International Journal of Science and Research (IJSR)

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Estimation of Pigments from Seaweeds by Using

Acetone and DMSO
T. Vimala1*, T. V. Poonghuzhali2
1
Department of Plant Biology & Plant Biotechnology, Quaid-e-Millath Govt. College (w), Chennai-600002, India
2
Department of Botany, Queen Mary’s College, Chennai-600004, India

Abstract: Seaweeds are macroalgae inhabiting marine water with 50% major productivity using pigments. These pigments are
important both in classification and biodiversity studies. Solvents play a major role in the process of extracting the pigments. In our
present study, the pigments of the algal species were determined using acetone and DMSO for extracting various pigments and were
examined comparatively. Acetone is an efficient solvent for pigment extraction but the study showed that DMSO was an equally good
solvent for extraction of pigments especially in case of brown algae. However, acetone extraction of pigments was more efficient in
green and red algae than DMSO extraction. The better of the two solvents used in this study, appears to be DMSO, because it does not
require maceration, centrifugation or filtration.

Keywords: Acetone, DMSO, pigments, chlorophyta, phaeophyta, rhodophyta.

1. Introduction have received particular attention as they have been found to

exhibit various beneficial biological activities such as
Seaweeds are the most abundant photosynthetic species that antioxidant, anticancer, anti-inflammatory, anti-obesity, anti-
contain different groups of light harvesting and photo- angiogenic and neuroprotective activities. Therefore, various
protective pigments. The pigments are important in natural pigments isolated from marine algae have attracted
classification and biodiversity studies and hence impart their much attention in the fields of food, cosmetic and
value as reserve food and other valuable products. There are pharmacology (Pangestuti and Kim, 2011). From this
three groups of photosynthetic pigments in macroalgae. perspective, investigations on carotenoids and chlorophylls
They are chlorophylls, carotenoids and phycobiliproteins are an important part of studies focused on economic
(Rowan 1989). Based on pigments the macroalgae are applications and in research more particularly directed to
classified into three major groups viz., Green algae ecological issues. However, these studies require the
(Chlorophyta), Brown algae (Phaeophyta) and Red algae extraction and analysis of pigments, which are quite unstable
(Rhodophyta). not only at high temperatures, but also when exposed to light
and to oxygen (Borsarelli; Mercadante, 2010), an obstacle to
The lipid-soluble Chlorophyll a is found in all the development of reliable, reproducible analysis protocols.
photosynthetic macroalgae, while chlorophyll b is found in In our present study, the pigments of the algal species were
Chlorophytes, and the chlorophylls c1 and c2 are found in determined using acetone and DMSO for extracting various
Phaeophytes (Rowan 1989). Fucoxanthin absorbs blue-green pigments and were examined comparatively.
light between 400-560 nm in the spectrum (Rowan 1989).
Carotenoids are separated into carotenes and xanthophylls 2. Materials and Methods
(Rowan 1989). The xanthophylls -fucoxanthin, violaxanthin,
antheraxanthin and zeaxanthin are found in the Phaeophytes, 2.1 Sample Collection
zeaxanthin is also found in the Rhodophytes, while lutein,
neoxanthin, violaxanthin and zeaxanthin are found in the Live and healthy specimens were collected along the
Chlorophytes (Rowan 1989). The carotene β, β-carotene is Rameshwaram coast of Tamilnadu during low tides in the
found in Rhodophytes (and to a certain extent in month of April, 2015. The seaweeds were then washed
Phaeophytes and Chlorophytes) (Rowan 1989). thoroughly with seawater to remove extraneous materials
Phycobiliproteins are water-soluble pigment-proteins and is and brought to the laboratory in plastic bags containing
characteristic of red macroalgae (Rowan 1989; Zhao et al. water to prevent evaporation.
2011). They absorb efficiently in the green to red part of the
light spectrum (500-650 nm in vitro) (Rowan 1989). There 2.2 Sample Preparation
are three major phycobiliprotein groups; allophycocyanins,
phycocyanins, phycoerythrins (Rowan 1989; Jeffrey et al. The samples were gently rinsed with fresh water to remove
2005; Zhao et al. 2011). salt, sand and epiphytes, epizoones, animal castings,
calcareous and other adhering detritus matters. The samples
Recently, the importance of diverse bioactive compounds were identified following the standard books. The seaweeds
found in seaweeds is growing rapidly and researchers have collected were identified as Chlorophyceae- Ulva reticulata
revealed that marine algal originated compounds exhibit Forsskål; Phaeophyceae- Sargassum ilicifolium (Turner)
various biological activities (Barrow & Shahidi, 2008; C.Agardh 1820, Sargassum polycystum C.Agardh ,
Wijesekara, Yoon, & Kim, 2010). Among functional Turbinaria conoides (J.Agardh) Kützing 1860 ,
ingredients identified from marine algae, natural pigments Hydroclathrus clathratus (C.Agardh) M.Howe ;
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Rhodophyceae- Amphiroa sp. J.V. Lamouroux, 1812, measured in spectrophotometer to estimate the fucoxanthin
Gracilaria salicornia (C. Agardh) Dawson 1954, Champia content.
parvula (C.Agardh) Harvey, Portieria hornemannii Fucoxanthin (mg g-1) = [A470 −1.239 (A631+A581−0.3 ×A664)
(Lyngbye) P.C.Silva. The seaweeds were then cut into small − 0.0275(A664)] /141
pieces, weighed and subjected to pigment analysis. Where, A = Absorbance at particular wavelength
V = Total volume of the pigment extract
2.3 Extraction Using 80% Acetone (Arnon1949) W = Weight of the sample used for extraction

