Weekly Science Research Journal

Original Article

Extraction of carotenoid and thin layer chromatography (TLC), GC-MS, FT-IR and HPLC

analysis of pharmaceutically important pigment astaxanthin from a new strain of Haematococcus

pluvialis

S. Elumalai , B. Infant Santhose and G. Rajesh KannaS. Elumalai , B. Infant Santhose and G.

Rajesh Kanna

FromPG and Research, Department of Plant

Biology and Plant Biotechnology,Presidency College (Autonomous),

Chennai, Tamil Nadu, India.

Article Is Published On September 2014 Issue & Available At

www.weeklyscience.org

10.9780/ 2321-7871/1202013/53DOI :

ABSTRACT

Introduction:

Carotenoids are bio-pigments naturally available in many prokaryotic and eukaryotic microorganisms which cannot be synthesized biologically by one self including animal but, required for our basic nutritional supplement. Different types of carotenoid pigments are available with different colors especially yellow, orange, red, and purple etc. includes approximately 700 different types (Cabral et al., 2011 and Li et al., 2012). Due to the availability of different varieties of colors; these carotenoid pigments are of great commercial value as colorants, as antioxidants due to free radical scavenging activity alternatively as anti-tumor, carotenoid pigments primarily plays role in the production of vitamin-A synthesis as a precursor and for morphogenesis of retinoid compounds. Thus, carotenoid plays wide applications in food colorants, nutrient supplements, animal feed and cosmetics manufacturing industries (Altamirano et al., 1992; Ananvoranich et al., 1995; Bakker et al., 1997; Cabral et al., 2011; Li et al., 2012).

Some of the pharmaceutically as well as commercially important carotenoid pigments are lycopene, á-carotene, ß-carotene, lutein, zeaxanthin and ß-cryptoxanthin which are needed most abundant for blood plasma in

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Astaxanthin is an important pharmaceutical compound obtained from different sources of micro algae. But Haematococcus pluvialis alone plays a significant role in high astaxanthin accumulation comparing with other sources. Even though astaxanthin extraction from Haematococcus pluvialis is plausible, since more number of new strains of Haematococcus pluvialis still needs effective study on astaxanthin accumulation. This study reports on astaxanthin accumulation, extraction and its analysis from a new strain of Haematococcus pluvialis. Optimization for complete isolation of carotenoids involves various extraction methods, and astaxanthin has been given great importance. When compared to all the methods of extraction, treatment with HCl banks high amount of astaxanthin due to the consistent corrosion of cell wall. The results from Thin layer chromatography especially on astaxanthin and its mono- and di-esters were similar in comparison with other studies from the same organism of different strain. Polyunsaturated fatty acid and saturated fatty acid occupies high percentage quantitatively with low amount of monounsaturated fatty acids comparatively from GC-MS analysis. The HPLC result coincides apparently with the HPLC chromatogram of the previous studies of astaxanthin from Haematococcus pluvialis.

Haematococcus pluvialis; astaxanthin; extraction; GC-MS; FT-IR; HPLC.

Keywords:

humans (Peng et al., 2011). An alternative natural way of production of pigments is still to be furnished rather than artificial chemical production, because of its damaging effects to the environment and high cost (Niziri et al., 2014). Elucidating biochemical pathway can escort us to produce bountiful amount of pigments in the upcoming future.

Astaxanthin (3, 3’-dihydroxy-ß, ß’-carotene-4, 4’-dione) (C H O ), is an orange-red 40 52 4

carotenoid pigment especially found in marine fishes and crustaceans. But rather than marine sources, microalgae play a vital role in astaxanthin production and accumulation quantitatively. Along with astaxanthin its relative pigments can also synthesized as mixtures, which are violaxanthin, zeaxanthin, canthaxanthin, neoxanthin, a-carotene, ß-carotene, echinenone and antheraxanthin etc.

