IJCRR

IJCRR - 8(15), August, 2016

Pages: 27-32

Date of Publication: 11-Aug-2016

ISOLATION AND CHARACTERIZATION OF PHOSPHOLIPIDS OF ERI-PUPAL OIL FROM PUPAE GROWN ON CASTOR AND TAPIOCA LEAVES

Author: Thumu Ravinder, Shiva Shanker Kaki, Konda Reddy Kunduru, Sanjit Kanjilal, B.V.S.K. Rao, Sarat Kumar Swain, R.B.N Prasad

Category: Healthcare

Abstract:Objective: The aim of the present study was isolation and characterization of phospholipids from eri pupal oils.
Methods: Silkworm lipids were extracted from pupae fed on castor and tapioca leaves and further separated in to individual class of lipids by column chromatography. Fatty acid composition of each lipid class was studied along with positional distribution of fatty acids in major phospholipids by enzymatic hydrolysis.
Results and Conclusion: The eri pupal oils were found to contain 96.72-97.33% of neutral lipids, 0.51-0.21% of glycolipids and 2.65-2.39% phospholipids. Phosphatidylethanolamine (PE) was the predominant classes of phospholipid present (65.65 and 64.29%) in tapioca and castor fed pupal oils respectively. Phosphatidylcholine (PC) was observed in 19.50 and 19.22% respectively in tapioca and castor fed pupal oils. Cardiolipin (CL) and phosphatidylinositol (PI) are observed in minor levels. Positional distribution of fatty acids showed that, \a-linolenic acid (ALA) was found to be more in sn-2 position of PE. Thus pupal oil offers a good natural source of PE with enriched ALA.

Keywords: Eri silkworm pupae, Lipid classes, Phospholipids, Positional distribution, Fatty acid composition

Full Text:

INTRODUCTION
Eri silk is obtained from non-mulberry silkworms and is known for its elegance and is mostly produced in the Northeastern parts of India. Silkworms are reported to show association with the host plants on which they feed for the necessary nutrients required for their metabolic activities and also for silk production1 . The host plants reported for growing of eri silkworms are mainly castor (Ricinus communis Linn.) and tapioca (Manihot utilissima Phol.) followed by some other species also2 . Besides production of silk, sericulture also produces large quantities of silkworm pupae which are thrown as sericulture waste or used as fertilizer and as a constituent of chick and fish feeds3-4. The desilked silkworm pupae are reported to have high nutritional value because of the presence of high protein and fat and these are considered as a source of nutritive components and protein supplements5-7. The silkworm pupae have been reported to be a good source of alpha linolenic acid (ALA) and the oil is reported to contain high amount of phosphorous3 . The study on nutritional and toxicological evaluation of silkworm oil revealed that eri pupal oil is safe and nutritionally equivalent to common vegetable oils, which increase its potential as source of ALA8 . The neutral lipids of eri pupae were isolated and characterized previously and the phosphorous content was reported to be 3-3.5%9 . In mulberry silkworm oil, phospholipid content of 10% was reported where the major PLs identified were PE and PC10. Lecithins from vegetable oils like rice bran11, corn12, rapeseed13, sunflower14, cottonseed and peanut15 are known to be good sources for phospholipids such as PC, PE and PI. In milk phospholipids, PE is the major component where as PC, sphingomyelin are almost in equal portions16. Studies on isolation and characterization of phospholipids from vegetable oils are abundant, whereas PLs from other sources is very rare. Literature reports are also available on fatty acid composition of total lipids of silkworm pupae Bombyx mori L 17. However, there is no report on the detailed study on phospholipids obtained from eri silkworm oil. Therefore, the present investigation was aimed at the determination of phospholipid composition in lipids extracted from pupae fed on both castor and tapioca leaves.

