74
FARMACIA, 2009, Vol.LVII, 1
QUANTITATIVE ANALYSIS OF BIO- ACTIVE
COMPOUNDS IN HIBISCUS SABDARIFFA L.
EXTRACTS. II. QUANTITATIVE ANALYSIS AND
BIOLOGICAL ACTIVITIES OF ANTHOCYANINS
SUHAD S. HUMADI1, VIORICA ISTUDOR2
1
University of Baghdad, Faculty of Pharmacy
University of Medicine and Pharmacy “Carol Davila”, Faculty of
Pharmacy, Traian Vuia 6, sect. 2, 020956, Romania
2
Abstract
Anthocyanins are naturally occurring compounds that impart color to fruits,
flowers, leaves and other plant organs. They are probably the most important group of
visible plant pigments besides chlorophyll. Apart from imparting color to plants,
anthocyanins also have an array of health-promoting benefits, as they can protect against a
variety of oxidants through a various number of mechanisms. This article reviews their
biological activities and also presents a comparative study of some analytical methods used
in the determination of anthocyanin content in different extracts. The study results revealed
that the measurement using the original standard curve method is the best, simplest and the
most accurate one among the other examined methods.
Rezumat
Antocianozidele şi antocianii sunt compuşi de origine vegetală care dau culoarea
fructelor, frunzelor şi altor organe ale plantelor. Antocianozidele, alături de clorofilă,
flavone, carotenoide şi aurone se numără printre pigmenţii vegetali vizibili cu ochiul liber.
Ele aduc o serie de beneficii sănătăţii, deoarece pot proteja organismul împotriva unei mari
varietăţi de oxidanţi, prin diferite mecanisme. Acest articol menţionează activităţile
biologice ale antocianozidelor şi prezintă un studiu comparativ al unor metode de analiză
folosite la determinarea conţinutului de antociani, în diferite extracte de Hibiscus sabdariffa
obţinute cu apă, alcool diluat, cu sau fară adaos de acid clorhidric. Între toate metodele
testate, metoda standard, care foloseşte curba construită cu o substanţă de referinţă (în cazul
de faţă clorura de cianidol) oferă cele mai bune rezultate.
Keywords: anthocyanins; pigments; Hibiscus sabdariffa; quantitative analysis
Introduction
The roles of anthocyanin pigments as medicinal agents have been
well-accepted in folk medicine throughout the world, and, in fact, these
pigments are linked to an amazingly broad-based range of health benefits.
For example, anthocyanins from Hibiscus spp. have historically been used
in remedies for liver dysfunction and hypertension; and bilberry (Vaccinium
myrtillus) anthocyanins have an anecdotal history of their use, for vision
disorders, microbial infections, and diverse other health disorders [1, 2]. But
while the use of anthocyanins for therapeutic purposes has long been
FARMACIA, 2009, Vol.LVII, 1
75
supported by both anecdotal and epidemiological evidence, it is only in
recent years that some of the specific, measurable pharmacological
properties of isolated anthocyanin pigments have been conclusively verified
by rigorously controlled in vitro, in vivo, or clinical research trials [3].
Extended researches have been carried on proving the anti-oxidant [4], antiinflammatory [5], anti-thrombotic [6], antimutagenic and antineoplasic
activities of anthocyanins [7, 8]; in addition, their role in improving the
immune system [9] and their activity against cardiovascular diseases has
been extensively studied [10, 11]. More over it was found that anthocyanins
prevent the diabetic complications including retinopathy [12, 13],
neuropathy, arteriopathy [14, 15] and participate in the maintenance of the
normal microcirculatory function including normal capillary filtration of
albumin and its uptake by the lymphatic system [16].
The quantitative analysis of anthocyanins in different plant extracts
have been reported by many authors. This article compares two different
methods, with their modifications performed on different extracts of Hibiscus
sabdariffa L. (family Malvaceae), in order to select the best procedure
followed in the determination of anthocyanins content in the chosen plant.