Biological synthesis and accumulation of astaxanthin pigment was reported in Haematococcus pluvialis and Chlorella zofingiensis, in which H. pluvialis quantitatively produce high yield of astaxanthin (30 mg/g of biomass) comparatively low (< 1 mg/g) in C. zofingiensis (Lorenz and Cysewski, 2000; Brinda et al., 2004; Kamath et al., 2008 and Ip and Chen, 2005). H. pluvialis has been studied for high accumulation of astaxanthin of about 2 to 3% of its cell content which is higher than any other sources (Grung et al., 1992; Yuan and Chen, 1998; Lorenz and Cysewski, 2000). Haematococcus pluvialis (Droop, 1955 and Donkin, 1976) belongs to the order volvocales, family Chlorophyceae, unicellular, motile, freshwater, and bi-flagellate zoospore producing green microalga (Margalith, 1999). H. pluvialis is still a major and fruitful source for astaxanthin production industrially.

When compared to all the carotenoid pigments, astaxanthin plays a key role by scavenging 500 fold stronger oxidative free radicals than vitamin-E and 38 fold more than ß-carotene (Kurashige et al., 1990; Shimidzu et al., 1996). Astaxanthin accumulation in H. pluvialis is directly proportional to stress factors such as N starvation and high light 2

intensity (Boussiba and Vonshak, 1991; Kobayashi et al., 1997; Fabregas et al., 1998). Therefore, artificial induction of astaxanthin synthesis can be feasible for high yield in microalga H. pluvialis.

Several methods of carotenoid extraction were available using solvents such as 90% acetone, DMSO (Dimethyl sulphoxide) and acid treatment includes HCl treatment to cleave the thick cell wall of cyst cells to extract maximum amount of astaxanthin from the biomass of H. pluvialis. Optically pure compounds of astaxanthin (5 %), astaxanthin monoesters (70 %), astaxanthin diesters (10 %), ß-carotene, canthaxanthin and lutein can be extracted from the total carotenoid of H. pluvialis (Renstrom et al., 1981).

Fatty acid methyl ester analysis using Gas chromatography (GC) implies the occurrence of saturated, mono and polyunsaturated fatty acids in the ratio of 5:3:2 in astaxanthin monoester and 4:3:3 in astaxanthin diester respectively (Sindhu and Sherief , 2011). Palmitic acid (C: 16: 0) and (C: 18: 1, C: 18: 2 and C 18: 3) C: 18 family are the principal fats along with astaxanthin esters. More than 20 % of fatty acids and 80 % of unsaturated fatty acids will occur in the red cyst cells of H. pluvialis (Ping et al., 2008).

Implementation of mass spectrometric analysis in nutrient and food research is now well established and developed in relation to techniques and to enable good quality control (Careri et al., 2002). High performance liquid chromatography (HPLC) analysis for the determination of astaxanthin is the most common method in feed companies in Norway and Chile. Canadian food inspection agency also registered Haematococcus alga meal in salmonid feeds (Reg. no. 990535) and US food and Drug administration (21CFR 73.185) using HPLC confirmation (Lorenz, 2001).

FT-IR spectroscopy is one of the most efficient method of prediction and confirmation of chemical functional groups present in the sample based on the vibrations of atoms of a molecule. Structural analysis of astaxanthin from H. pluvialis was done and described using FTIR by Kim et al. (2008).

In this present study, carotenoid extracts of a newly isolated Haematococcus pluvialis from high altitude region (Prabhakaran et al., 2014), was analyzed and investigated for astaxanthin and its esters. Investigation processes includes Thin layer chromatography (TLC), Gas chromatography (GC), High performance liquid chromatography (HPLC) and Fourier transmission infra red spectrometry (FTIR), the results were interpreted.