500 mg of seaweed was kept in a pestle and mortar with 10 3. Results and Discussion
ml of 80% acetone and it was ground well and the
homogenate was centrifuged at 3000 rpm for 15 minutes and The major photosynthetic pigments, total chlorophyll and
the supernatant was stored. The pellet was re-extracted by carotenoid content were estimated from fresh seaweeds. The
repeated washing with 5 ml of 80 % acetone till it became chl a content (Table.1) extracted using 80% acetone ranged
colourless. All the extracts were pooled and utilized for from 4.29 to 21.99 µg/ml with minimum in the red seaweed
pigment quantification. The process was followed for all the Amphiroa sp. and maximum in the green seaweed
samples. U.reticulata, whereas , chl a content extracted using DMSO
ranged from 0.96 to 21.44 µg/ml minimum in the red
2.4 Extraction of pigments using DMSO (J. D. Hiscox & seaweed Amphiroa sp and maximum in the brown algae
G. F. Israelstam, 1979) S.ilicifolium.

500 mg of dry sample was taken, cut into small pieces and Table 1: Chl a (µg/ml) content extracted using 80%
suspended in test tubes containing 2 mL of dimethyl Acetone and DMSO
sulphoxide (DMSO). Test tubes were incubated at 60° C for Sample 80% Acetone DMSO
20 min in a water bath. The supernatant was decanted and U.reticulata 21.99±4.01 3.17±1.03
another 3 mL of DMSO was added to the residue and H. clathratus 12.52±3.02 18.12±2.03
incubated at 60° C for 20 min. The supernatants were pooled S.ilicifolium 19.38±3.03 21.44±3.01
and the volume was made up to 10 mL by adding DMSO. S.polycystum 9.93±2.03 17.20±2.02
The chlorophyll extract was transferred to a cuvette and the Turbinaria sp 5.61±1.02 20.94±3.01
absorbance was read in a Spectrophotometer at 645 and 663 Amphiroa sp 4.29±1.02 0.96±0.82
nm against DMSO blank Chlorophyll a, b, total chlorophyll G.salicornia 7.03±2.02 1.60±0.38
and chlorophyll a/b ratio were calculated.
C.parvula 6.94±0.78 5.29±1.02
P. hornemonii 12.66±3.05 4.66±0.56
2.5 Estimation of Chlorophyll
± Standard Error
The amount of chlorophyll present in the algae was
estimated by the method of Arnon (1949). Absorbance was
measured at 645 nm and 663 nm in a spectrophotometer.
The chlorophyll content was determined by using the
following formula
Arnon’s (1949) equations:
Chlorophyll a (µg/ml) = 12.7 (A663) - 2.69 (A645)
Chlorophyll b (µg/ml) = 22.9 (A645) - 4.68 (A663)
Total chlorophyll (µg/ml) = 20.2 (A645) + 8.02 (A663)

Where, A = Absorbance at respective wave length = Volume

of extract (ml), W = Fresh weight of the sample (g).