Materials and methods
All the experimental procedures were performed in order to
calculate the anthocyanin content in different Hibiscus sabdariffa extracts
calculated as cyanidin chloride or cyanidin-3-glucoside equivalents.
Plant Material
The plant samples were provided by the Department of
Pharmacognosy, Faculty of Pharmacy, University of Medicine and
Pharmacy, Bucharest.
Instruments
UV/Vis spectrophotometer Jasco V-530 (Jasco, Japan) with PC-HP
845x UV-Visible System (Jasco, Japan) and 1 cm quartz cells were used for
all absorbance measurements. PH –meter (Metrohm Swiss made/716 DMS
titrino); centrifuge (Universal 16, Hettich/ Zentrifugen); rotary evaporator
(Buchi, R-210/215)
Reagents and solutions
Ethyl acetate, iso-amyl alcohol, methanol, and ethanol were
provided by Fluka-chemika. For standard chemicals we used Quercetin
(Sigma, Germany). We also used during the study: concentrated
hydrochloric acid, potassium chloride 0.025 M (in distilled water) and
sodium acetate 0.4 M (in distilled water).
76
FARMACIA, 2009, Vol.LVII, 1
Preparation of Plant Extracts
Six grams of the dried Hibiscus sabdariffa calyces were extracted
separately with 150 mL of three different extracting solvents: methanol (E1), water (E-2) and acidified ethanol (40% ethanol with 1% HCl, E-3), using
reflux apparatus at 500C for one hour. The resulted filtrate was made off up
to 150 mL with the same extracting solvent (sol. A for each of the three
extracting solvents E-1, E-2 and E-3). Solution A was then divided in three
parts, as follows:
Part one [17]: 50 mL of sol. A was hydrolyzed with a solution of 4
N HCl in a ratio of (1:1) for 30 minutes. The resulted hydrolyzed solution
was first partitioned with ethyl acetate (15 mL x 3) to extract the flavonoids;
the ethyl acetat layer was washed with distilled water (10 mL x 3),
evaporated to dryness under reduced pressure and re-dissolved in ethanol to
a final volume of 25 mL. This solution is denoted as sol. 1. The remained
hydrolyzed layer was secondly partitioned with iso-amyl alcohol (15 mL. x
3) to extract the anthocyanidins. The iso-amyl alcohol layer was evaporated
to dryness and finally the residue was re-dissolved in 0.01 % HCl in ethanol.
This solution was denoted as sol. 2
Part two: 50 mL of sol. A was hydrolyzed with 2 N HCl in a ratio
of (1:1) for 8 hours. The resulted hydrolyzed solution was treated using the
same method mentioned in part one, resulting in sol. 3 (the red
anthocyanidins part).
Part three [18]: 50 mL of sol A was centrifuged at 2000 rpm for 10
min at 4 C. The collected aliquots were evaporated to dryness using a rotary
evaporator at 400C under vacuum condition, then re-dissolved in 0.01% HCl
in distilled water to a final volume of 25 mL. This solution is sol.4.
0
Quantitative analysis of anthocyanins
Two methods with their modifications are examined in this section
in order to select the best procedure followed for the determination of
anthocyanin content in different Hibiscus extracts.
The pH-Differential method [19]
To perform this method, two types of buffers should be prepared:

pH=1 buffer
This buffer is prepared by dissolving 1.86g of KCl in 980 mL
distilled water. The pH is adjusted to pH=1 by the drop wise addition of
HCl, then the volume is completed to 1L with distilled water.

pH=4.5 buffer
FARMACIA, 2009, Vol.LVII, 1
77
54.43g of sodium acetate is dissolved in 970 mL distilled water.
The pH is adjusted to 4.5 through the addition of HCl drop by drop, and
then the volume is completed to 1L using distilled water.
