Effect of time after harvest on chlorophyll concentration in spinach leaves

Kyle Scholtz

Barker College

Chlorophyll-a and chlorophyll-b are chemical compounds found in photosynthetic plants and its application in medicine is becoming widely accepted. This report aimed to reveal how time after harvest affects chlorophyll-a and chlorophyll-b concentration. Spinach leaves where stored over different time periods and dimethylsulfoxide was used to extract chlorophyll from leaf tissue. A colourimeter was used to determine chlorophyll-a and chlorophyll-b concentration. The results demonstrated that chlorophyll-a concentration had no significant difference after each harvest time, but concentration of chlorophyll-b did have a statistical difference after each harvest time. Concentration of chlorophyll-b was found to be higher 48 hours after harvest compared to instantly after harvest which resulted in the rejection of the null hypothesis.

Abbreviations

Chlorophyll-a (Chl-a), Chlorophyll-b (Chl-b), dimethylsulfoxide (DMSO).

Literature review

Chlorophyll is a chemical compound or green pigment which is found within the cells of the thylakoid membrane of the chloroplast. Chl-a and Chl-b are present in higher plants whereas chlorophyll c, d and e are found in photosynthetic algae. Chlorophyll reflects and absorbs certain wavelengths of light; its primary role is to absorb light to use for photosynthesis. Plants use the energy collected from the chlorophyll to convert carbon dioxide and water into glucose and oxygen. The glucose produced through photosynthesis is used for energy and the oxygen produced is released into the atmosphere. Without chlorophyll, plants would be unable to undergo photosynthesis and hence would be unable to synthesise carbohydrates.

The process of photosynthesis is written in the equation: 6CO2 + 6H2O → C6H12O6 + 6O2.

The two main types of chlorophyll are Chl-a and Chl-b, Chl-a is generally found in higher concentration then Chl-b by a 3:1 ratio but it varies between species and can be influenced by a number of factors including pre- and post-harvest treatment and agroclimatic conditions (Ferruzzi & Blakeslee 2007). Chl-a and Chl-b have different roles in the process of photosynthesis, and they have different structures. Both Chl-a and Chl-b are similarly shaped with hydrophobic tails and hydrophilic heads. The structure of a chlorophyll molecule includes a porphyrin ring in the centre of the molecule, as seen in Figure 1. Chl-a and Chl-b differ by one atom in a side chain on the third carbon, Chl-b contains an aldehyde group (-CHO) whereas Chl-a contains a methyl group (CH3), as seen in Figure 1. Chl-a is the primary pigment of photosynthesis whereas Chl-b is an accessory. Chl-a absorbs light from the orange-red and violet-blue regions of the visible spectrum whereas Chl-b absorbs light from blue areas of the visible light spectrum.

Figure 1: Structure of chlorophyll (Source: May, 1999).

It is known that as plants lose the green colour, otherwise known as degreening, it is a result of the degradation of chlorophyll. None of the genes which encode for the catabolic enzymes responsible for the breakdown of chlorophyll have been isolated but chlorophyll degradation has been proven to have multiple degradative pathways (Matile, Hörtensteiner & Thomas, 1999). A study conducted by Yamauchi and Watada (1991) concluded that the degradation of

chlorophyll in spinach was regulated through the Peroxidase-hydrogen peroxide pathway which opens the porphyrin ring of the chlorophyll molecule as seen in Figure 2. The opening of the porphyrin ring results in the colour loss of spinach leaves. Chlorophyll has many pathways of degradation and it is not the same in every plant, some plants rely on chlorophyllase which results in the release of the phytol chain in chlorophyll to form chlorophyllide (Yamauchi & Watada, 1991). The main chlorophyll degradative reactions and the derivatives formed are summarised in Figure 3.

Figure 2: Peroxidase-hydrogen peroxide pathway. (Source: Kaewsuksaeng, 2011)

The benefit of chlorophyll to humans has been extensively researched in the medical field and it has been found that chlorophyll and its derivatives, as seen in Table 1, have many medical benefits. When chlorophyll is digested, it is exposed to an acidic environment resulting in conversion to metal free pheophytins. Chlorophyll derivatives are then absorbed by intestinal cells and enter blood circulation which allows the derivatives to act through a wide range of mechanisms, including being a modifier for genotoxic effect and antioxidant activity. A genotoxin is an agent or chemical which can cause chromosomal or DNA damage. A study conducted by Waters et al. (1996) assessed the antimutagenicity profile for chlorophyll and their research concluded that chlorophyll is antimutagenic to range of direct- and indirect-acting mutagens (Waters et al. 1996). A study conducted by Osowski et al. concluded that consumption of chlorophyll in food does not significantly protect against mutagenic compounds but a derivative of chlorophyll, chlorophyllin, which does not occur naturally, can act as a binding agent against mutagenic compounds if it is used as a supplement (Osowski et al. 2010). Chlorophyllin was found to have applicable therapeutic measures for individuals exposed to aflatoxin, as chlorophyllin was found to be effective in preventing liver cancer (Egner, Kensler & Muñoz, 2003).

