roughly 15x higher than resveratrol. Given the estimated low proportion of resveratrol in the tested samples, the results show non-zero inhibition levels (between 0.03-1.3mm) which
strongly indicate the potential of polyphenols as antifungal in the right concentration. In absence of an exact concentration, an inhibition area equal to that of industry standards (such as KHCO3) can be achieved by increasing the maximum concentration of the initial generated samples by 2.5-fold. If successful, this opens a new avenue for sustainable applications of fungicides in horticulture, as compounds sourced from food products may pose lower health risks for surrounding ecosystems.
This experiment was primarily limited by equipment, the total yield by mass of the polyphenols could not be determined or optimised. The seeds of the grape were used as literature has shown grape seeds contain in the highest concentration of polyphenols (60-70 wt%) in V. vinifera (Nawaz et al. 2006). However, Xie et al. (2010) observed that polyphenols extracted in the seed usually consist of proanthocyanins, epicatechin or other flavonoids, while resveratrol is primarily found in the skin. Thus, it is likely that the level of inhibition may be significantly greater when extracting polyphenols from the skin due to a greater presence of resveratrol. Furthermore, it was impossible to delineate between types of polyphenols present in the solution, such as resveratrol or flavonoids, limiting the generalisability of this study.
Although not as effective as synthetic fungicides, the results suggest the potential of polyphenols as an organic antifungal agent due to the correlation between inhibition and concentration. However, further research needs to be conducted regarding the presence of polyphenolics such as resveratrol hypothesised in the discussion. Testing other parts of the grape such as skin, with potentially high contents of resveratrol, may continue to validate polyphenols as a potential fungicide
Conclusion
In conclusion, this study has demonstrated a positive linear association between the concentration of polyphenols extracted from V. vinifera and inhibition against B. cinerea. These results indicate that the hypothesis can be cautiously accepted. However, results also show that the efficacy of polyphenols are significantly less than industry standard fungicides such as KHCO3. This trend may be explained by the low presence of antifungal compounds in V. vinifera and the structural adaptations of B. cinerea has developed to resist plant-based pathogen responses. study used live samples of B. cinerea to test the level of inhibition on a practical level, which remains critical in the search for an alternative to synthetic and ecologically damaging fungicides. Further, research into the presence of compounds such as resveratrol in other parts of V. vinifera and its potential as an antifungal agent may reveal new mechanisms in plant-pathogenic response, forming new frontiers in molecular biology and biochemistry.
Amidst the growing global food crisis, efficient and safe horticultural practices remain critical in the maintenance of the global food supply chain. Further research identifying the structure and mechanisms of plant derived secondary metabolites such as polyphenols are required to balance the demands of an exponentiating population with the harmful side effects of modern agriculture.
Acknowledgements
I would like to thank Dr Alison Gates for her insightful contributions during the writing and practical components of this study. I would also like to thank the Barker College and the lab staff for providing the adequate equipment to perform the experiment.
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