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RESEARCH ARTICLE

 

Isolation and Purification of Lutein from Indian Spinach Basella alba

 

Ashwini Prabhu1, Kadar Sajida Abdul2, Punchappady-Devasya Rekha1*

1Yenepoya Research Centre, Yenepoya University, Mangalore – 575 018, Karnataka, India

2 Department of Postgraduate Studies and Research in Biosciences, Mangalore University, Mangalagangotri – 574 199, Karnataka, India

*Corresponding Author E-mail: rekhapd@hotmail.com

 

ABSTRACT:

Lutein is a pharmacologically important phytochemical belonging to the class of carotenoids widely distributed in marigold flowers and green vegetables. It is industrially important for its biological activities such as antioxidant, immunostimulatory and macular protective properties. In this study, isolation and purification of lutein from Indian spinach, Basella alba was carried out. For the extraction of lutein, petroleum ether- acetone extract of spinach leaves was prepared which yielded 2.3 mg/100g of lutein. Preparative TLC of the extract yielded lutein with a Rf value of 0.12. Lutein isolated was subjected to spectral characterization by UV-Visible and FT-IR spectroscopy. The peaks obtained corresponded well with those reported for standard lutein. Results of the study indicated that B. alba leaves can be used as a source for obtaining lutein.

 

KEYWORDS: Basella alba, Lutein, Solvent extraction, Spectral characterization.

 

 

INTRODUCTION:

Plants produce secondary metabolites termed in general as ‘phytochemicals’ having the potential to combat diseases such as cancer, heart stroke or metabolic syndrome. They are non-essential nutrients and possess certain properties like antioxidant, antimicrobial and immune modulating activities1. Carotenoids, constituting an important class of phytochemicals, are a group of red, yellow and orange pigments that have extended conjugated double - bond systems2 and belong to the tetraterpenoid group3. Carotenoids are derived from isoprenoid precursors and are basically divided into two groups: the carotenes- acyclic or cyclic hydrocarbons and the xanthophylls – oxygenated derivatives of carotenes4. The widely distributed antioxidant property of the carotenoids is due to its double bond which reacts with ROS to scavenge the radicals5.

 

 

 

 

 

 

Received on 12.08.2015          Modified on 24.08.2015

Accepted on 15.09.2015        © RJPT All right reserved

Research J. Pharm. and Tech. 8(10): Oct., 2015; Page 1379-1382

DOI: 10.5958/0974-360X.2015.00247.4

 

Among carotenoids, there has been a special interest in lutein and zeaxanthin, due to its enormous health benefits6. Lutein is found in vegetables, such as kale, spinach, and broccoli. Currently, commercial scale isolation of lutein is from marigold flowers7. It has physiological role in improving vision and in eye protection from harmful UV light8 and to quench singlet oxygen, a highly reactive free radical that can damage DNA9. During inflammation, lutein in immune tissues is depleted and the level of depletion is dependent on dietary lutein intake10. There are many applications of lutein which include pigmentation of poultry products, drugs and cosmetics, coloration of food and prevention of age related macular degeneration11.

 

Basella alba is commonly known as Ceylon spinach, Malabar spinach, Indian spinach and is a widely cultivated cool season vegetable with succulent stem and leaves12. Leaves are fleshy, ovate or heart-shaped. Mainly leaves and stems are used for consumption as well as for medicinal purpose13. Major phytoconstituents reported in B. alba include alkaloids, tannins, flavonoids, saponins, phenolic compounds and phytosterols14. Leaves are rich in proteins, fats, vitamins A, C, E and K, folic acid, riboflavin, niacin, thiamine and minerals such as calcium, magnesium and iron. B. alba also contains the flavonoid kaempferol at a concentration of 1.4 mg/100 g15. At present, lutein extraction is limited to a few sources like marigold flowers and microalgae, wherein the process is tedious and expensive16-17. B.alba leaves are known to contain significant amounts of lutein18. However, no efforts were made towards its exploitation for isolating lutein. Hence, in this study, we report the isolation and purification of lutein from the leaves of B. alba.

 

MATERIALS AND METHODS:

Chemicals:

Iodine crystals, potassium iodide, picric acid, copper acetate, α-naphthol, copper sulphate, sodium hydroxide, sodium potassium tartarate, ninhydrin, ferric chloride, gelatin, hydrochloric acid, sulphuric acid, acetic anhydride, petroleum ether, diethyl ether, acetone, acetic acid  and silica gel R254 were purchased from Merck (India).

 

Plant Material and Extraction:

The leaves of B. alba were collected from local farms of Mangalore (India). They were washed, shade dried and powdered using an analytical mill (Ika, Germany). The powder was stored at 4˚C in air-tight container until further analyses. Two methods were followed for extraction. In the first method, 100 g of spinach powder was suspended in 1000 ml of diethyl ether and methanol mixture (2:1) and was agitated on a shaker for 24 h. The filtrate was collected and concentrated to obtain the extract. In the second method, 100 g of leaf powder was extracted with 1000 ml of petroleum ether and acetone mixture (1:1) using soxhlet apparatus for 8 h. The extract was concentrated to yield dark green colored thick paste.

 

Evaluation of phytoconstituents in the extract:

B. alba extract was subjected to preliminary phytochemical screening to detect the presence of various phytoconstituents in the extract19.