Procedure
1 mL of sol. 4 was leveled off to 10 mL with the buffer solution of
pH 1. A second 1 mL of sol. 4 was diluted to 10 mL using buffer solution of
pH 4.5. This procedure was performed on each of the three extracting
solvents. The flasks are left at room temperature for 15 min., and then the
absorbance was read at λ = 520 nm, and λ =700 nm. Distilled water was
used as a blank.
The anthocyanins are calculated as cyanidin-3-glucoside equivalents,
mg/L, using the equation:
A x MW x DF x 103
εxl
where A = (A520nm – A 700nm) pH 1.0 – (A 520nm – A 700nm)
pH 4.5; MW (molecular weight) = 449.2 g/mol for cyanidin-3-glucoside
(cyd-3-glu); DF = dilution factor (1:10); l = path length in cm; ε ( = 26900
molar extinction coefficient, in L · mol–1 · cm–1, for cyd-3-glu; and 103 =
factor for conversion from g to mg.
The Standard Curve method
This method depends on the preparation of cyanidin chloride
standard curve. The anthocyanins are calculated as cyanidin chloride
equivalents by means of the standard curve equation obtained.
Preparation of the standard curve
The first step here is the preparation of the standard cyanidin
chloride by dissolving 0.1g of quercetin, using 5 mL of conc. ethanol in a
100 mL volumetric flask. We added 2.5 g of Magnesium strips, and 16 mL
of conc. HCl (added step wise). The color will change from yellow to dark
violet-red color. The volume was completed to 100 mL using 50% ethanol;
this is sol B.
The second step was the preparation of the standard curve. We took
0.5 mL, 0.75 mL, 1 mL, 1.25 mL, 1.5 mL, 1.75 mL, 2 mL, 2.25 mL, 2.5 mL,
2.75 mL, and 3 mL of sol. B, and we put them separately in a series of 10 mL
volumetric flasks, completing the volumes to 10 mL with 50% ethanol. We
left the flasks for 10 minutes and read the absorbance at λ = 520 nm.
Measurement of samples
First: 5 mL of sol. 2 (for each of the three extracts) were diluted
with 0.01 % HCl in ethanol to 25 mL. Secondly: 5 mL of soln. 3 were
78
FARMACIA, 2009, Vol.LVII, 1
diluted with 0.01% HCl in ethanol to 25 mL. Finally, 2 mL of sol. 4 were
diluted with 2 % HCl in distilled water to a volume of 25 mL. All the flasks
were left for 10 min, and then the absorbance of each was read at λ = 520 nm.
The blanks used were 0.01%HCl in ethanol, and 2% HCl in water respectively.
Statistical analysis
The results of the spectrophotometric analysis were expressed as
Mean ± standard deviation (SDOM) upon three independent analyses.
Results and discussion
The pH Differential Method (AOAC Official Method 2005.02) is
based on the structural change of the anthocyanin chromophore between pH
1.0 and 4.5 and that “pure,” anthocyanins have very little or no absorbance
in pH 4.5 buffer (only the polymeric or degraded anthocyanins will absorb
at this pH), thus, the difference in absorbance at the λ-max 520 nm of the
pigment is proportional to the concentration of pigment. The reason for
measuring the absorbance at 700 nm is to correct for haze. Absorbance
should be measured at λ-max of the pigment solution, and the pigment
content should be calculated using the molecular weight (MW) and molar
extinction coefficient of the major anthocyanin in the sample extract
(cyanidin for Hibiscus flos).
Therefore the anthocyanin content of Hibiscus is customarily
calculated as the content of cyanidin-3-glucoside (MW = 449.2 g/mol) using
a molar extinction coefficient of 26900 L · mol–1 · cm–1.
The other method used in the anthocyanin quantitative analysis is
the use of cyanidin chloride standard curve (figure 1, and table I) with the
expression: Ab = A + B x Conc. (A= 0.0171; B= 0.0368); r2 = 0.9994 (fig. 1
and table I).
