Extraction and degradation of chlorophyll a and b from Alternanthera sessilis

The use of Alternanthera sessilis, which is commonly known as Mukunuwenna in Sri Lanka as a source of chlorophyll was examined. The extraction of chlorophyll was carried out using buffered 80 % (v/v) aqueous acetone. The optimum operating conditions such as solvent to A. sessilis ratio, extraction temperature and extraction time were found to be 5 mL/g, 50 °C and 45 minutes, respectively. The yield of chlorophyll a and chlorophyll b under these optimum operating conditions were 659 and 261 μg/g of A. sessilis, respectively. Mechanical grinding of A. sessilis gave a higher yield as compared to blanching and drying. Refrigeration at 15 °C was found to be ideal for storing of fresh A. sessilis up to 3 days without a considerable loss of chlorophyll content. Chlorophyll extraction could be modelled successfully using basic mass transfer equations up to 30 °C. It failed above this temperature due to the degradation effect. Kinetic study on the degradation of chlorophyll extracted from A. sessilis confirmed first order reaction model and the effect of temperature on the rate constant was also adequately modelled by the Arrhenius equation.


INTRODUCTION
Chlorophyll is the green pigment found in plants, and is formed in the chloroplasts of the plant cells influenced by sunlight.This pigment is necessary for the process of photosynthesis.Chlorophyll exists mainly as chlorophyll a and chlorophyll b.
Chlorophyll and chlorophyll derivatives, which are known as chlorophyllin, are widely used in industry as a & Borrmann, 2009).Various therapeutic properties of chlorophyll such as anti-inflammatory activity, acceleration of wound healing, immune modulator properties and body deodorisation in geriatric and ileostomy patients have been reported (Chernomorsky & Segelman, 1988).Furthermore, the anti-mutagenic and anti-carcinogenic activities of chlorophyll and its derivatives have been proposed (Matney, 1980;Dashwood et al., 1991;Sarkar et al., 1994;Ferruzi et al., 2002;Ferruzzi & Blakeslee, 2007).Due to the anti-oxidant, anti-atherogenic, anti-inflammatory and detoxification properties of chlorophyll and its derivatives, it is used in medicines and food supplements (Chernomorsky et al., 1999;Kamat et al., 2000;Fernandes et al., 2007;Ferruzzi & Blakeslee, 2007).Chlorophyll can also be used to produce fuel additives to enhance the combustion characteristics of carbonaceous fuels (Jordan, 1998).

Extraction and degradation of chlorophyll a and b from Alternanthera sessilis
The potential of the photo-conversion efficiency of a chlorophyll derivative in dye-sensitised solar cells have also been studied (Wang et al., 2005).
The production of chlorophyll from algae and higher plants is a key topic of scientific and commercial interest.Chlorophyll is widely extracted for industrial applications from stinging nettle, spinach, algae (Macias-Sanchez et al., 2007), silkworm excreta, alfalfa, pine needles, other pasture grasses and plant harvest by-products (Marquez & Borrmann, 2009).

March 2016
Journal of the National Science Foundation of Sri Lanka 44 (1) Alternanthera sessilis (L.) R. Br. ex D.C., which is commonly known as sessile joy weed or dwarf copperleaf around the world, while it is known as Mukunuwenna (Sinhala) or Ponnannkannjkkirai (Tamil) in Sri Lanka, was selected because of the low cost, ready availability and medicinal and nutritional value.A. sessilis is a perennial plant belonging to the class Magnoliopsida of the family Amaranthaceae.It is widespread throughout the tropics and subtropics.This plant is commonly used as a leafy vegetable and a medicine in Sri Lanka. A. sessilis is used in Ayurveda for the treatment of biliousness, chronic congestion of liver, acute and chronic pyelitis, cystitis, gonorrhea, and snake bite in Sri Lanka (Gayathri et al., 2006).Further, its antimicrobial properties, wound healing abilities (Jalalpure et al., 2008) and the ability to increase the production of milk in nursing mothers (Jayaweera, 1981) have also been reported.
The objective of the present study was to evaluate A. sessilis as a potential source of extractable chlorophyll and to examine the ideal extraction, pre-processing and storage conditions.The applicability of general mass transfer equations in modelling the chlorophyll extraction was also examined.
There are a few different forms of chlorophyll that occur naturally.Since the most widely distributed forms in the terrestrial plants are chlorophyll a and b, the study was focused on those two forms.Various solvents such as aqueous acetone, methanol, ethanol and dimethylsulfoxide (DMSO) are used for the extraction of chlorophyll (Shoaf & Lium, 1976;Sartory & Grobbelaar, 1984;Simon & Helliwell, 1998;Porra et al., 1999).Although methanol, ethanol and DMSO proved to be superior to acetone (Sartory & Grobbelaar, 1984;Barnes et al., 1992), 80 % acetone was selected as the solvent in the present study since it is the most suitable solvent for the selected quantification method (Lichtenthaler, 1987;Wellburn, 1994), produces a more stable product, is cheaper and less toxic (Thompson et al., 1999).

