Olowosulu et al., Nig. Journ. Pharm. Sci., March, 2008, Vol. 7 No. 1, P.

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Nigerian Journal of Pharmaceutical Sciences Vol. 7, No. 1, March, 2008, ISSN: 0189-823X All Rights Reserved

SPECTROPHOTOMETRIC ANALYSIS OF HIBISCUS SABDARIFFA COLOURANT AT DIFFERENT PH VALUES

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A. K. Olowosulu*, 2P. A. Akpa, 2U. V. Eze and 2M. U. Adikwu

Department of Pharmaceutics and Pharmaceutical Microbiology, Ahmadu Bello University, Zaria, Nigeria Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka, Nigeria *Author for Correspondence: 08028440822, akolowo@yahoo.co.uk

ABSTRACT Spectrophotometric analyses of the natural colourant from Hibiscus sabdarifa were carried out at different pH levels. The results show that H. sabdarifa extract (colourant) was stable at the experimental pH range of 4.7 9.47. This was indicated by the similar peaks of absorption maxima at the different pH values. Hibiscus sabdarifa extract, with further studies could be used as a colourant in pharmaceutical formulations. Keywords: Spectrophotometric analysis, Hibiscus sabdariffa, colourant, pH effect

INTRODUCTION The use of colourants in cosmetics, foods and drugs dates back to antiquity. Colours are so common in our environment that we are not always aware just how much we depend on them. As colourants abound, so are the reasons for using them. Colours could be used as a means of identification, improvement of aesthetics, and protection from various factors of degradation, warning and concealment, to mention but a few (Marmion, 1984). The use of these colourants, both natural and synthetic in foods, drugs and cosmetics went on unrestricted for quite some good number of years. However, with the discovery of some fraudulent acts of marketing the same colourant under different names and also the discovery of some dangerous and undesirable
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properties of some colourants, specifications and restrictions have been placed on the manufacture, sale and use of these colourants in foods, drugs and cosmetics. Some colourants were found to be carcinogenic, teratogenic, degrade to toxic metabolites and various unwanted products. Consequent upon these, colourants used in foods, drugs and cosmetics were required to meet with the following specifications (Bainbridge, 1926). i) Arsenic contents must be less than 1/700,000. ii) Must be free from heavy metals according to authorized test. iii) Must be structurally true to type. iv) The allowable amount of contamination with other dyes varies between 1.5% and 3%.

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Decomposed dyes and other organic impurities must be reduced to a minimum. vi) Insoluble matter must in no case exceed 0.3%. The carcinogenic effect is most common with synthetic colourants. Thus the use of natural colourants is most desirable. This study is geared towards the evaluation of a natural colourant from Hibiscus colourant for use in food and drugs. The emphasis on pH is to see the possibility of using it in different formulations of food, cosmetic and drug at different pH levels. Hibiscus belongs to a family of plants called malvaceae of which over three hundred species have been described (Trease and Evans, 1995). A tea prepared from the dry calyxes and flower

v)

popularly called Zobo drink in Nigeria is widely consumed for its refreshing qualities. This is prepared by boiling the dried calyxes and flowers in some quantities of water for some time. The concentrate is strained through a muslin cloth and allowed to cool. Flavours and sweeteners are added to taste and the product consumed best as a refreshing cold drink. Hibiscus (red calyxes) yields its colour easily to water. It is highly soluble in water that it does not need great effort or special technique to get it into solution. It is used in colouring wines, ice creams, butter, marmalade, refreshing beverages, pudding and cakes calyxes are used in the West Indies and elsewhere in the tropics to colour and flavour rum (Ndu, 2003). The effect of pH changes on the colourant is the main focus of this study.