MATERIALS AND METHODS
Pupae fed on castor and tapioca leaves collected from Shadnagar, Ranga Reddy district, Telangana and Rampachodavaram, East Godavari district, Andhra Pradesh, India were supplied by Central Silk Board, Bengaluru, India. Fatty acid mixtures, references PE, PC, PI, CL and snake venom (Naja naja Atra) phospholipase A2 source were purchased from M/s Sigma Chemicals, St Louis, USA. Silica gel (60-120 mesh) for column chromatography was purchased from Acme Synthetic Chemicals, Mumbai, India. Pre-coated thin layer chromatography (TLC) plates (silica gel 60F254) were procured from Merck, Darmstadt, Germany. HPLC grade solvents procured from M/s Merck, Mumbai, India. All other analytical reagent-grade chemicals and solvents were purchased from M/s SD Fine Chemicals, Mumbai, India. Total Lipid Extraction The matured pupae grown on castor and tapioca leaves were dried at 80-90°C for 6-8 hours using vacuum oven, followed by the oil extraction using Soxhlet extractor with hexane as reported earlier9 . The crude total lipids of castor and tapioca pupal oils were dried under reduced pressure, stored for further use. Separation of Lipid classes Total lipids (TL) of eri silkworm oils were separated in to neutral lipids (NL), glycolipids (GL) and phospholipids (PL) using column chromatography18 by the elution with chloroform, acetone and methanol respectively. GLs and PLs were qualitatively identified by TLC using chloroform/methanol/ water (65:25:4, v/v/v) with suitable spray reagents19-20. The phospholipid mixtures obtained from column chromatography of castor (0.619 g) and tapioca (0.687 g) fed pupal lipid extracts were further individually re-chromatographed to separate individual phospholipids. The column was eluted with a gradient of 5-10, 20, 40 and 50% of methanol in chloroform to obtain CL, PE, PC and PI respectively21. The separated phospholipids were identified by comparison with individual standards and confirmed by spray reagents20. The isolated yields of phospholipids were found to be in the following amounts. For castor based PLs: CL (0.058g, 9.37%), PE (0.398g, 64.29%), PC (0.119g, 19.22%), PI (0.042g, 6.78%) and for tapioca based PLs: CL (0.060g, 8.73%), PE (0.451g, 65.65%), PC (0.134g, 19.50%), PI (0.041g, 5.96%). Positional Distribution of Fatty Acids Positional distribution of fatty acids in individual phospholipids of both was carried by phospholipase A2 mediated regiospecific hydrolysis as described by Christie22. Briefly, PE (50 mg) was dissolved in 4 ml of diethyl ether and to this 300 µL of snake venom solution (6 mg of snake venom in 1 ml of borate buffer; pH 7.0) was added and shaken vigorously for 1 hour. After complete hydrolysis, the ether solution was evaporated under nitrogen. The mixture containing liberated free fatty acids (FFA) and lysophospholipids were separated by column chromatography with the elution of gradient methanol in chloroform23. PC, PI were also hydrolyzed in a similar manner and the fatty acid composition was determined22-23. Fatty Acid Methyl Esters (FAME) All the lipid fractions isolated were converted to FAME using 2% sulfuric acid in methanol24 and the FFA was treated with diazomethane. All the FAMEs were analyzed by GCFID for fatty acid composition. Gas Chromatography (GC) GC was performed on Agilent 6890N series Gas Chromatograph equipped with a flame ionization detector (FID) on a split injector. A fused silica capillary column (DB-225MS, 30 m x 0.25 mm i.d. J and W Scientific, USA) was used for separation. The oven temperature was programmed at 160°C for 2 min, increased to 230°C at 5°C/min and hold for 20 min at 230°C. The injector and detector temperatures were maintained at 230 and 250°C respectively. Nitrogen used as carrier gas with a flow rate of 1 mL/min. Identification of fatty acids was carried out by comparing with the retention time of respective commercial standards.

HPLC analysis of Phospholipids The phospholipid mixtures separated by column chromatography were qualitatively analyzed by Agilent HPLC chromatograph equipped with a quaternary pump and an evaporative light scattering detector (ELSD2000, Alltech, Deerfield, IL, USA). The drift tube temperature was set at 50°C and the nitrogen gas flow set at 1.5 L/min. The castor and tapioca phospholipids of 1 mg/mL concentration was separated on a SunFireTM Prep Silica column (5 μm, 4.6 x 250 mm; Sunfire columns, Waters, Ireland) at a mobile phase flow 0.5 mL/ min. A binary gradient mobile phase composed of eluent A [chloroform/ methanol/ammonium hydroxide (80:19.5:0.5, v/v/v)] and eluent B [chloroform/methanol/ammonium hydroxide/water (60:34:0.5:5.5, v/v/v/v)] was used for elution as follows25: 0-10 min, 95% B; 10-15 min, 100% B and 15-20 min, 95% B. Identification of phospholipids was carried out by comparing the retention times of the respective commercial standards. All the analysis was carried out in duplicate.