Materials
Mature and healthy A. sessilis samples were collected from a farm in Kesbewa, Sri Lanka.All parts of the plant except the roots were taken to prepare the samples.Double distilled acetone (analytical grade) was used as the solvent.A buffer solution of pH 7.8 was prepared with sodium dihydrogen phosphate and disodium hydrogen phosphate.

Analytical technique
A dual beam recording UV-visible spectrophotometer (Shimadzu: model 1800) with 1 cm square glass cuvettes was used to determine the chlorophyll concentration.A Panasonic MX-AC-300 mixer-grinder was used for mechanical grinding of A. sessilis and REMI R-4C laboratory centrifuge was used to centrifuge the extract.
Aqueous acetone with a concentration of 80 % (v/v) was used as the solvent and 2.5 mM phosphate buffer of pH 7.8 was added to the solvent.Fresh A. sessilis (5 g) were cut into pieces and ground using the mixer-grinder.The ground A. sessilis was added to the solvent and placed in a water bath.The extraction was carried out in a dark environment with frequent agitation.After the extraction, samples were centrifuged for 10 min at 2,000 rpm and the supernatant was separated for analysis of chlorophyll.
Chlorophyll concentrations were determined using the spectrophotometer according to the procedure used by Wellburn (1994) and Lichtenthaler (1987).Absorption spectrum was recorded in the UV-visible spectrophotometer.The solutions were diluted using the solvent (80 % acetone) to obtain the absorbance values in the range of 0.1 to 0.7.The concentrations of chlorophyll a, b and a plus b were calculated using the following formulae (Lichtenthaler, 1987;Wellburn, 1994).

Optimum solvent volume to A. sessilis ratio
Chlorophyll was extracted by following the analytical techniques described previously.Solvent volume to A. sessilis weight ratio was changed from 3 to 10 mL/g.Temperature of the water bath was kept at 30 °C and the extraction time was selected as 300 min in order to provide sufficient time for the extraction of chlorophyll.

Optimum temperature of extraction
Solvent volume to A. sessilis ratio was taken as 5 mL/g based on the results of the previous experiment.Five water baths were maintained at temperatures 20, 30, 40, 50 and 60 °C.The extraction time varied from 60 to 300 min with 60 min interval.Five samples were tested for each temperature and extraction time.

Optimum time for extraction
A solvent volume to A. sessilis ratio of 5 mL/g and an extraction temperature of 50 °C was selected based on the results of the previous experiments.The extraction time varied from 15 min to 60 min with 15 min intervals, and then up to 300 min with 60 min intervals.

Effective storage conditions for A. sessilis
In order to determine the effective method of storage for harvested A. sessilis, six commonly used storage conditions were tested (Table 1).

Effective method of pre processing of A. sessilis
Five different commonly used pre-processing methods were used to disintegrate the structure of the plant cells as described in Table 2.