Figure 3: Derivatives of chlorophyll. (Source: Ferruzzi & Blakeslee, 2007)

Table 1:Chlorophyll and its derivatives used in medicine (Source: Mishra, Bacheti & Husen, 2011).

Chlorophyll Category Derivatives

Natural chlorophyll Chlorophyll a, b, c, d, e Metal free chlorophyll derivatives Pheophytin, Pyropheophytin

Metallochlorophyll derivatives

Zn-Pheophytin Zn-pyropheophytin Chlorophyllide Pheophorbide Cu(II)chlorin e 4 Cu-chlorin e 6 Cu-chlorin e 4 ethyl ester

A study conducted by Barnes et al. (1992) concluded that the use of Dimethyl Sulfoxide (DMSO) as a solvent is an effective way of extracting chlorophyll from plants due to its amphiphilic properties. After incubation in DMSO, further extraction resulted in no additional chlorophyll and there was no green colour left in plant tissue which concluded that through incubating plant tissue in DMSO, the complete extraction of chlorophyll can take place. The use of DMSO as a means to extract chlorophyll proved to be more effective than previous methods such as using 80% acetone as incubation in DMSO as it resulted in no chance in the ratio of Chl-a and Chl-b (Barnes et al. 1992).

Through knowing how time after harvest affects chlorophyll concentration, the degradation pathways of chlorophyll can be better understood. The degradation of chlorophyll results in a variety of derivatives which are becoming widely accepted for their application in the medicinal field. Through understanding how time after harvest affects chlorophyll concentration, a better understanding of when chlorophyll is most useful can be formed

Scientific research question

How does time after harvest affect the concentration of chlorophyll-a and chlorophyll-b extracted from spinach?

Scientific hypothesis

As time after harvest increases, chlorophyll concentration will decrease.

Methodology

Preparation

A spinach plant (swiss chard variety) was grown from the beginning of December 2020, up until the end of March 2021. The plant was grown outdoors under a netting to protect against animals and environmental stresses. The plant was grown in favourable conditions and was provided adequate sunlight, water and cooling when necessary. Four sets of three spinach leaves were harvested and stored in complete darkness at room temperature. Each set of leaves were stored over different time periods, 0 hours, 24 hours, 48 hours and 96 hours. After each set of leaves were stored for the required time period the extraction of chlorophyll occurred.

Extraction of chlorophyll-a and chlorophyll-b (carried out for all time points)

Plant tissue from each leaf was cut into small pieces, approximately 5mm x 5mm, using a sterilized scalpel. 0.1g of the plant tissue, one sample from one leaf and two samples from the other two leaves (5 samples) were weighed and then placed in separate test tubes. 10ml of DMSO solvent was added into each test tube which was shaken for five seconds. The test tubes were placed into a test tube rack and incubated in a water bath with a constant temperature of 600 -650C for 60 minutes which allowed for decolourisation of leaf tissue. After the 60minute incubation period, when the leaves were fully decolourised, the test tubes were removed from the water bath and cooled at room temperature for 30 minutes. This method was completed for each time point.

Colourimetry (Carried out for all time points):

A PASCO colourimeter was used alongside Spark Vue data logging software. A clean quartz cuvette was filled with DMSO solvent and the colourimeter was calibrated at 650 nm. A sample from each test tube was removed using a dropper the solution was placed in separate, clean quartz cuvettes. Each cuvette was placed in the colourimeter and absorbance was measured at 650 nm for each sample and results were recorded.

Beer-lamberts law (Equation 1) was used to calculate Chl-a and Chl-b concentration. The extinction coefficient (ε) used for Chl-a was 18.47 and the extinction coefficient used for Chl-b was 50.81. (Inskeep & Bloom 1985).

A = εlc

Equation 1: Beer-lambert law.

