Spectrophotometric analysis of Hibiscus sabdariffa colourant at ...

Olowosulu et al., Nig. Journ. Pharm. Sci., March, 2008, Vol. 7 No. 1, P. 36 – 40
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
1 A. K. Olowosulu*, 2 P. A. Akpa, 2 U. V. Eze and 2 M. U. Adikwu
1 Department of Pharmaceutics and Pharmaceutical Microbiology, Ahmadu Bello University, Zaria, Nigeria
2 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
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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Olowosulu et al., Nig. Journ. Pharm. Sci., March, 2008, Vol. 7 No. 1, P. 36 – 40
v) 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
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.
Beer’s 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 Beer’s 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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Olowosulu et al., Nig. Journ. Pharm. Sci., March, 2008, Vol. 7 No. 1, P. 36 – 40
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.
Table 1: Effect of pH on the peak absorption values
pH values
Peak Absorption (nm)
4.7 520
5.5 520
7.0 520
8.7 519
9.47 520
1.5
1.2
1
Absorbance
1
0.5
0
0 500 1000
Wavelength(nm)
Absorbance
0.8
0.6
0.4
0.2
0
0 500 1000
Fig.1 Sample scan of zobo extract
in buffer
(pH 4.7)
Absorbance
Wavelength
Fig.2 Sample scan of zobo extract in
buffer
(pH Absorbance 5.5)
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Olowosulu et al., Nig. Journ. Pharm. Sci., March, 2008, Vol. 7 No. 1, P. 36 – 40
1. 2
1
0.8
0.6
0.4
0.2
0
0 200 400 600 800
Wavel ength(nm)
Absorbance
1.2
1
0.8
0.6
0.4
0.2
0
0 200 400 600 800
Wavelength (nm)
Fi g.3 Sampl e scan of Zobo extr act i n buf f er (pH 7.0)
Fig.4 Sample scan of zobo extract in buffer
(pH 8.7)
Absor bance
Absorbance
2.5
2
Absorbance
1.2
1
0.8
0.6
0.4
0.2
0
0 200 400 600 800
Absorbance
1.5
1
0.5
Wavelength(nm)
0
0 2 4 6 8
Fig.5 Sample scan of zobo extract in buffer
(pH 9.74)
Abs orbance
-0.5
Concentration (mg/ml)
Beer's plot for Zobo extract in water
Fig 6: Beer’s plot for H. sabdariffa
extract in water
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Olowosulu et al., Nig. Journ. Pharm. Sci., March, 2008, Vol. 7 No. 1, P. 36 – 40
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 2 nd 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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