journal of chemical and pharmaceutical research, 2012… · 2016-09-28 · journal of chemical and...

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Journal of Chemical and Pharmaceutical Research, 2012, 4(2):1312-1318

Research Article ISSN : 0975-7384 CODEN(USA) : JCPRC5

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Qualitative chromatographic analysis of sugars present in non-edible rind portion of custard apple (Annona Squamosa. L)

S. Chandraju*1, R. Mythily 1 and C. S. Chidan Kumar2

1Department of Studies in Sugar Technology, Sir M. Vishweshwaraya Post-graduate Centre, University of

Mysore, Tubinakere, Mandya, Karnataka, India 2Department of Chemistry, G. Madegowda Institute of Technology, Bharathi Nagar, Karnataka, India

______________________________________________________________________________ ABSTRACT Custard apples have been part of human diet for ages due to its nutritional and medicinal values. But consumption of these fruits generates peel wastes that could bring about environmental pollution if not properly managed. Towards recycling of wastes and avoiding littering and waste-related environmental degradation, this study was carried out to explore the sugar components of custard apple peels with a view to establishing their raw material potentials. Selected samples are cut into small bits, dried, powdered and were subjected to sensitive extraction procedure developed using the mixture Methanol –Dichloromethane - Water (MDW) (0.3:4:1v/v) and MeOH-H2O phase was assayed for sugar analysis. The extracted sugars were put through chemical characterization procedures for purposes of separation and identifying its components. The various standard sugars were spotted using the solvent system n-butanol - acetone - diethylamine - water (10:10:2:6, v/v) in the cellulose layer for TLC analysis indicated the presence of lactose, sucrose, galactose and glucose. Keywords. Sugars, Custard apple peels, Separation, Identification, Waste Management. ______________________________________________________________________________

INTRODUCTION

Custard apples are the fruits of tropical tree, Annona reticulata, of the family Annonaceae. Also known by different names like bullock's heart or bull’s heart because of its heart shaped fruits, and is known as the ‘aristocrat of fruits’. The fruit is mostly heart shaped, sometimes irregular or oblong with a shallow depression at the base [1]. They have a yellow green or brownish, thin but tough knobby skin with a yellow coloring between the nodules on the skin when fully ripen. They have a creamy white, somewhat granular, sweet, soft edible flesh, and hard, dark brown or black, smooth and glossy non-edible seeds [2]. Only the creamy white flesh is to be eaten and the seeds are discarded. The composition includes considerable amount of carbohydrates, small amount of protein. Low in sodium and good source of potassium, which helps to maintain normal blood pressure, reduces the risk of hypertension, transmit nerve impulses to muscles, and improves muscle contraction [3]. Moderate source of minerals like calcium, iron, potassium, phosphorus, and magnesium, which play a very important role in maintaining proper metabolic activities of the body. Moderate source of vitamins like thiamine, riboflavin, niacin, and Vitamin B6, which are essential to maintain the normal activities of the body, and enhance the production of energy from the food. Moderate source of Vitamin A, which enhances the eye sight, fights against acne, and results in smooth radiant skin. Moderate source of Vitamin C, a natural water soluble antioxidant, which enhances the body immune system, increases the elasticity of skin, blood vessel increase the absorption of iron from the intestines and

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prevents bruising of the skin. Good source of dietary fiber [4]. It adds roughage to the contents of the intestines, promotes satiety, promotes the health of the colon, and also helps in relieving constipation, hemorrhoids, diverticular disorders, and also helps weight loss and its maintenance. Vitamins A and C are natural antioxidants that scavenge the free radicals from the body and protect the body from degenerative and inflammatory diseases. Custard apples promote digestion. They are also used to replace the body with minerals during diarrhea and dysentery [5,6].