Kinetics of chlorophyll degradation
Four samples of fresh A. sessilis were prepared for extraction by the method Pre 1 (Table 2).Solvent to A. sessilis ratio of 5 mL/g, temperature of 50 °C and time of 45 min were selected as the extraction conditions based on the results of previous experiments.The extracted chlorophyll samples as solutions were used to examine the degradation kinetics at four different temperatures; 15, 30, 40 and 50 °C.This temperature range was selected based on the normal storage and transportation conditions and the possible processing temperatures.Each sample was tested for chlorophyll a and b for 0 -6 hours at 1 hour

Storage Description condition Env 1
Fresh A. sessilis was stored in a closed container in a freezer section of a refrigerator, where the average temperature was 5 ± 2 °C and relative humidity (RH) was 50 ± 2 %.
Env 2 Fresh A. sessilis was stored in a closed container in a cooler section of a refrigerator, where the average temperature was

Optimum temperature of extraction
The concentration of chlorophyll a and b in the extract with various extraction temperatures is shown in Figure 2.
Chlorophyll extraction improves with the increase in temperature up to 50 °C.The degradation of chlorophyll may become prominent at high temperatures and prolong extraction.Therefore 50 °C was selected as the optimum temperature with extraction time not exceeding 2 hours.
intervals.The average of duplicated experiments was used for the analysis.

Optimum solvent volume to A. sessilis ratio
Figure 1 indicates that the maximum chlorophyll concentration was obtained at a solvent to A. sessilis ratio of 5 mL/g.However, the extracted weight of chlorophyll was found to be increasing up to a ratio of 8 mL/g.This may be due to the dual effect of increase in the rate of extraction and dilution of the solution.Up to a solvent to A. sessilis ratio of 5 mL/g, the rate of extraction is significantly higher than the dilution.However the dilution effect dominates above 5 mL/g.

Method Description
Pre 1 Fresh A. sessilis was cut (1 inch pieces) and then ground for 5 minutes using the mixer-grinder.
Pre 2 Fresh A. sessilis was blanched by soaking the samples in hot water at a temperature of 95 °C for one minute and then immediately dipping it in cold water at a temperature of 15 °C for two minutes (Chandrika et al., 2006).Then the samples were cut into pieces of one inch length, approximately.
Pre 3 Fresh A. sessilis was blanched following the procedure in Pre 2 and then ground for 5 minutes using the mixer-grinder.
Pre 4 Fresh A. sessilis was ground using mortar and pestle.
Pre 5 Fresh A. sessilis was dried in an oven for six hours at a temperature of 40 °C and then ground manually.

Table 2:
Tested pre processing methods for the determination of the effective pre processing method extraction time and 1 minute homogenisation time) (Cubas et al., 2008).The contents of chlorophyll a and chlorophyll b were found to be 6.4 and 6.8 mg per 100 g of fresh green beans, respectively.Furthermore Khalyfa et al. (1992) have reported that the extraction of chlorophyll from 100 g of fresh spinach leaves with methanol resulted in 38 mg of chlorophyll a and 13 mg of chlorophyll b, while extraction with acetone resulted 61 mg of chlorophyll a and 29 mg of chlorophyll b (Khalyfa et al., 1992).Comparatively, the amount of chlorophyll a, b and a plus b extracted from A. sessilis were 65.95, 26.1 and 92.05 mg of chlorophyll per 100 g of fresh A. sessilis, respectively.Although the amount of chlorophyll b extracted from A. sessilis was slightly lower than the amount extracted from spinach using acetone, both the amounts of chlorophyll a and a plus b extracted from A. sessilis were higher than the reported values for green beans and spinach.3. The results suggest that A. sessilis contains a considerably higher amount of chlorophyll compared to other higher plants and vegetables.

Mass transfer model for chlorophyll extraction
Mass transfer of chlorophyll from A. sessilis to solvent was modelled by assuming that the rate of mass transfer from the solid surface to the liquid was the limiting factor.The following general mass transfer equation was used (Geankoplis, 2002).
Where;  By integrating, Where, C a0 is the initial solute concentration.
Since C a0 = 0; Where, is a constant as all the samples were prepared from the same material, the solvent volume was kept constant and the extraction process was carried out at a constant temperature.Finally the mass transfer equation is simplified to, The experimental results given in section 'Optimum temperature of extraction' were smoothed with Loess (quadratic) method using MatLab™.The smoothed concentrations of chlorophyll a and b were plotted against the extraction time and these data were used in curve fitting tool of MatLab™ to find the values of C as and K in mass transfer model corresponding to each temperature.Figure 4 shows both the predicted [using equation (7)] and smoothed experimental concentrations of chlorophyll a. Similar trends were observed for chlorophyll b as well.The correlation coefficients R 2 (coefficient of determination) and sum of squares due to error (SSE) values corresponding to the plots for each temperature are given in Table 4.The R 2 and SSE values for temperatures 20 and 30 °C indicate better correlations of actual and theoretical data.For temperatures above 30 °C, the predicted and actual data were significantly different.This may be due to the degradation of chlorophyll at elevated temperatures, which was not accommodated in the mass transfer model.