1 .3 0 5 0 4
1
Abs
0 .5
0
0
1 0
2 0
C o n c .[ % ]
Figure 1
Cyanidin chloride standard curve
3 0
3 2 .5
FARMACIA, 2009, Vol.LVII, 1
79
Table I
Cyanidin chloride calibration curve parameters
Calibration curve: Linear
Expression: Abs = A + B x Conc.( Factors: A= 0.0171; B = 0.0368
Coefficient r 2 = 0.9994
Standard blank = 0.0358
Sample No.
Conc. (mg /100 mL)
Absorbance
1
5.0000
0.2182
2
7.5000
0.2963
3
10.0000
0.3817
4
12.5000
0.4680
5
15.0000
0.5589
6
17.5000
0.6499
7
20.0000
0.7422
8
22.5000
0.8578
9
25.0000
0.9327
10
27.5000
1.0389
11
30.0000
1.1239
This method is applied on three types of sample preparation (sol. 2,
sol. 3, and sol. 4) to indicate the suitable sample preparation that should be
followed in performing this method.
Table II shows the results for both methods of analysis calculated
as mg per 100g dried plant, using the following formula:
(R x DF x V x 100)/W, where
R= result obtained from the standard curve equation; DF= dilution
factor; V= volume of stock solution; 100 = for 100 grams dried plant; W =
weight of plant used in experiment in (mg).
Extract
E-1
E-2
E-3
pH-Differential
method, Sol. 4
15.5 mg%± 0.208
34.8mg% ±0.045
66.58mg%±0.085
Table II
Representative results of anthocyanin quantitative analysis
Standard curve
Standard curve
Standard curve
method Sol. 4
method Sol. 2
method Sol. 3
46.86mg%±0.045 13.35mg%±0.031
2mg%0.021
58.97mg%±0.028 16.61mg%±0.012 10.15mg%±0.011
587.1mg%±0.035
98.13%±0.042
7mg%±0.032
It is obvious from the above data that the standard curve method
applied to the unhydrolyzed sample extract is the best compared to the other
procedures examined in this study as it measures both the monomeric and
polymeric anthocyanins (although the pH differential method, is widely
applied in researches concerning the quantitative analysis of anthocyanins)
The pH method does not give the exact quantity of anthocyanins, as it
highlights only the monomeric anthocyanins absorbed at pH=1. On the other
80
FARMACIA, 2009, Vol.LVII, 1
hand, the limited stability of the buffers prepared makes it difficult to run
this method. Application of the standard curve method on each of sol. 2
and sol. 4 also gave low results; this is attributed to the fact that
anthocyanins are light and heat sensitive [20] which is noticed for sol. 4, as
hydrolysis extended for 8 hours.
Conclusions
The present study revealed that the quantitative measurement of
anthocyanins determined using the standard curve method applied to the
unhydrolyzed plant extract is the best among the other examined
procedures. Further more it was also noted from the results that the acidified
ethanol is the best extracting solvent for anthocyanins.
References
Smith M., Marley K., Seigler D., Singletary K., Meline B. - Bioactive properties
of wild blueberry fruits; J. Food Sci., 2000, 65, 352-356
2. Wang C., Wang J., Lin W., Chu C., Chou F., Tseng T. - Protective effect of
Hibiscus anthocyanins against tert-butyl hydroperoxide-induced hepatic toxicity in
rats; Food Chem. Toxicol., 2000, 38(5), 411–416
3. Tsuda T., Horio F., Uchida K., Aoki H., Osawa T. - Dietary cyanidin 3-O-beta-Dglucoside-rich purple corn color prevents obesity and ameliorates hyperglycemia
in mice; J. Nutr., 2003, 133(7), 2125–2130
4. Wang H., Cao G., and Prior, R. L. - Oxygen Radical Absorbing Capacity of
Anthocyanins; J. Agric. Food Chem., 1997, 45, 304-309
5. Borissova P., et al. -Anti-inflammatory effects of flavonoids in the natural juice
from Aronia melanocarpa, rutin, and rutin-magnesium. Complex on an
experimental model of inflammation induced by histamine and serotonin; Acta.