Results

Table 2: Mean concentration of Chl-a and standard deviation for each group.

Group Time after harvest (hours) Mean concentration (mg/L) σ

1 0 19.1364 2.3678 2 24 18.768 1.5768 3 48 22.4336 3.391 4 96 21.091 3.6041

(mg/L)

concentration Chl a

25

20

15

10

5

0 Chlorophyll-a

0 24 48 72 96

Time after harvest (Hours)

Figure 4: Mean concentration of Chl-a versus time after harvest.

Table 3: Mean concentration of Chl-b and standard deviation for each group.

Group Time after harvest (hours) Mean concentration (mg/L) σ

1 0 6.618 0.2066 2 24 7.0702 0.5078 3 48 8.4186 0.9074 4 96 7.4936 1.5612

b Concentration (mg/L) Chl 25 20 15 10 5 0 Chlorophyll-b

0 24 48 72 96

Time after harvest (Hours)

Figure 5: Mean concentration of Chl-b versus time after harvest.

H0 = That there is no statistical difference between the mean chlorophyll concentration after each harvest time. HA = That there is a significant difference between the mean concentration of chlorophyll after each harvest time.

Table 3: Mean concentration of Chl-b and standard deviation for each group.

0 hours (T1) 24 hours (T2) 48 hours (T3) 96 hours (T4)

Average 19.136 18.768 22.433 21.091

Standard deviation 2.3678 1.5768 3.391 3.6041

F-statistic 1.81412

Alpha Value 0.05

P value 0.185179

Analysis p value > Alpha value, therefore values are not statistically significant. H0 = That there is no statistical difference between the mean chlorophyll concentration after each harvest time. HA = That there is a significant difference between the mean concentration of chlorophyll after each harvest time.

Table 4: ANOVA test output for comparing concentration of Chl-b extracted after different times after harvest.

0 hours (T1) 24 hours (T2) 48 hours (T3) 96 hours (T4)

Average 6.618 7.0702 8.4186 7.4936

Standard deviation 0.2066 0.5078 0.9074 1.5612

F-statistic 3.307

Alpha Value 0.05

P value 0.047164

Analysis p value < Alpha value, therefore values are statistically significant.

Table 5: Post-Hoc Tukey test p-values for comparing the mean concentration of Chl-b extracted at each time point after harvest

Treatment Pairs Qstat Tukey HSD p-value Tukey HSD analysis

T1:T2 M M 1 2 = 6.62 = 7.07 1.07 0.872 insignificant

T1:T3

T1:T4 M M 1 3 = 6.62 = 8.12 4.27 0.036 significant (p < 0.05) M M 1 4 = 6.62 = 7.49 2.07 0.479 insignificant

T2:T3

T2:T4 M2 M3 =7.07 = 8.42 3.20 0.150 insignificant M M 2 4 = 7.07 = 7.49 1.00 0.891 insignificant

T3:T4 M3 M4 = 8.42 =7.49 2.19 0.433 insignificant

Discussion

This report explored how time after harvest affects Chla and Chl-b concentration in spinach. As seen in Tables 2 and 3, the ratio of Chl-a to Chl-b was approximately 3:1 which aligns with previous studies, (Ferruzzi & Blakeslee, 2007). An ANOVA statistical analysis was completed for Chla, as seen in Table 4. The p-value calculated for Chl-a was 0.185179 and the alpha value used was 0.05. The calculated p-value was greater than the alpha value, hence the null hypothesis cannot be rejected, (Ho = There is no significant statistical difference between the means of each group.). This suggests that time after harvest has no significant affect on Chl-a concentration in spinach. However, due to time pressure and limited recourses the longest time period after harvest where Chl-a concentration was measured was 96 hours which decreases the reliability of the experimental results. The extinction coefficient used to calculate Chl-a concentration was 18.47 which is the extinction coefficient used when absorbance is measured at 647 nm (Inskeep & Bloom 1985). The colourimeter used in the experiment was unable to measure absorbance at 647 nm but absorbance was measured at 650 nm and 18.47 was still the value used as the extinction coefficient which decreased the accuracy of the results.