MATERIALS AND METHODS

The following materials were used as procured without further purification: hydrochloric acid and sodium chloride (Merck). Distilled water was obtained from an all glass still. Preparation of Hibiscus Extract The hibiscus extract was prepared by soaking the calyxes in distilled water for about twentyfour hours. After extraction, the solution was filtered through a fine mesh muslin cloth to obtain a clear, deeply pink to dark red coloured solution. The solution was then concentrated by boiling off some quantities of the solvent. The dried material was obtained by lyopholization. The material obtained was stored in amber-coloured bottles to prevent darkening which often occurs when the material is exposed to light. Absorption spectra of Hibiscus extract at different pH values A 0.2-%w/v concentration of the colourant was made in aqueous solution and pH values adjusted to 4.7, 5.5, 7.0, 8.7 and 9.47 respectively. These solutions were scanned in turn at intervals of 1 nm using UV Vis

spectrophotometer (Pye-Unicam, England). Their peak absorbances were all determined. Beers plot of Hibiscus colourant solution A stock solution of Hibiscus (zobo) colourant (1% w/v) was made by dissolving 1 g of the solid extract in 100 ml of distilled water. A serial dilution of the stock was made to obtain concentrations of 1 mg/ml, 2mg/ml, 3mg/ml, 4mg/ml and 5mg/ml. The maximum absorbances of these solutions were determined using the UV VIS spectrophotometer at 580 nm (Pye-Unicam, England) and plotted against the concentration to obtain a standard plot.

RESULTS AND DISCUSSION

A straight line passing through the origin was obtained for the Beers plot (Fig.6). This show that light absorbance of standard solution of hibiscus colourant extract can be used in quantitative analysis of the crude extract. Table 1 shows the absorption peaks of the hibiscus extract solution prepared at different pH values. Hibiscus colourant solution, within the limits of pH range chose, showed consistency in its wavelength of the peak of

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absorption of light spectrum (Figs 1, 2, 3, 4 and 5). This shows that a single chromophore is contained in the hibiscus extract. Had it been composed of several chromophores, it would have shown different wavelengths of peak absorption. Results also indicate the stability of the colourant within the stipulated pH range. Different colours have their different and characteristic wavelength of peak absorption. It was found that hibiscus colourant extract was stable at the experimental pH-range of 4.7 9.47. The spectrophotometer analysis did not show

significant instability as a result of the pH modifications. Due to the adverse effects of some synthetic colourants, which include carcinogenicity, tetratogenicity and various other toxic effects (Anstead, 1991), natural colourant such as Hibscus sabdariffa can be used. Natural colourants have wider margin of safety than synthetic colourants. In conclusion it can be stated that Hibiscus sabdariffa colourant, with further studies, could be used as a colourant in pharmaceutical formulations especially syrup.

pH values 4.7 5.5 7.0 8.7 9.47

Table 1: Effect of pH on the peak absorption values Peak Absorption (nm) 520 520 520 519 520

1.2

1.5 Absorbance
Absorbance

1 0.8 0.6 0.4 0.2

1 0.5 0 0 500 Wavelength(nm) Fig.1 Sample scan of zobo extract in buffer (pH 4.7) Absorbance 1000

0 0 500 Wavelength Fig.2 Sample scan of zobo extract in buffer (pH 5.5) Absorbance 1000

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1. 2

0.8

0.6

0.4

0.2

0
0 20 0 40 0 60 0 80 0

1.2 1 0.8 0.6 0.4 0.2 0 0 200 400 600 800 Wavelength (nm)

Wavel ength(nm)

Fi g. 3 Sampl e sc an of Zobo ex t r act i n buf f er (pH 7. 0)

Absorbance

Fig.4 Sample scan of zobo extract in buffer (pH 8.7)

A bsor bance

Absorbance

2.5

1.2 1 0.8 0.6 0.4 0.2 0 0 200 400 600 800

Absorbance

1.5 Absorbance

0.5

Wavelength(nm) Fig.5 Sample scan of zobo extract in buffer (pH 9.74) Absorbance

0 0 -0.5
Concentration (mg/ml)

Beer's plot for Zobo extract in water

Fig 6: Beers plot for H. sabdariffa extract in water

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REFERENCES
Anstead, D. F. (1991) Cosmetic colours. In: In. Handbook of Cosmetic Science, Pergamon Press, New York, Pp. 101-118. Bainbridge, W.C. (1926) Development of the food colour industry in the United States. Ind. Eng. Chem. USA, Pp.1329-1331.

Marmion, D M. (1984) Handbook of U.S. Colourants for Foods, Drugs and Cosmetics 2nd Ed., John Wiley and sons, U.S.A., P. 3. Ndu, O. (2003). M. Pharm. Thesis, University of Nigeria, Nsukka. Trease, G.E. and Evans, W. C. (1995). A Textbook of Pharmacognosy, W B Sauders. London, Pp. 201.

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