Physiol. Pharmacol. Bulg., 1994, 20(1): 25-30
6. Morazzoni P., Magistretti MJ. - Activity of Myrtocyan®, an anthosyanoside
complex from Vaccinium myrtillus (VMA), on platelet aggregation and
adhesiveness; Fitoterapia, 1990, 61, 13–21
7. Glade M. J. - Food, nutrition, and the prevention of cancer: a global perspective;
American Institute for Cancer Research/World Cancer Research Fund, American
Institute for Cancer Research, Nutrition, 1999, 15, 523-526
8. Shih P. H., Yeh, C. T., and Yen G. C. - Effects of anthocyanidin on the inhibition
of proliferation and induction of apoptosis in human gastric adenocarcinoma cells;
Food Chem. Toxicol., 2005, 43, 1557-1566
9. Bub A., Watzl B., Blockhaus M., Briviba K., Liegibel U., Muller H., Pool-Zobel
B. L., and Rechkemmer G. - Fruit juice consumption modulates antioxidative
status, immune status and DNA damage; J. Nutr. Biochem., 2003, 14, 90-98
10. Tsuda T. - Dietary cyanidin 3-0-beta-D-glucoside increases ex vivo oxidative
resistance of serum in rats; Lipids, 1998, 33(6), 583-8
11. Andriambeloson E. - Natural dietary polyphenolic compounds cause endotheliumdependent vasorelaxation in rat thoracic aorta; J. Nutr., 1998, 128 (12), 2324-33
1.
FARMACIA, 2009, Vol.LVII, 1
81
12. Scharrer A., Ober M. - Anthocyanosides in the treatment of retinopathies; Klin
Monatabl Augenheilkd, 1981, 178, 386-389
13. Perossini M., et al. - Diabetic and hypertensive retinopathy therapy with
Vaccinium myrtillus anthocyanosides (Tegens): Double-blind placebo controlled
clinical trial; Ann Ophtalmol. Clin. Ocul., 1987, 113, 1173
14. Cantuti-Castelvetri I., Shukitt-Hale B., and Joseph J. A. - Neurobehavioral
aspects of antioxidants in aging; Int. J. Dev. Neurosci., 2000,18, 367-381
15. Andres-Lacueva C., Shukitt-Hale B., Galli R. L., Jauregui O., Lamuela-Raventos
R. M., and Joseph J. A.- Anthocyanins in aged blueberry-fed rats are found
centrally and may enhance memory; Nutr. Neurosci., 2005, 8. 111-120
16. Cohen-Boulakia F. - In vivo sequential study of skeletal muscle capillary
permeability in diabetic rats: effect of anthocyanosides; Metabolism 2000, 49(7),
880-5
17. Harborne JB -Photochemical Methods: A Guide to Modern Techniques of Plant
Analysis; Ed Chapman and Hall, London 1998; 112-116
18. Pu Jing M.S. - Purple corn anthocyanins: chemical structure, chemo-protective
activity and structure/function relationships. PhD thesis, The Ohio State
University; USA, 2006; p. 96
19. Jungmin Lee et al. - Determination of Total Monomeric Anthocyanin Pigment
Content of Fruit Juices, Beverages, Natural Colorants, and Wines by the pH
Differential Method: Collaborative Study- J. of AOAC International 2005, 88 (5)
16-19
20. Wrolstad R. E., Durst R. W., Giusti M. M., and Rodriguez-Saona L. E. - Analysis
of anthocyanins in nutraceuticals, Quality Management of Nutraceuticals, C. T.
Ho, and Q. Y. Zheng, eds.Washington D.C.: Am. Chem. Soc. 2002, 4, 42-62
21. Suhad S. Humadi, Viorica Istudor, Quantitative analysis of bio- active compound
in Hibiscus sabdariffa L. extracts. I. Quantitative analysis of flavonoids,
Farmacia, 2008, 56, 699-707
Manuscript received: 02.09.2008