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2. Materials and Methods

2. 1. Extraction of carotenoid

2. 2. Thin layer chromatography (TLC)

2. 3. GC-MS

2. 4. HPLC

2. 5. FTIR

The airlift photo-bioreactor culture of H. pluvialis about 3 l optimized BBM medium (Prabhakaran et al., 2014) yielded approximately 2 g of dried orange-red colored biomass. Around 20 mg of biomass was homogenized almost fine powder using mortar and pestle and carotenoid was extracted using different methods to optimize the method of extraction for achieve high yield of astaxanthin. Solvents include acetone, DMSO (Dimethyl sulphoxide) and methanol and acid treatment involves 2N concentration of HCl, formic acid, citric acid, acetic acid and tartaric acid were optimized for astaxanthin accumulation (Sarada et al., 2006).

Different wavelength frequencies were used to analyze extractability percentage of astaxanthin under absorbance at 470, 480, 645 and 661 nm respectively by utilizing UV-visible spectrophotometer (Hitachi U-2900). Percentage of extractability was calculated based on the formula given below (Kobayashi et al., 1997).

Percentage of Extractability = free astaxanthin (µg mL-1) / total astaxanthin (µg mL-1) × 100

The carotenoid extract from H. pluvialis was subjected to TLC for qualitative analysis of presence of astaxanthin and its allied products such as astaxanthin monoesters and astaxanthin diesters. Acetone, petroleum ether and chloroform (mobile phase) were optimized for separation using commercially available TLC sheets (Stationary phase) (TLC Silica gel 60 F from MERCK). The whole experiment was performed in a 254

closed glass TLC chamber.

Carotenoid extract was made by using 90 % acetone especially for GC and MS analysis. Fatty acid methyl esters (FAME) were quantified using GC-MS-QP 2010 (SHIMADZU) in VF-5ms column with 30.0 m × 0.25 mm and film thickness of 0.25 µm. GC Conditions such as oven temperature 70°C for 3 min., Injector temperature of 240°C with split ratio 10, Helium (99.9995 % purity) as carrier gas with column flow of 1.51 ml/min. and injection volume of 1 µl. Column temperature was raised to 300°C for 9 min.

Mass spectrum conditions such as Ion source temperature 200°C, Interface temperature-240°C, scan range from 40-1000 m/z, MS start time: 5 min and end time: 35 min, Ionization: EI (-70ev) and scan speed of 2000 were concerned. NISTO8s, WILEY8 and FAME softwares were used as MS library for FAME identification and analysis.

Carotenoid extract from H. pluvialis was subjected to High performance liquid chromatography (HPLC) (Shimadzu LC analysis solutions, Japan) using C column with 18

injection volume 20 µl, 25 cm × 4.6 mm with diameter 5 µm under room temperature. The mobile phase was combination of an isocratic solvent system consists of dichloromethane, acetonitrile and methanol in the ratio of 20:70:10 with low rate of 1.0 ml/min.

All the separated carotenoids were analyzed at 450 nm and 474 nm using UV-visible detector (Shimadzu, Kyoto, Japan). Retention times were used to identify peaks and quantify components in comparison with the spectra of standard and reference chromatographs respectively.

Carotenoid extract of H. pluvialis was further analyzed in IR spectrum (using electromagnetic radiation) to predict the presence of astaxanthin based on its molecular structure. FT-IR (Lark) was used to analyze the carotenoid sample at range of measuring

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-1spectrum from 4000 to 450 cm ; the peaks were measured in terms of percentage of transmittance and results were recorded.

When compared to all the optimized methods of extraction, treatment with 0.2 N of HCl yielded about 85.24 % of astaxanthin from carotenoid extraction. Extractability efficiency was studied among solvent and acid treatment in which acid treated extract yields maximum amount of astaxanthin in HCl treatment (85.24 %) whereas solvent treated extract yields comparatively low astaxanthin (65.19 %) in DMSO treatment.

Among solvent treated extraction DMSO acquired more amount of astaxanthin than any other solvents as such case occurs in acid treated extraction also in which HCl treated extraction yielded high astaxanthin (Table 1).

Mobile phase (running solvent) was optimized and resulted that acetone, petroleum ether and chloroform are in the ratio of 2:2:1 for efficient separation of carotenoid. Astaxanthin, astaxanthin monoesters and astaxanthin diesters are separated and the relative front (Rf) value were recorded 0.63, 0.77 and 0.82 respectively (Fig. 1).