2.6 Estimation of Carotenoid

The amount of Carotenoid was estimated by the method of

Kirk and Allen, 1965.The same chlorophyll extract was
measured at 480 nm in spectrophotometer to estimate the
carotenoid content.

Carotenoids (μg/g.fr.wt.) = A480 + (0.114 × A663) ─ (0.638 Figure 1: Chl a content extracted using 80% Acetone and
× A 645) DMSO
Where, A = Absorbance at respective wave length
The chl b content (Table.2) extracted using 80% acetone
2.7 Estimation of Fucoxanthin ranged from 0.55 to 19.75 µg/ml with minimum in the red
seaweed Amphiroa sp. and maximum in the brown seaweed
The amount of fucoxanthin was estimated by the method of H. clathratus, whereas, chl b content extracted using DMSO
Seely et al, 1972. The same chlorophyll extract was ranged from 2.25 to 12.40 µg/ml with minimum in

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Amphiroa sp and maximum in the brown algae Turbinaria ± Standard Error
sp.

Table 2: Chl b (µg/ml) content extracted using 80%

Acetone and DMSO
Sample 80% Acetone DMSO
U.reticulata 18.01±3.02 3.76±2.03
H. clathratus 19.75±3.01 7.46±3.01
S.ilicifolium 13.63±2.03 3.86±1.02
S.polycystum 6.48±1.03 5.18±1.02
Turbinaria sp 3.82±1.04 12.40±2.03
Amphiroa sp 0.55±0.62 2.25±0.52
G.salicornia 4.09±1.12 2.96±1.02
C.parvula 1.54±0.44 3.31±0.22
P. hornemonii 2.94±0.32 3.08±0.73
± Standard Error

Figure 3: Total Chlorophyll content extracted using 80%

Acetone and DMSO

Carotenoid content (Table.4) extracted using 80% acetone

ranged from 0.13 μg/g (Amphiroa sp ) to 1.71μg/g
(S.polycystum). However, DMSO extraction showed a range
between 0.02 μg/g (Amphiroa sp) and 1.75μg/g
(S.ilicifolium). Similar findings have been reported by N.
Kumar J.I. et.al., (2009) . According to them the highest
carotenoids were present in S. Polycystum belonging to the
Phaeophyta group while the lowest carotenoid content was
observed in Chlorophyta group
Table 4: Carotenoid (μg/g) content extracted using 80%
Acetone and DMSO
Sample 80% Acetone DMSO
U.reticulata 1.38±0.54 0.14±1.02
Figure 2: Chl b content extracted using 80% Acetone and
H. clathratus 1.35±1.02 0.93±0.33
DMSO
S.ilicifolium 1.18±1.03 1.75±1.04
S.polycystum 1.71±1.01 0.93±0.52
Total chlorophyll content (Table.3) extracted using 80%
Turbinaria sp 1.69±1.03 1.55±1.01
acetone ranged from 4.83 to 39.99µg/ml with minimum in
Amphiroa sp 0.13±0.71 0.02±0.02
Amphiroa sp and maximum in U.reticulata whereas, total
chlorophyll extracted using DMSO ranged from 3.21 to G.salicornia 0.38±0.42 0.09±0.03
33.32 µg/ml with minimum in Amphiroa sp and maximum C.parvula 0.35±0.02 0.09±0.34
in Turbinaria sp. P. hornemonii 0.35±0.72 0.11±0.13
± Standard Error
N. Kumar J.I. et.al., (2009) reported that Chlorophyll (Chl a,
b and total) content extracted using acetone shot up in
species of Chlorophyta followed by Phaeophyta and
Rhodophyta. Carotenoid content was recorded greater in the
members of Phaeophyta than Rhodophyta and Chlorophyta.
The present study shows similar findings.

