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Isolation & identification of flavonoid 'rutin' from Indian plantation white sugars.

Introduction

Colour is main criteria to define the quality of sugars, lower the colour value, higher the quality of sugars. A colourless sugar has a more saleable value than coloured one. Sugars directly produced from cane juice for consumption generally possess colour having high ICUMSA colour value (>100). By knowing the nature of colour this may help in the improvement in our refining process to produce, better quality sugar. These colourants are different in origin such as from sugarcane plant (xanthophylls, chlorophylls, carotenes, flavonoids etc.) or may be during processing (melanins, melanoidins, HADP, caramels, strecker degradation products etc.) The enzymes present in cane juice may react with flavonoids or polyphenolic acids during milling of sugarcane of generate polymeric colour 'melanins' while melanoidins, caramels or HADP have not much significant role in colour formation. Flavonoids yellow coloured colourants are more stable than other type of colourants and pass over to the final stage of processing. According to Smith and Poton [1] the group of flavonoids is most critical to sugar colour generation.

Flavonoids are a group of about 4000 naturally occurring polyphenolic compounds found universally in sugar plant & in food plant (Harbone, 1986). These are primarily responsible for the colour generation and widely distributed. These compounds (flavonoids) appear to play vital role in colour generation in various grades of sugar crystals.

Flavonoids could not be extracted sufficiently by normal methods due to the high impurity content and time taking processes, therefore it is necessary to develop a more effective and reliable method for the isolation and identification of flavonoids. In a taxonomic study [2] of the saccharum revealed that the presence of many O & C-glycosides of tricin and iso-orientin. Many others were identified but show yellow to orange colour with and without ammonia in UV light. Kaempferol & Umbelliferone were detected in refined sugars [3], tricin-T-glucosyl glucoside from mill syrup and several others have been identified by HPLC technique in sugar factory products (5,8).

Flavonoids are water soluble so enter into the mixed juice during processing while others such as chlorophylls, xanthophylls and carotenes are water insoluble hence removed in clarification. Flavonoids & their glycosides due to soluble in water pass through the entire process into the sugar crystals.

White sugar manufactured in India by double carbonation & double sulphitation process is off-white in appearance which is directly consumed.

Therefore the present investigation deals with the determination of free as well as combined flavonoids and phenolic (colourants) in the various grades of sugar crystals.

Experimental

The sugar sample used in the experiment was taken from south-west region of India. This paper described the procedures used are

* Polarization and colour

* Chromatography

* Colourants recovery of from sugar

* Fractionation of colourants.

* Spectroscopy.

Polarization & Colour

To Determine pol percent of sugar, a digital polarimeter [Bellingham+Stanley AP-220] was used. The ICUMSA colour was measured at 420nm in a 4cm cell by the recommended standard procedure [9].

Chromatography

Double Distilled water was used in all chromatographic experiments and reagents used were analytical grade. All vacuum evaporations were done at 40[degrees]C.

A polystyrene cross-linked adsorbent resin Amberlite XAD-4 [pore dia = 40A surface area--725[m.sup.2]g] was used to recover colourants from sugar solution. The size of resin column was 30x600mm filled up to a height of 350mm. The resin column was prepared in methanol and then washed free of methanol with water.

The Gel column used for the fractionation of colourants was filled with sufficient 'Sephadex G50-150', in the form of a slurry to occupy a settled volume filled in the same size column upto about 400mm height.

The so obtained fractious were subjected to TLC on 20x20cm cellulose glass plates using n-butanol-acetic acid-water (4:1:5) solvent system. The spots were developed by fuming with N[H.sub.3] vapours and viewed under UV lamp (254 & 350nm). Paper chromatography was done using same solvent system on whatman no. 1 paper.

Colourants Recovery from Sugar

Make 20[degrees] Bx sugar solution, filtered and pH was adjusted to about 3 with concentrated HCl. This suppressed the ionization of colourants which would otherwise have inhibited their adsorption on the resin. The flow rate of sugar solution was 2-3ml/min.

Now column is washed with distilled water to remove residual salts & sugar. A mixture of methanol : ammonia : water (40 : 4 : 56) was used for colourants recovery. The elution rate was 2-3ml/min. the deadsorbed colourants solution solidify under vacuum and dried over [P.sub.2][O.sub.5] in a desicator.

Fractionation of Colourants by Gel Permeation Chromatography

The separation of colourants was done on a molecular basis. Solid colourants was dissolved in 150ml water, add 1-2 drops of concentrated HCl to precipitate any polymeric colourants. Filter the colourants solution now ready for adsorption on gel column at a flow rate of 1ml/3min. Elute the colourants with mixture of water and methanol (40%), 5 ml fractions were collected and subjected to TLC on cellulose plates. Pure fractions were completely evaporated & investigated for identification.