The fatty acid methyl ester profile represents plethora of Polyunsaturated fatty acids and Saturated fatty acids engulfing about 49.12 % and 44 % respectively from overall fatty acid profile and rest of them shared by Monounsaturated fatty acids (6.98 %) (Fig. 2). About nine individual Fatty acids were identified from the H. pluvialis extract in this study.

When in comparison with all the fatty acids Hexadecanoic acid, ethyl ester (C:16:2) (Fig. 3) accumulated more area in terms of percentage (17.75 %), 1-Docosanol (C:22:1) (Fig. 4) placed next with 16.45 %. These two fatty acids aggregated more than any other fatty acids analyzed. Ethyl Oleate (C:20:2) (11.65 %) (Fig. 5), Cyclopentane, 1-phenyl-3-(-phenyl-1-trimethylsily methylene) (C:17:2) (10.92) (Fig. 6), Nonadecane, 9-Methyl- (C:20:0) (9.96 %) (Fig. 7), Heptadecane (C 17:0) (9.71 %) (Fig. 8), Ethanol, 2-(9-octadecenyloxy)-, (Z) (C:20:2) (8.8 %) (Fig. 9), 2-Hexadecanol (C:16:1) (7.78%) (Fig. 10) and 5-Octadecanol (C:18:1) (6.98 %) (Fig. 11) are the serial of fatty acids in decreasing order of percentage. C:16:2 and C:22:1 are the fatty acids shows high area percentage which are commonly called as Palmitoleic acid and Docosanoic acid or Behenic acid respectively (Table 2).

High performance liquid chromatography (HPLC) analysis of extracted cartotenoid sample of H. pluvialis is a fruitful study by attaining a single peak at the retention time of 5.751 min. but the peak is obviously broad starting from the retention time 5.6 to 6.4 min. The obtained HPLC chromatograph of this study was compared with available typical

TMchromatogram of commercial Naturose or Bioastin powder product (BioAstin/ NatuRose Technical Bulletin #015, Cyanotech Corporation Lorenz, 2001) of astaxanthin from H. pluvialis. Result reveals that the peaks belongs to Di-Cis-Astaxanthin (5.508 min.), Trans Astaxanthin (5.750 min.) and 9-Cis-Astaxanthin (6.375 min.) from typical reference seems relatively similar with the present study by attaining retention time TR ranging from 5.6 to 6.4 min (Fig. 12).

This present HPLC results apparently similar with the HPLC chromatograph of H. pluvialis in which Astaxanthin esters occurs at the retention time between 5.5 to 14.5 min. (Ranga rao, CFTRI, 2011). And HPLC result obtained relatively similar with one more reference of saponified esters of astaxanthin from H. pluvialis (Jian-Ping Yuan, University of Hong Kong, 1999).

The FTIR results from carotenoid extract of H. pluvialis showed several absorbance

3. Results

3.1 Extraction of carotenoid

3. 2. Thin layer chromatography (TLC)

3. 3. GC-MS

3. 4. HPLC

3. 5. FTIR

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-1 -1peaks ranging from 3793 cm to 513 cm (Fig. 13). Those peaks from its analysis through IR chart confirmed the presence of the following functional groups in a compound.

-1Absorbance peaks at spectrum frequencies 3427 cm corresponds to hydroxyl group (-OH) -1 -1(3200 to 3400 cm ), 2925 and 2854 cm both confirms the presence of Methyl group

-1(Aliphatic) (-CH) (2925 and 2850 cm respectively). Presence of ketone group (-C=O) was -1 -1confirmed by the peak at 1718 cm (cyclic 6-membered) (1720 cm ). Alkenes

-1(Monosubstituted alkenes, dienes) (C-C, C=C) at 1634 cm resulted in this study correlates -1 with C=C and C-C stretch of IR chart (1640-1680 and 1645 respectively). Peak at 1384 cm

predict the presence of methyl group as C-H in umbrella formation (CH ). Aromatic bends 3

were analyzed from the peaks at 857 and 783 cm especially C-H bends as Para--1

disubstituted benzene and Meta-disubstituted benzene respectively. All the peaks with its respective functional group were illustrated in Table 3.