Determination of the effective storage condition for A. sessilis
The weight of extracted chlorophyll a plus b obtained for different storage conditions are presented in Figure 5.The loss of chlorophyll is significantly low with refrigeration as compared to other storage conditions.
The results indicate no significant difference between refrigeration at 15 and 5 °C.Drying also reduces the loss of chlorophyll in long term storage, as compared to storing in ambient conditions.Moisture content of the dried samples at 40 and 70 °C were found to be 84 and 55 %, respectively.This may be the reason for better preservation of chlorophyll after drying at 70 °C as compared to drying at 40 °C.
Effective method of pre processing of A. sessilis Figure 6 depicts that mechanical grinding (Pre 1) has given the highest yield, while grinding by mortar and pestle (Pre 4) has given the second highest yield.Pre processing may result in the degradation of chlorophyll and this effect is more significant with blanching (Pre 2 and 3) and drying (Pre 5).Further, the extraction of chlorophyll from A. sessilis may be promoted by disintegration of cell walls as in the case of grinding (Pre 1 and 4).Mechanical grinding (Pre 1) has given more yield than grinding by mortar and pestle (Pre 4) because of the better disintegration of cells by the motor driven grinding.

Degradation kinetics of chlorophyll extracted from A. sessilis
Chlorophyll degradation follows a first-order model (Hayakawa & Timbers, 1977  The plot of the logarithm of rate constant against 1/T shows a good correlation (Figure 8) with the Arrhenius equation with a minimum R 2 value of 0.9985.Therefore the values for 0 A and 0 E were calculated based on those data (Table 7).The 0 E values listed in Table 7 are for solutions of chlorophyll and that may be the reason for having very low values in comparable to the values found in literature on pastes and purees.

CONCLUSION
The optimum operating conditions for the extraction of chlorophyll from A. sessilis using 80 % (v/v) acetone were found to be: solvent volume to A. sessilis weight ratio of 5:1 mL/g, extraction temperature of 50 °C and extraction time of 45 minutes.
Comparison with other higher plant materials and vegetables suggests that A. sessilis is a good source for the extraction of chlorophyll.Under the above mentioned optimum conditions, the maximum extractable amount of chlorophyll a and chlorophyll b were 659.5 and 261 µg per gram of fresh A. sessilis, respectively.General mass transfer equation adequately describes the chlorophyll extraction using acetone as the solvent at temperatures up to 30 °C but fails above this temperature due to degradation effects.Kinetic studies on the degradation of chlorophyll extracted from A. sessilis confirmed that chlorophyll degradation follows the first order kinetics and the temperature dependence of the rate constant can be modelled by the Arrhenius equation.
The study on the effect of storage conditions suggests that fresh A. sessilis retain the chlorophyll content without considerable loss under ambient conditions for one day.Refrigeration and/or freezing are better alternatives when compared with drying, for lengthy storage times.Mechanical disintegration of A. sessilis by grinding was found to be a better pre-processing technique as compared to blanching and drying for obtaining higher yields in the extraction of chlorophyll.
C a = 12.25 A 663.2 − 2.79 A646.8  ...(1) Where A 646.8 and A 663.2 are the absorbance at wave lengths 646.8 and 663.2 nm, respectively.C a and C b are the concentrations of chlorophyll a and b in plant extract in micrograms per millilitre, respectively.Concentrations of the extracts were calculated by multiplying the concentrations of diluted samples with the respective dilution factor.The weights of chlorophyll a, b and a plus b were calculated by multiplying the concentrations of chlorophyll a, b and a plus b with the volume of the sample.Experiments were triplicated and the average value was taken.The weights have been expressed on wet basis unless stated otherwise.