RESULTS AND DISCUSSION
The present study describes the separation and characterization of lipid classes from eri pupae (Samia Cynthia ricini) grown on castor and tapioca leaves. Total lipids were extracted from pupae and were found to be in the range of 18-20 % as reported earlier26. The extracted total lipids were separated into 3 lipid classes by column chromatography and the data is shown in Table 1.

The fatty acid composition of total, neutral, glyco and phospholipids of both varieties were determined by GC and are given in Tables 2 and 3. It was observed that, ALA was the major fatty acid followed by palmitic (16:0) and oleic (18:1) in all tapioca lipids compared to castor fed lipids which could be due to the influence of host plants as reported earlier9 . The ALA content in castor and tapioca fed PLs was observed 30.51% and 38.59% respectively. Previous studies on phospholipids from mulberry silkworm oil reported 40% of ALA10. Among other fatty acids, stearic (18:0), oleic and linolenic (18:2) acids were observed in greater amounts in both PLs and GLs of both varieties compared with neutral lipids. However, ALA content was observed to be low in PLs and GLs compared with NLs in both pupal oils. Long chain saturated fatty acids like arachidic (20:0) and behenic (22:0) acids were found slightly higher amounts in polar lipids compared with NLs of both pupal lipids. The phospholipids from both silkworm oils were identified by HPLC and quantified by column chromatography into CL, PE, PC and PI. The results showed that the castor and tapioca phospholipids are major source for PE (64-65%). Such a high PE content was earlier observed in the phospholipids of an obligate intracellular parasitic bacterium, Rickettsia prowazeki, where the PE reported 60-70%27. It was reported that mammalian and plant tissues have lesser occurrence of PE than PC, where as in bacteria PE is the principal phospholipid present28. In vegetable oil phospholipids, only castor seed oil was reported to contain high amounts of PE29. The fatty acid compositions of individual phospholipid classes and their hydrolysis products were determined by GC and are given in Tables 4 and 5. In cardiolipin isolated from castor and tapioca PLs, palmitic acid was the major fatty acid followed by oleic, stearic, ALA and other fatty acids. ALA content was more in PE of both pupal oils, followed by stearic, oleic, palmitic and other fatty acids compared to other PLs of both varieties.

The results (Table 4 and 5) indicate that, ALA was present in higher amounts at sn-2 position of tapioca PE (51.74%) compared to castor PE (45.56%). Saturated fatty acids were majorly located at sn-1 and unsaturated fatty acids were predominantly located at sn-2 position for all the phospholipid classes. A higher content of ALA and their predominance at sn-2 position increases the nutritional importance of the lipid9 . Hence, the phospholipids of castor and tapioca fed oils are rich source of ALA with predominant distribution at sn-2 position. In addition, phospholipids are a major component of cell membranes and required for signal transduction, metabolic regulation and maintenance of living cells30. The higher content of PE with ALA can be helpful for formation of fluid membrane structures which can have potential applications in signal transduction and other biological applications.

CONCLUSIONS
In this study, eri pupal oil is shown as a novel source for phosphatidylethanolamine containing ALA. The extracted oil from eri pupae was separated into different lipid classes and further characterization of phospholipids is reported for first time. With a few exceptions, castor and tapioca leaf fed eri silkworm pupal oils showed similar characteristics in lipid classes, fatty acid composition and positional distribution of fatty acids in phospholipids. The phospholipid fraction of eri pupal lipids with a high amount of PE could be a useful product for pharma and food applications.

ACKNOWLEDGEMENT
Authors acknowledge the immense help received from the scholars whose articles are cited and included in references of this manuscript. The authors are also grateful to authors / editors / publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed.

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