Extraction of astaxanthin from red encysted cells of H. pluvialis plays a very crucial role due to the presence of thick algaenan-type cell wall. In such cases some other treatment is required for extraction of astaxanthin, solvent involving method of extraction is the commonly practiced method for astaxanthin extraction (Montsant et al., 2001).

?Kobayashi et al. (1997) exemplified about 40 % acetone treatment at 80 C for 2 min. acquired about 70 % of astaxanthin followed by selective removal of chlorophyll. Bubrick (1991) extracted astaxanthin by homogenizing dried biomass of H. pluvialis at very low temperature with butylated hydroxyl toluene. DMSO treatment along with drops of acetic

?acid by heating at 70 C for 10 min. yielded astaxanthin from H. pluvialis (Boussiba et al., 1992).

Low concentration acid treatment with dried biomass of thick walled H. pluvialis can yield high amount of astaxanthin rather than solvent methods because, acid treatment can corrode the thick cell wall in order to provide almost all the pigments from it. Various acids were treated and among all the acids Hydrochloric acid (HCl) alone yielded 90 % of astaxanthin from the dried biomass of H. pluvialis (Sarada et al., 2006). Treatment with HCl for about 15 to 30 min. can yield more astaxanthin at room temperature after successful sonication (Mendes-Pinto et al., 2001). Supercritical fluid extraction of astaxanthin from H. pluvialis yielded about 87.42 % can be useful for food and pharmaceutical industries (Wang et al., 2012).

Successful extraction of astaxanthin from dried biomass of H. pluvialis has been proved in our study and which is directly correlates with the study done by Sarada et al. (2006). Both the study reveals that 0.2 N of HCl treatment yielded approximately 80 % of astaxanthin. TLC observation of free astaxanthin, astaxanthin mono and diesters from this present study apparently showed the presence of all the above mentioned pigment with esters at relative front (Rf) value of 0.63, 0.77 and 0.82 respectively. The TLC result of this study obviously relative with some other results reported early. Nagaraj et al. (2012) separated carotenoid extraction from H. pluvialis and Rf are 0.37, 0.58 and 0.73 with respective to astaxanthin, astaxanthin mono and diesters. Carotenoid extracts from shell waste of Arabian red shrimp Aristeus alcocki, Ramadan 1938 provides three distinct bands with Rf value of 0.33, 0.60 and 0.78 which respect to astaxanthin, astaxanthin mono and diesters respectively (Sindhu and Sherief, 2011). Fatty acid profile of H. pluvialis extract indicates the presence of C: 16: 0 and C: 18: 0 with 19.2 % to 23.5 % and 6.9 % to 13.6 % respectively (Lei et al., 2012). But in contrast to the above mentioned study, present results found C: 16: 2 (Palmitoleic acid) and C: 22: 1 (Docosanoic acid) are quantitatively 17.75 and 16.45 % respectively. Among all the fatty acids Palmitoleic acid was reported as in the spiny lobster Panulirus japonicas extract. Fatty acids profile of P. japonicus reported that saturated fatty acids 45.55 %, monounsaturated fatty acids 30.25 % and PUFAs 29.3 % by Maoka and Akimoto (2008), when compared to our study saturated fatty acids alone related to this result with 44 %. Unsaturated fatty acids alone accumulates about 56 % of the total fatty acids includes 49.12 % of Polyunsaturated, 6.98 % of monounsaturated fatty acids rest of them shared by Saturated fatty acids in this study. The unsaturated fatty acid profile in this study approximately matches with 35.5 to 52.9 % of unsaturated fatty acid of H. pluvialis extract among total fatty acid (Chen et al., 2004). Heptadecane (C: 17: 0) found about 9.71 %

4. Discussion

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among the total fatty acid in this study, in some other studies cis-10heptadecanoic acid (C: 17: 1) can be detectable under nitrogen limited conditions or in relatively very low rate (Damiani et al., 2010).