Spectroscopic Study

A double beam spectrophotomer (Perkin-Elmer) was used to record UV spectra of compound using spectroscopic grade methanol. The reagents used for observing UV shifts were of analytic grade and measured according to method reported by Mabry, Markham & Thomas [6].

The H-NMR spectra were recorded on a Bruker-DSR spectrophotometer at 300 MHz using DMSO-[d.sub.6] solvent. The compound must be completely dried before recording the spectra.

Results and Discussion

This method was used to determine the isolation and presence of single colourants (such as rutin) also, TLC, UV & NMR spectral studies have provided new dimensions to the chemistry of flavonoids.

The sugar sample used was off-white in appearance with a pol percent 97.92 and ICUMSA colour 243. the solid colourants yield was 210 mg/kg. twenty fractions (10 ml each) were collected by gel permeation chromatography of the colourants. TLC (BAW) revealed yellow spots in 1, 2, 3, 4 & 5 fractions. All these fractions were purified by paper chromatography and eluted with 40% methanol. Again these purified fractions were subjected to paper chromatography (BAW) with an authentic sample of rutin, reveled to be rutin ([R.sub.f] = 0.45) [7]. It gives deep purple colour in UV light. After fuming with ammonia vapours in UV light detected as a yellow spot.

The UV spectrum of the compound exhibited two major absorption peaks 266 nm and 361 nm which matched with that reported for rutin [6].

[FORMULA NOT REPRODUCIBLE IN ASCII]

The final confirmation was done b6y NMR spectrum of the compound. It is agreement with the reported rutin spectra [6].

The investigation reveals the presence of flavonoids in sugar crystals manufactured directly from cane juice. The distribution of flavonoids was different in different region of India [10]. This may indicate regional differences since the sugar were said to be from different areas of India which is an interesting consideration.

Rutin and other flavonoids are highly coloured in the acidic medium due to formation of oxonium salts.

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In Flavonoids Due to oxidation of one aromatic ring into quinoid form, shows different colour in different colour in different mediums such as red chelates absorption shows finding of flavonoids.

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[FIGURE 1 OMITTED]

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Acknowledgement

The Author Viesh Kumar is highly thankful to Government of India (Ministry of Consumer Affairs, Food and Public distributions) for giving Senior Research Fellowship. We are also thankful to Director, CDRI Lucknow for providing the NMR facility.

References

[1] Smith, P. and Paton, N.H. (1985) Sugar cane flavonoids. Sugar Technol. Rev. 12 : 117-142.

[2] Williams, C.A., Harborne, J.B. and Smith, P. (1974) The taxonomic significance of leaf flavonoids in Saccharum and related genera. Phytochernistry 13 : 1141-1149.

[3] Farber, L. and Carpent'r, F.G. (1971) Identification of pigments that persist into refined sigar. Proc. 14th Cong. ISSCT : 1589-1600.

[4] Linecar, D., Paton, N.H. rnd Smith. P. (1978) Techniques for the isolation of cane sugar cclorants. Proc. Tech. Session Cane Sugar Refining Research 81-90.

[5] Paton, N.H. and Smith., P. (1983) Colorant adsorption in the refinery. mt. Sugar J. 85 : 102-105.

[6] Mabry, T.J., Markham, K.R. and Thomas, M.B. (1970) The Systematic Identification of Flavonoids. Springer-verlag. New York.

[7] Harborne, J. B. (959) The chromatograph of the flavonoid pigments. J. Chrornatog 2 : 581-604.

[8] Colombo, R., I ancas, F.M. and Yariwal, J.H. (2006) Determination of fiavonoids in cultivated s arcane leaves, bagasse, juice and in transgenic sugarcane by liquid chromatography- UV detection. f. Chromatog. A 1103 : 118-124.

[9] Chen, J.C.P. (1985) Cane Sugar Hand Book. John Willey-Inter Science Publication (1 11h Edition).

[10] Dwivedi, S. Bhatt, S. (2010) Flavonoids from direct consumption off-white sugars. International Sugar Journal .CXII: 287-290.

(1) Vikesh Kumar, (2) Om Kumari, Surabhi Singh, (3) Rashmi Upadhyay and (3) M.R. Tripathi

(1) National Sugar Institute, Kanpur-208017, India

(2) Department of Chemistry, K.K. P.G. College, Etawah-206001, Uttar Pradesh, India

(3) Department of Chemistry, D.A.V. P.G. College, Kanpur-208001, Uttar Pradesh, India

E-mail: vikeshkumarnsi@yahoo.in
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Author:Kumar, Vikesh; Kumari, Om; Singh, Surabhi; Upadhyay, Rashmi; Tripathi, M.R.
Publication:International Journal of Applied Chemistry
Article Type:Report
Date:Jan 1, 2012
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