Prior to early 1980’s, phytochemical studies of the plant family Annonaceae mainly focused on the isolation of numerous secondary metabolites including isoquinoline alkaloids, poly-phenols, aromatic compounds, essential oils and terpenes [7]. Annonaceous acetogenins can be found in the seeds, leaves, twigs, barks, and root of the plants. They are found to have potent and diverse biological effects such as cytotoxic, antitumor, anti-malarial, pesticidal, and insecticidal activities [8-15]. In 1982, Cole and coworkers isolated and characterized the first acetogenin, Uvaricin, from the roots of Uvaria accuminata (a member of the Annonaceae family). It contains a set of bis-adjacent tetrahydrofuran (THF) rings flanked by two hydroxyl groups and a terminal unsaturated lactones ring, which was connected to the THF moiety through an unbranched alkyl chain. Uvaricin was demonstrated to have anti-tumor properties in the in vivo PS System (P-338 lymphocytic leukemia in mice) [16, 17]. After uvaricin, during the past quarter century, more than 400 related compounds have been isolated by preparative HPLC, counter current chromatography, or chromatography on silica gel. Research in the field of Annonaceous acetogenins dealing with the isolation, structural elucidation, semi-synthesis or total synthesis, mechanism of the cytotoxic action, and the structure-activity relationship studies has been growing rapidly [12, 14, 18]. Processing of custard apple peels into sugars is a sure way of transforming these wastes with great potential for environmental pollution into a resource with great potential for economic prosperity, and also for securing the public health impacts of safer and healthier environment, likely to be obtained from the indirect waste management option so offered. In this regard, the possibility of converting these waste peels to industrial raw materials is being considered, and forms the central interest behind the conception of this study. Exploring and exploiting the abundant sugar components, seemed to be an additional way to evaluate the underlying economic values of these due to the special roles it plays in food and beverage industries.

EXPERIMENTAL SECTION

Extraction Selected samples are sliced, dried under vacuum at 60 °C for 48 h and powdered. 100.0 g of raw material was extracted with doubly distilled water 75mL, 15mL of 0.1N sulphuric acid and kept on hot plate for about 1 h at 60 °C. Contents are cooled and stirred well with magnetic stirrer for 30’. Neutralized with AR barium hydroxide and precipitated barium sulphate is filtered off. The resulting syrup was stored at 4 °C in the dark. The syrup was treated with charcoal (coir pith) and agitated for 30’ followed by Silica gel (230-400 mesh) packed in a sintered glass crucible for about 2cm thickness connected to suction pump, where rota vapour removed the solvent of the filtrate. The residue was placed in an air tight glass container covered with 200 ml of boiling 80% ethanol. After simmering for several hours in a steam bath, the container was sealed and stored at room temperature. For analysis, sample was homogenized in a blender for 3-5 min at high speed and then filtered through a Buchner funnel using a vacuum source replicated extraction with 80% EtOH (2 x 50mL) each time and the whole syrup was concentrated. Methanol - Dichloromethane - water (0.3:4:1, v/v), Sample tubes fed with the mixture were loosely capped, placed in a water bath for 5 sec, and left at room temperature for 10’ and placed in separating funnel, agitated vigorously by occasional release of pressure, results two phases. The organic phase was discarded which removes the organic impurities and the methanol: water phase was assayed for sugar. The residues were oven-dried at 50 °C overnight to remove the residual solvent, and stored at –2 °C for analysis [19-23].

Instrumentation The mixture was separated in 26’ by reversed phase HPLC on an Adsorbosphere column-NH2, (250 x 4.6 mm column) using both isocratic and gradient elution with acetonitrile/water and detected using Waters ELSD 2420. In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser. The separated fractions were subjected to UV analysis using Agilent 8453 coupled with Diode array detector. HPLC–MS analysis was performed with LCMSD/Trap System (Agilent Technologies, 1200 Series) equipped with an electrospray interface. The MS spectra were acquired in positive ion mode. The mobile phase consisted of 0.10% formic acid in hplc grade deionized water (A) (milli-q-water (subjected to IR radiation under 3.5 micron filters) and Methanol (B) taken in the stationary phase of Atlantis dc 18 column (50 x 4.6mm - 5µm). The gradient program was as follows: 10% B to 95% B in 4’, 95% B to 95% B


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in 1’, 95% B to 10% B in 0.5’ followed by 10% B in 1.5’ at a flow rate of 1.2 mL min-1. The column oven temperature was kept at 40°C and the injection volume was 2.0 µL. Product mass spectra were recorded in the range of m/z 150-1000. The instrumental parameters were optimized before the run [19-23].

Preparation of chromatoplate Thin layer chromatography was performed for the concentrated separated fraction using Cellulose MN 300 G. The fractions obtained were subjected to one dimensional chromatogram on a cellulose layer plate. Each plate was activated at 110 °C prior to use for 10’. Standard samples Pure samples D (-) Arabinose, D (-) Ribose, D (+) Xylose, D (+) Galactose, D (+) Glucose, D (+) Mannose, L (-) Sorbose, D (-) Fructose, L (+) Rhamnose, D (+) Sucrose and D (+) Maltose, D (+) Lactose were used as standard. One – dimensional chromatography 10 mg of each sugar and the separated fractions were dissolved in 1ml of deionised water. 1µL of each sugar solution was applied to the chromatoplate with the micropipette in the usual manner. The chromatoplate was placed in the chamber containing the developing solvent. The solvent system used was n-butanol - acetone - diethylamine - water (10:10:2:6, v/v). The plates were developed in an almost vertical position at room temperature, covered with lid [24-27]. After the elution, plate was dried under warm air. The plate was sprayed with 5% diphenylamine in ethanol, 4% aniline in ethanol and 85% phosphoric acid (5:5:1v/v). The plate was heated for 10’ at 105 °C. While drying coloured spots appear. The Rf values relative to the solvent are reported above.