The HPLC chromatogram of this study found comparably relative with the typical HPLC standards of commercially available astaxanthin (BioAstin/NatuRoseTM) from Cyanotech Corporation (Lorenz, 2001). The TR of carotenoid extraction belongs to H. pluvialis showed astaxanthin esters in the range of 5.5 to 14.5 min. (Ranga rao, Ph. D., CFTRI; 2011) which was almost similar with HPLC results of this study. The third similar HPLC result was the saponified mixtures of astaxanthin esters of H. pluvialis (Yuan, Ph. D., Hong Kong University; 1999).

Apart from all the similar results above mentioned, HPLC of carotenoid extract from H. pluvialis of this study also have some other additional comparable results from other study with different sources of carotenoid extracts. The TR range from 3.3 to 7.7 min. illustrate the presence of astaxanthin, canthaxanthin, apocarotenoic ester, torularhodin and ß-carotene synthesized by Sporobolomyces ruberrimus H110 (Razavi et al., 2006), which correlates with the HPLC result of carotenoid extraction from H. pluvialis of this study where TR found from 5.5 to 6.4 min. Sarada et al. (2006) exemplified the presence of free astaxanthin in the carotenoid extract from H. pluvialis within the range of TR from 5 to 8 min. when compared to present the study it has proven the presence of free astaxanthin in carotenoid extract of H. pluvialis of present study.

Two more report which supports this study, Katsuda et al. (2004) and Miao et al. (2006) showed the occurrence of astaxanthin and its esters in TR range from 2.5 to 9 min. and astaxanthin, siphonaxanthin, echinenone and canthaxanthin in the TR range from 5 to 15 min. respectively from similar source of carotenoid extraction from H. pluvialis.

Carotenoid extract from H. pluvialis predicted for astaxanthin has confirmed the presence of astaxanthin and its esters in this study. Functional groups such as hydroxyl (OH) and ketone group (C=O) are the major groups for identification of astaxanthin because such groups can easily reacts with other molecules and changed to some other forms. Presence of methyl groups (C-H) confirms the presence of aliphatic chain of astaxanthin

-1and alkenes (C-O, C=O) in relation with the astaxanthin esters. Methyl group at 1385 cm forms the umbrella formation (CH ) as in the aliphatic chain and aromatic rings of 3

astaxanthin structure. Bend formations at aromatic rings in either ends of aliphatic chain of astaxanthin structure were confirmed by aromatic bends of benzene ring due to the

-1spectrum occurs at 857 and 783 cm . The aliphatic group of FTIR spectrum from carotenoid extract from H. pluvialis (Kim et al., 2008) apparently matches with the present FTIR analysis of carotenoid extract of H. pluvialis.