 

Preparative Thin Layer Chromatography (TLC):

For this, 5 mg of the extract was redissolved in 50 µl dichloromethane and spotted on silica gel plates. Compounds were eluted using petroleum ether: diethyl ether: acetic acid (80:20:1) as a mobile phase. From the developed plates, Rf values were compared to that reported in the literature. The spot corresponding to lutein was recovered from TLC plate, redissolved in dichloromethane and concentrated to obtain lutein.

 

Spectral Analysis of the Pigment:

For spectral analysis, 1 mg of isolated lutein was dissolved in 1 ml of dichloromethane. Taking dichloromethane as reference, the sample was screened for absorbance maxima by scanning in the wavelength range of 400-700 nm. Spectral characterization of the sample was carried out using UV-visible spectrophotometer (UV-1800, Shimadzu, Japan).

Fourier Transform Infrared Spectrometric (FT-IR) analysis:

The lutein recovered from the TLC plates was subjected to FT-IR analysis. FT-IR spectrum of lutein was recorded in the range of 500 – 4000 cm-1 using Shimadzu IR Prestige-21 spectrophotometer. The peaks obtained were compared with the peaks reported for lutein in the literature.

 

Purification of lutein using column chromatography:

For this, 2 g of the spinach extract was chromatographed using was silica gel (60-120 mesh) column and eluted using petroleum ether – diethyl ether mixture (80:20). The elute was concentrated to give yellow semisolid, which upon TLC, yielded lutein with a similar Rf value reported for standard lutein.

 

RESULTS AND DISCUSSION:

The first method employed for extraction, upon phytochemical screening was found to be unsuitable as it did not show the presence of phenolics and flavonoids in abundance. Screening of B. alba petroleum ether – acetone extract for phytochemicals indicated the presence of saponins, phytosterols, phenolics and flavonoids in large quantities.  In this investigation, lutein was separated using TLC at Rf value of 0.12 and it matched with the results obtained by Kadeem29 in the solvent system petroleum ether, diethyl ether and acetic acid in the ratio 80:20:1 (Figure 1).

 

Rf – 0.12
 

Figure 1. Identification of lutein from B. alba extract using TLC

 

 

 

Figure 2. Photometric spectrum of lutein isolated from B. alba

 

Figure 3. FT-IR spectra of lutein isolated from B. alba

 

The spectral analysis for lutein revealed a peak at 447.5 nm in dichloromethane which was matching with the results obtained by Craft and Soares21. The characteristic spectra of lutein in dichloromethane is represented in figure – 2.

 

The FT-IR spectral analysis for lutein revealed a broad peak at 3394 cm-1 indicating O–H stretch. The peaks at 2925, 2825 cm-1 indicated C–H stretch; 1654 cm-1 indicated –C=C– stretch; 1617, 1513 cm-1 indicated C–C stretch for aromatics; 1444 cm-1 indicated C–H bend; 1177, 1103, 1074, 1036 cm-1 indicated C–O stretch for alcohols; 889, 816 cm-1 indicated C–H for aromatics; 733, 698 cm-1 indicated C–H rocking (Figure 3). Similar results were reported in a study carried out by Joseph 22.

 

From the results of this study, it is evident that B. alba is a rich source of lutein that can be separated easily using petroleum ether – diethyl ether solvent system. The method used for extraction in this study is relatively economical with high yield.  Extraction of lutein from B. alba may have advantage over extraction using marigold flower petals23 or microalgae like Murielopsis sp. and Scenesdesmus almeriensis24. Extraction of lutein from marigold flowers is tedious process involving extensive column chromatography techniques while, that from microalgae is an expensive process because of its specific growth requirements.  The study carried out on the isolation and purification of lutein has enormous significance. This study focuses on extraction of lutein from easily available plant material B. alba to offer high yield and high affordability. Lutein being an antioxidant, protects the cells from damage caused by free radicals. Besides, lutein has a number of biological actions including intercellular communication, inhibition of cell transformation, inhibition of the monocyte mediated inflammatory response, immune enhancement, inhibition of LDL resistance to oxidation and macula protection25.  Studies revealed that higher intake of lutein can reduce the risk of lung cancer26. Daily intake of 6 mg lutein is required to prevent the onset of age-related macular degeneration according to a study by El-Sayed et al 27. The individual carotenoid content is considered as high and very high  if the yields are 0.5-2 mg/100g and >2 mg/100g respectively28. Extraction of lutein from B. alba yielded 2.3 mg/100 g of the carotenoid, which is considered as relatively higher yield. B. alba can be a reliable source for lutein extraction as it can be cultivated throughout the season with no much nutrient inputs. This makes it an interesting candidate for extracting lutein.

 

CONCLUSION:

Lutein is a neutraceutically important natural product with enormous health benefits. This study explores into the possibility of exploiting B. alba for the isolation of lutein and shows promising findings. Hence, we conclude that B. alba can be used as an alternate source for lutein extraction. Also, B. alba, being used in the regular diet, can provide this carotenoid in significant amounts meeting the physiological requirements.

 

ACKNOWLEDGEMENT:

The author Ashwini P acknowledges the postdoctoral fellowship provided by Yenepoya University, Mangalore.

 

CONFLICT OF INTEREST:

Authors declare that they have no conflict of interest.

 

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