An ANOVA statistical analysis was also completed for Chl-b, as seen in Table 5. The calculated p-value for Chl-b was 0.047164 which is smaller than the alpha value used (0.05). p-value < alpha-value, hence the null hypothesis can be rejected, and the alternative hypothesis can be accepted, (HA = There is a significant statistical difference between the means of each group.). This suggests that time after harvest does affect Chl-b concentration in spinach. The calculated f-ratio was 3.307 which indicated that there was an overall difference between the sample means. A Post-Hoc Tukey test, as seen in Table 6, was conducted and it concluded that the variance between T1 (0 hours) and T3 (48 hours) were statistically different. This was evident because the p-value calculate was 0.03696 and the value for Q was calculated as 4.27 which indicates a statistically significant difference between the two groups. Chl-b concentration increased between T1 and T3 which was different to Chl-a concentration which had no statistically significant differences between groups. A possible reason for these results is that the ratio of Chl-a to Chl-b is a 3:1 ratio, hence there is much lower concentration of Chl-b. This can possibly lead to slightly different results as changes in Chl-b concentration can be more statistically significant due to the lower concentration. It is also possible that this result is an outlier but due to the large sample size (20 samples), it is unlikely to be an outlier. Similar to Chl-a, the coefficient used to calculate the concentration of Chl-b (50.81) is the value used when absorption is measured at 647 nm. This source of error has the same affect on Chl-b as it has on Chl-a.

With the exception of the value calculated for Q between T1 and T3 for Chl-b, the majority of the results concluded that time after harvest does not affect chlorophyll concentration. The concluded results where unexpected as the hypothesis stated that time after harvest would affect chlorophyll concentration. It is possible that the duration of the experiment was a source of error as chlorophyll concentration was only calculated at four time points. Through increasing the length of the experiment and adding more time points, a better understanding of how time after harvest affects chlorophyll concentration can be formed. It is possible that results did not align with literature because chlorophyll may take longer to degrade then what time allowed for in the experiment. Through increasing time after harvest, a larger dataset could have been collected and more reliable results could have been collected. It was due to limited time that a larger number of time points could not have been recorded.

In the extracted solutions, other pigments may havebeen present, hence the pigments present may have absorbed some of the light. This is a possible source of error which could have impacted the accuracy of the experimental results. Multiple sources of error such as not having an accurate colourimeter and limited time resulted in inaccuracies throughout the experimental results. By measuring absorbance at 650 nm instead of 657 nm, inaccurate results were recorded. Limited time resulted in a small data set which negatively impacts the reliability of the experimental results.

In order to develop a deeper understanding of how time after harvest affects chlorophyll, further studies must be conducted. To extend on results concluded from this report, a wider range of time points will be needed, alongside a more accurate colourimeter to ensure accurate results. Through understanding how time after harvest affects chlorophyll levels, yield of natural chlorophyll can be increased through knowing when chlorophyll is at its highest concentration. This knowledge can be applicable to the medicinal field as chlorophyll is becoming increasingly accepted in medicine. Chlorophyll is becoming increasingly significant in science because medical applications of chlorophyll are increasing and through a better understanding of chlorophyll and how it works, its application to medicine can soon become better understood.

Conclusion

There has been significant potential found in the use of chlorophyll in medicine. This experiment investigated how time after harvest affects Chl-a and Chl-b concentration in spinach leaves. DMSO was used to extract chlorophyll from spinach leaf tissue which allowed for absorption of the solutions to be recorded. The concentration of Chl-a recorded at each time point did not reveal any statistically significant differences which resulted in the null hypothesis being accepted. Chl-b revealed a statistically significant difference between Chl-b concentration measured at T1 and T2. Chl-b concentration increased between T1 and T2 but further research will need to be conducted to further understand how time after harvest affects chlorophyll concentration.

The results collected revealed that between 0 hours after harvest and 96 hours after harvest, Chl-a concentration did not significantly change. Chl-b however did reveal a statistically significant difference between timepoints as chlorophyll concentration was recorded to be higher 48 hours after harvest compared to instantly after harvest. The results addressed the research question and revealed how time after harvest affects chlorophyll concentration, but only at four different time points. Due to limited time the longest period spinach was stored at was 96 hours which only allowed chlorophyll concentration to be recorded up until 96 hours. There is need for further research in multiple areas surrounding this report including further research on the degradation pathway of chlorophyll, finding more accurate equations for determining chlorophyll concentration and recording how chlorophyll concentration is affected by time after harvest through using a larger data set. Chlorophyll has the potential for widespread medical use however further research must be conducted before it can be better understood.

Acknowledgments

I would like to thank Dr Katie Terrett for her extensive assistance in the completion of the report. I would also like to thank Mr Robert Paynterfor his supervision when the experiment was completed.

References

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