Figure 2 :Figure 1 :
Figure 2: Effect of extraction temperature on concentration of chlorophyll a + b; solvent to A. sessilis ratio 5 mL/g Barnes et al. (1992) have compared DMSO and acetone as solvents for the extraction of chlorophyll from several lichens and higher plants.The highest amount of chlorophyll extraction was observed from the lichen Ramalina farinacea collected from El Robledo, and the higher plant Trifolium repens out of the plants under investigation in their study using both the solvents (Barnes et al., 1992).The highest chlorophyll content found from the lichens and higher plants in this study and the comparative values for chlorophyll extracted from A. sessilis are given in Table N a -Rate of solute dissolving to the solution (mol/s) A -Surface area of particles K L -Mass transfer coefficient C a -Concentration of solute at time t C as -Saturation solubility of solute in solution V -Volume of the solution Comparison of the yield of chlorophyll extracted from A. sessilis with other plants Hojnik et al. (2007) have studied the isolation of chlorophyll from stinging nettle (Urtica dioica L.) using different preservation techniques and storage conditions.The yield was found to be 0.8 mg of chlorophyll per gram of fresh nettle at a temperature of 20 °C and an extraction time of one hour with a solvent to nettle ratio of 10 L/kg(Hojnik et al., 2007).Comparatively, the yield of chlorophyll a plus b was 0.9205 mg of chlorophyll per gram of fresh A. sessilis.Cubas et al. (2008) used N,N-dimethylformamide to extract chlorophyll in green beans (Phaseolus vulgaris L.) of cultivar Emerite.They have reported a maximum chlorophyll content of 13.3 mg of chlorophyll per 100 g of fresh green beans (for five extractions, a 90 minute

Figure 3 :
Figure 3: Effect of extraction time on chlorophyll concentration; temperature 50 °C, solvent to A. sessilis ratio 5 mL/g

Figure 5 :Figure 6 : 1 −EC
Figure 5: Effect of storage condition on the yield of chlorophyll a + b.The weight is presented in milligrams per 1 g of fresh A. sessilis

Figure 7 :
Figure 7: The relationship between the concentration of chlorophyll and time at 15 o C (ED -experimental data, MD -model data)

Figure 8 :
Figure 8: The relationship between the rate constant and the temperature (ED -experimental data, MD -model data)

Table 1 :
Tested storage conditions for the determination of effective storage conditions for harvested A.Therefore, considering the economy of operation, 5mL/g was selected as the optimum solvent to A. sessilis ratio, under 30 °C operating temperature and a time duration of 300 minutes.The corresponding concentrations obtained for chlorophyll a, b and a plus b were 87.34, 41.33 and 128.67 µg/mL, respectively with a chlorophyll a to b ratio of 2.12, and the weight of chlorophyll a plus b was 643.36 µg/g.
(1)sh A. sessilis was dried in an oven for six hours (until weight becomes a constant) at a temperature of 40 ± 2 °C and then stored in a closed transparent container, where RH was 76 ± 2 % Env 6Fresh A. sessilis was dried in an oven at a temperature of 70 ± 2 °C until weight becomes a constant (about four hours) and then stored in a closed transparent container, where RH was 76 ± 2 % sessilis March 2016 Journal of the National Science Foundation of Sri Lanka 44(1)

Table 3 :
Comparison of the chlorophyll content of A. sessilis with some of the plant materials used byBarnes et al. (1992) Figure 4: Comparison of experimental (ED) and model (MD) data of concentration of chlorophyll a with extraction time at five different temperatures Extractior time (hrs) Journal of the National Science Foundation of Sri Lanka 44(1) March 2016

Table 4 :
Correlations of actual and predicted data

Table 5
shows the coefficients of mass transfer model at 20 and 30 °C.The increase of K values with the increase in temperature agrees with the general characteristic of leaching behaviour for most of the solvent extraction processes.

Table 5 :
Coefficients of mass transfer model

Table 6 :
The rate constants at various temperatures

Table 7 :
Activation energy and A 0 of chlorophyll constant k was calculated.The calculated rate constant values at four different temperatures have been tabulated in Table6with the corresponding R 2 values.