Figure 1: UV inactive spectrum of the Separated Fractions

RESULTS AND DISCUSSION

It was found that the extracted separated components are UV inactive as in (Fig-1). The Mass Spectrum of fractions 1, 2, 3 and 4 were detected at 0.636 and 0.666, 0.525 and 0.702’, 0.578’, 0.606 and 2.637’ respectively. The MS report recorded at the appropriate time as per MSD for Fraction1 scanned between the time periods 0.507:0.600’ gave m/z values 126.9, 163.0, 343.2, 360.0, 365.0, 374.0 and 0.600: 0.878’ gave m/z values 126.9, 163.0, 342.2, 365.0, 365.0, 375.1. Fraction 2 scanned between the time periods 0.480: 0.546’ gave m/z values 115.1, 145.1, 175.9, 279.2, 312.1, 366.0, 365.0, 707.2 and 0.573: 0.812’n gave m/z values 111.2, 145.1, 279.2, 312.1, 360.0, 365.0, 707.2. Fraction 3 scanned between the time periods 0.493:0.772’ gave m/z values 112.9, 145.1, 163.0, 164.1, 180.1,

S. Chandraju et al

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202.9.Fraction4 scanned between the time period 163.0, 180.1, 198.0, 360.0 and 112.1. This concludes that, these masses corresponds to Hexose, and disacchariwhose masses are 180.1 and 342.2 (Figs 2, 3, 4 & 5).

Fig

Figure

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202.9.Fraction4 scanned between the time period 0.507: 0.798’ and 2.495: 2.760’ gave m/z values 112.9, 145.1, 163.0, 180.1, 198.0, 360.0 and 112.1. This concludes that, these masses corresponds to Hexose, and disacchariwhose masses are 180.1 and 342.2 (Figs 2, 3, 4 & 5).

Figure 2: Mass report of Separated Fraction 1


J. Chem. Pharm. Res., 2012, 4(2):1312-1318 ______________________________________________________________________________

gave m/z values 112.9, 145.1, 163.0, 180.1, 198.0, 360.0 and 112.1. This concludes that, these masses corresponds to Hexose, and disaccharides

S. Chandraju et al

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Figure

Fig Thin layer chromatographic analysis Four separated and purified sample fractions mentioned as F 1, F 2, F 3 and F4 in the chromatogram shown in (Figmatching with four standard sugars. R

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Figure4: Mass report of Separated Fraction 3


Thin layer chromatographic analysis Four separated and purified sample fractions were spotted in the cellulose layer and the eluted species were mentioned as F 1, F 2, F 3 and F4 in the chromatogram shown in (Fig- 6). The fractions obtained were found to be matching with four standard sugars. Rf value for the analytical grade samples shown in Table

J. Chem. Pharm. Res., 2012, 4(2):1312-1318 ______________________________________________________________________________

re spotted in the cellulose layer and the eluted species were The fractions obtained were found to be

value for the analytical grade samples shown in Table-1.


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Figure 6: Developed thin layer chromatogram over a cellulose layer, (La – Lactose, So – Sorbose, Ar- Arabinose, Rh – Rhamnose, Ri – Ribose, Xy-Xylose, Gal – Galactose, Gl - Glucose, Man – Mannose, Fr -

Fructose, Su – Sucrose and Mal –Maltose).

Table 1. Rf values matching of the analytical standard samples and the separated samples

Sugars Rf ( Scale of Rf =1)

Fraction matching

Lactose 0.18 F1 Maltose 0.24 - Sucrose 0.35 F2

Galactose 0.36 F3 Glucose 0.41 F4 Mannose 0.47 - Sorbose 0.46 - Fructose 0.46 -

Arabinose 0.46 - Xylose 0.53 - Ribose 0.63 -

Rhamnose 0.70 -

CONCLUSION

The array of sugar contents of custard apple peel were extracted, it will amount to huge economic waste to consider these peels as waste garbage meant only for the waste bin. Therefore, there is need to explore ways of reducing the environmental menace that may arise from huge seethapal peel wastes. The significance of this study could be seen from the fact that: successfully accomplishing the objectives of this study would mean a secured and safer environment for our enjoyment, boost to job/employment opportunities for the unemployed, increase in productivity and buoyancy of industrial economy as well as general economic growth.