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5. Acknowledgement

6. References

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31.Nagaraj, S., Rajaram, M. G., Arulmurugan, P., Baskaraboopathy, A., Karuppasamy, K., Jayapriya, K. R., Sundararaj, R. and Rengasamy, R. 2012. Antiproliferative potential of astaxanthin-rich alga Haematococcus pluvialis Flotow on human hepatic cancer (HepG2) cell line. Biomedicine and Preventive Nutrition, 2: 149-153.32.Naziri, D., Hamidi, M., Hassanzadeh, S., Tarhriz, V., Zanjani, B. M., Nazemyieh, H., Hejazi, M. A. and Hejazi, M. S. 2014. Analysis of carotenoid production by Halorubrum sp. TBZ126; an extremely halophilic archeon from Urmia lake. Advanced Pharmaceutical Bulletin, 4 (1): 61-67.33.Peng, J., Yuan, J. P., Wu, C.F. and Wang, J. H. 201. Fucoxanthin, a marine carotenoid present in brown seaweeds and diatoms: metabolism and bioactivities relevant to human health. Mar Drugs, 9 (10): 1806-1828.34.Ping, M. F., Yan, L. D., Wu, Z. C., Cheng, Z. J., Hong, G. Y., Jun, H. H. and Guang, L. Y. 2008. The synthesis of astaxanthin esters, independent of formation of cysts, highly correlates with the synthesis of fatty acids in Haematococcus pluvialis. Sci. China. Ser. C-Life Sci. 51 (12): 1094-1100.35.Prabhakaran, M., Elumalai, S., Santhose, B. I. and Kanna, G. R. 2014. Collection, isolation and identification of Haematococcus pluvialis Flotow from high altitude region of Pithoragarh district, Uttarkhand, India. Golden Research Thoughts, 3 (9): 1-6.36.Ranga Rao, A. 2011. Production of Astaxanthin from cultured green alga Haematococcus pluvialis and its biological activities. Ph. D. thesis. pdf. CFTRI, India.37.Razavi, S. H., Blanchard, F. and Marc, I. 2006. UV-HPLC / APCI-MS method for separation and identification of the carotenoids produced by Sporobolomyces ruberrimus H110. Iran. J. Chem. Chem. Engg., 25 (2): 1-10.38.Renstrom, B. and Liaaen-Jensen, S. 1981. Fatty acid composition of some esterified carotenols. Comp. Biochem. Physiol., 69B: 625-627.39.Sarada, R., Vidhyavathi, D., Usha, D. and Ravishankar, G. A. 2006. An efficient method of extraction from green alga Haematococcus pluvialis. J. of Agric. Food Chem., 53:7585-7588.40.Shimidzu, N., Gato, M. and Miki, W. Carotenoids as singlet oxygen quenchers in marine organisms. Fisheries Sci., 62: 134-137.41.Sindhu, S. and Sherief, P. M. 2011. Extraction, characterization, antioxidant and anti-inflammatory properties of carotenoids from the shell of Arabian red shrimp Aristeus alcocki, Ramadan 1938. The Open Conference Proceedings Journal. 2: 95-103.42.Wang, L., Yang, B. Yan, B. and Yao, X. 2012. Supercritical fluid extraction of astaxanthin from Haemaotcoccus pluvialis and its antioxidant potential in sunflower oil. Innovative Food Science and Emerging Technologies, 13: 120-127.43.Yuan, J. O. 1999. Characteristics and chromatographic separation of astaxanthin and its esters from the microalga Haematococcus pluvialis. Ph. D. thesis. pdf. University of Hong Kong.44.Yuan, J. P. and Chen, F. 1998. Chromatographic separation and purification of trans-astaxanthin from the extracts of Haematococcus pluvialis. J. Agric. Food Chem., 46:

3371–3375.

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Table 1: Extractability percentage of astaxanthin from different solvent and acid treatment methods

Table 2: Fatty acid profile from GC-MS of H. pluvialis extract

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2321-7871 th Vol-2, Issue-8, 4 Sept 2014

Treatment methods Total percentage of astaxanthin extracted

Solvent treatment (1 hr)

Acetone 15.26 %

Methanol 19.54 %

DMSO 65.19 %

Acid treatment (1hr)

2N HCl 85.24 %

2N Formic acid 9.57 %

2N Tartaric acid 19.83 %

2N Acetic acid 21.34 %

2N Citric acid 7.59 %

S. no. Fatty acid profile Mol. Wt in

Da.

Mol.

Formula

Fatty acid

classification

RT Area Area %

1. Heptadecane 240.46 C17H36 Saturated 14.7 9630080 9.71

2. Hexadecanoic acid, ethyl

ester

256.42 C16H32O2 Polyunsaturated 17.83 17587904 17.75

3. Nonadecane, 9-methyl 282.54 C20H50 Saturated 15.15 9878368 9.96

4. Ethyl Oleate 310.51 C20H38O2 Polyunsaturated 19.55 11542288 11.65

5. Ethanol, 2-(9-

octadecenyloxy)-,(Z)

312.53 C20H40O2 Poly unsaturated 16.65 8731792 8.8

6. Cyclopentane, 1-phenyl-3-(-

phenyl-1-trimethylsily

methylene)

288.45 C17H24O2Si Polyunsaturated 17.98 10821504 10.92

7. 5-Octadecanol 270.49 C18H38O Monounsaturated 13.38 6922640 6.98

8. 2-Hexadecanol 242.44 C16H34O Saturated 16.83 7719824 7.78

9. 1-Docosanol 326.6 C22H46O Saturated 19.72 16293056 16.45

Extraction of carotenoid and thin layer chromatography (TLC), GC-MS, FT-IR and HPLC analysis.............