Table 3: Total Chlorophyll (µg/ml) content extracted using

80% Acetone and DMSO
Sample 80% Acetone DMSO
U.reticulata 39.99±3.02 6.93±1.02
H. clathratus 32.25±2.03 25.56±3.03
S.ilicifolium 33.00±3.01 25.30±2.02
S.polycystum 16.41±2.02 22.38±4.03
Turbinaria sp 9.44±1.04 33.32±3.03
Amphiroa sp 4.83±0.94 3.21±0.63
Figure 4: Carotenoid content extracted using 80% Acetone
G.salicornia 11.12±1.03 4.56±0.62
and DMSO
C.parvula 8.48±1.22 8.59±1.11
P. hornemonii 15.59±3.02 7.74±2.02

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Amphiroa sp also showed a minimum quantity of the field. Unfortunately, the Chl red peaks are generally
Fucoxanthin extracted by acetone (0.05 μg/g) and DMSO broader and lower in methanol and ethanol. The peaks for
(0.41 μg/g) whereas S.ilicifolium showed maximum Chl b, Chl c1+c2, and Chl c2 and Chl d are not only lower
fucoxanthin content extracted both by acetone (13.8 μg/g) and broader in methanol and ethanol, the widened peak of
and DMSO (12.90 μg/g) respectively (Table.5). Chl a in these solvents tends to interfere more strongly with
the absorbance of the other Chlorophylls (Ritchie 2006).
Table 5: Fucoxanthin (μg/g) content extracted using 80%
Acetone and DMSO Acetone solvent gives very sharp Chlorophyll absorption
80% Acetone DMSO peaks but acetone is sometimes a poor extractant of
H. clathratus 6.81±1.01 5.87±1.02 Chlorophyll from some algae, particularly green algae.
S.ilicifolium 13.8±1.74 12.90±1.02 Acetone is known to have a lower extractability of
S.polycystum 7.62±1.02 7.10±1.62 chlorophylls from the protein matrix (Nakamura and
Turbinaria sp 5.16±1.03 8.44±1.32 Watanabe, 2001). Acetone, on the other hand, provides a
Amphiroa sp 0.98±0.13 0.54±0.42 stable environment. Porra et al. (1991) and Wright et al.
G.salicornia 0.05±0.61 0.41±0.32 (1997) have discussed the merits of dimethyl sulphoxide
C.parvula 2.25±0.63 0.71±0.02 (DMSO) used for chlorophyll extraction and assay, and
P. hornemonii 1.73±1.02 0.98±0.34 reported as efficient when pigment concentrations are low.
± Standard Error
Conclusion

Results from this experiment clearly indicate that extraction

of photosynthetic pigments by different solvents depends on
chemical nature of bio-molecules (cholorophyll-a,
chlorophyll-b, carotenoids and fucoxanthin). Investigation
revealed DMSO as best extracting solvent of chlorophyll-a
and b for brown algae. The better of the two solvents used in
this study, appears to be DMSO, because it does not require
maceration, centrifugation or filtration. DMSO is solid at
temperatures below 18 ºC and re-crystallizes slowly, but is
good for dealing with delicate tissues, such as those found in
seaweeds. Care should be taken when it is heated during
extraction. The choice of solvent for extracting pigments
from algae samples must take several factors into account:
toxicity, cost, the number of extractions and efficiency.

Though slight variations persists among the experimented

species even for same extractant solvent which can be
Figure 5: Fucoxanthin content extracted using 80% Acetone attribute to inherent physiological characteristics of
and DMSO individual species. Temporal and seasonal changes and local
geological condition can also be the reason for variations in
Analysis of photosynthetic pigments depends on the pigment concentrations in seaweeds, therefore further study
appropriate extraction process (Leeuwe et.al., 2006). Even in this context is recommended.
though acetone and methanol are widely used solvents for
extraction, DMSO is also used for pigment extraction
analysis. Acetone is an efficient solvent for pigment
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Author Profile
T.Vimala is working as Assistant Professor in Plant Biology and
Plant Biotechnology at Quaid-e-Millath Government College for
women, Chennai, India.

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