REFERENCES

[1] Marcel Dekker, Handbook of Food Analysis, 1996, 533–550. [2] Lima e Silva, Paulo Sergio, Araujo Sousa, Thiago de, Mariguele, Keny Henrique, Dantas Rocha de Lima, Khadidja, Barbosa e Silva and Paulo Igor, Caatinga (Mossoró,Brasil), 2009, 22, 88-93. [3] Rahul Thube, Suresh Purohit and Abhijit Gothoskar, World Applied Sciences Journal, 2011, 12, 364-371. [4] Shashirekha,N.; Revathy Baskaran, Lingamallu Jaganmohan Rao, Munusamy, R.;Vijayalakshmi and Somasundaram Rajarathnam, , 2008, 41,236-243. [5] Trevor Olesen and StevenMuldoon J.; Tree Structure and Function, 2009, 23,855-862. [6] Mariappan, V.; and Saxena. R C.; Journal of Economic Entomology, 1983, 77, 573-576. [7] Leboeuf,M.; Cave, A.; Bhaumik, PK.; Mukherjee, R.; Phytochemistry, 1980, 21,2783-2813 [8] Zafra-Polo, MC.; Gonzalez, MC.; Estornell, E.; Sahpaz, S.; Cortes,D.; Phytochemistry, 1996, 42, 253-271. [9] Zafra-Polo, MC.; Figadere,B.; Gallardo,T.; Tormo,J.; Cortes,D.; Phytochemistry, 1998, 48,1087-1117. [10] Zeng,L.; Ye,Q.; Oberlies, NH.; Shi,G.; Gu,ZM.; He,K.; McLaughlin,JL.; Nat.Prod.Rep, 1996, 13, 275-306.


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[11] Cave,A.; Figadere,B.; Laurens,A.; Cortes,D.; Herz,W.; Kirby,G.; Moore,RE.; Seglich,W.; Tamm,C.; In progress in the Chemistry of Organic Natural Product, Eds, Springer-Verlag:Wien, 1997, 70,81. [12] Alali,FQ.; Liu,XX.; McLaughlin,JL.; Nat.Prod, 1999, 62,504-540. [13] Tormo,JR.; Gallardo,T.; Gonzalez, MC.; Bermejo,A.; Cabedo,N.; Andreu, L.; Estornell,E.; Curr. Top. Phytochem, 1996, 2, 69. [14] Bermejo,A.; Figadere,B.; Zafra-Polo,MC.; Barrachina,I.; Estornell,E.; Cortes,D.; Nat.Prod.Rep, 2005, 22,269-303. [15] Rupprecht,JK.; Hui,YH.; McLaughlin,JL.; Nat.Prod, 1990, 53, 237-278. [16] Tempesta,MS.; Kriek, GR.; Bates,RB.; J.Org.Chem, 1982, 47,3151-3153. [17] Geran,RL.; Greenberg,NH.; MacDonald,MN.; Schumacher,AM.; Abbott,BJ.; Cancer Chemother.Rep, 1972, 3,9. [18] Maezaki,N.; Kojima,N.;Tanaka,T.; Synlett, 2006, 993-1003 [19] Chandraju, S.; Mythily, R.; Chidan Kumar, C S.; J. Chem. Pharm. Res, 2011, 3,312-321. [20] Chandraju, S.; Mythily, R.; Chidan Kumar, C S.; J. Chem. Pharm. Res, 2011, 3, 422-429. [21] Chandraju, S.; Mythily, R.; Chidan Kumar, C S.; Int J Cur Sci Res, 2011, 1, 125-128. [22] Chandraju, S.; Mythily, R.; Chidan Kumar, C S.; Asian. J. Chem, 2012, 24(5), 2170-2172; [23] Chandraju, S.; Mythily, R.; Chidan Kumar, C S.; Recent Research in Science and Technology, 2011, 3, 58-62. [24] Baldwin, E.; Bell, DJ.; Cole's Practical Physiological Chemistry, 1955, 189. [25] Schweiger, A.; Journal of Chromatography, 1962, 9,374. [26] Vomhot, DW.; and Tucher, TC.; Journal of Chromatography, 1963, 17, 300. [27] Lato, M.; Brunelli, B.; Ciuffins, G.; Journal of Chromatography, 1968, 26,34.

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