Table 3: FTIR analysis of functional groups from H. pluvialis extract

Fig. 1: TLC sheet showing astaxanthin and its derivatives from carotenoid extract of H. pluvialis

Fig. 2: Gas Chromatography (GC) of carotenoid extracts from H. pluvialis

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2321-7871 th Vol-2, Issue-8, 4 Sept 2014

S. no. Wave number cm-1 Stretch and bond Functional group

1 3427

O-H Hydroxyl group

2 2925

C-H Methyl group

3 2854

C-H Methyl group

4 1718

C=O Ketone group (cyclic 6-membered)

5 1634 C-C, C=C Alkenes (Monosubstituted Alkenes) 6 1384 C-H Alkyl, Methyl (CH3 umbrella formation)

7 857 C-H bends Aromatic bends (Para-disub. Benzene)

8 783 C-H bends Aromatic bends (meta-disubstituted Benzene)

Extraction of carotenoid and thin layer chromatography (TLC), GC-MS, FT-IR and HPLC analysis.............

Fig. 3: GC-Mass Spectrum of Hexadecanoic acid, ethyl ester (C:16:2) from carotenoid extract of H. pluvialis

Fig. 4: GC-Mass Spectrum of 1-Docosanol (C:22:1) from carotenoid extract of H. Pluvialis

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2321-7871 th Vol-2, Issue-8, 4 Sept 2014Extraction of carotenoid and thin layer chromatography (TLC), GC-MS, FT-IR and HPLC analysis.............

Fig. 5: GC-Mass Spectrum of Ethyl Oleate (C:20:2) from carotenoid extract of H. pluvialis

Fig. 6: GC-Mass Spectrum of Cyclopentane, 1-phenyl-3-(-phenyl-1-trimethylsily methylene (C:17:2) from carotenoid extract of H. pluvialis

Fig. 7: GC-Mass Spectrum of Nonadecane, 9-methyl- (C:17:2) from carotenoid extract of H. pluvialis

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2321-7871 th Vol-2, Issue-8, 4 Sept 2014Extraction of carotenoid and thin layer chromatography (TLC), GC-MS, FT-IR and HPLC analysis.............

Fig. 8: GC-Mass Spectrum of Heptadecane (C:16:1) from carotenoid extract of H. pluvialis

Fig. 9: GC-Mass Spectrum of Ethanol, 2-(9-octadecenyloxy)-,(Z) (C:20:2) from carotenoid extract of H. pluvialis

Fig. 10: GC-Mass Spectrum of 2-Hexadecanol (C:16:1) from carotenoid extract of H. pluvialis

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2321-7871 th Vol-2, Issue-8, 4 Sept 2014Extraction of carotenoid and thin layer chromatography (TLC), GC-MS, FT-IR and HPLC analysis.............

Fig. 11: GC-Mass Spectrum of 5-octadecanol (C:18:1) from carotenoid extract of H. pluvialis

Fig. 12: HPLC Spectrum of carotenoid extract from H. pluvialis

Fig. 13: FTIR spectrum of carotenoid extract from H. pluvialis

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2321-7871 th Vol-2, Issue-8, 4 Sept 2014

S. Elumalai PG and Research, Department of Plant Biology and Plant Biotechnology,Presidency College (Autonomous), Chennai, Tamil Nadu, India.

Extraction of carotenoid and thin layer chromatography (TLC), GC-MS, FT-IR and HPLC analysis.............