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QUANTITATIVE ANALYSIS OF BIO- ACTIVE
COMPOUND IN HIBISCUS SABDARIFFA L.
EXTRACTS. NOTE I
QUANTITATIVE ANALYSIS OF FLAVONOIDS
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
In the phytochemical extraction researches there has been an increasing demand
to find a simple, rapid, and precise method for the quantitative analysis of flavonoids
(flavones, anthocyanins, and tannins) in plant extracts. This paper describes the quantitative
determination of flavonoids (flavonols) calculated as quercetin equivalent in different
Hibiscus sabdariffa extracts analyzed using different spectrophotometric methods: ChristMüllers, Chang et al and some modified methods, to obtain extracts with the highest
flavonoid content showing hypoglycemic (proved by experiments performed on aloxan
induced diabetic rats) and anti-oxidant activities. The obtained results indicated that the
flavonoid percent determined using the Christ-Muller's method showed the lowest value
among all other methods, while the highest percentual value was analyzed via Chang et al
method used for the determination of both the free aglycone obtained by hydrolysis with 4
N HCl for 30 minutes and glycosylated form of flavonols.
Rezumat
În cercetarea fitochimică a extractelor, există o cerere din ce în ce mai mare de
metode de analiză cantitativă a flavonoidelor (flavone, antociani, taninuri) care să fie
simple, rapide, selective şi precise. În această lucrare autorii abordează determinarea
cantitativă a flavonozidelor din extractele de Hibiscus sabdariffa L. (Malvaceae) utilizând
mai multe metode spectrofotometrice: Christ-Müllers, Chang şi colab., şi câteva variante
ale acesteia. Obţinerea acestor extracte are ca scop evidenţierea fracţiunii cu cel mai bun
potenţial hipoglicemiant (verificat prin experiment pe şobolani cu diabet zaharat indus
experimental prin aloxan) şi antioxidant. Rezultatele obţinute arată că procentual,
conţinutul în flavone determinat prin metoda Christ-Müllers conduce la cele mai mici
valori, în timp ce prin metoda Chang şi colab. se obţin cele mai mari valori.
În concluzie, metoda Chang şi colab. este cea mai simplă, rapidă, şi precisă
pentru determinarea conţinutului în flavonoli (liberi şi glicozidaţi), precum şi extracţia cu
metanol şi hidroliza heterozidelor prin refluxare cu HCl 4 N, timp de 30 min.
 Flavonoids
 analytical methods
 Hibiscus sabdariffa
 aluminium chloride
 hydrolysis
 flavonol
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INTRODUCTION
There has been considerable interest in the flavonoid content of
foods and plants since the early 1980s when the studies of Steinmetz and
Potter [1] demonstrated a relationship between a diet rich in fruits and
vegetables and a reduced risk for chronic diseases. Because reduced risk did
not correlate with traditional nutrients, attention has focused on many nonnutrient, potentially bioactive compounds, of which the flavonoids
constitute one family [2].
Flavonoids are naturally-occurring polyphenolic compounds with a
C6-C3-C6 backbone. This group of plant pigments which are found in
fruits, vegetables, grains, bark, roots, stems, flowers, tea, and wine can be
chemically subdivided into six structural categories: flavones, flavonols,
flavanones, flavanonols, flavan-3-ols (catechins), and anthocyanidins. These
compounds (aglycones) are commonly glycosylated (at one or more sites
with a variety of sugars) and may also be alkoxylated or esterified. As a
result, over 5000 different flavonoids have been identified in plant materials
[3-6].
The methods that have been reported for the determination of
flavonoids are based on the aluminium chloride complex formation, which
is one of the most commonly used analytical procedures applied to the
flavonoid content determination in various plants [5-8]. In general,
aluminum chloride forms acid stable complexes with the C-4 keto group and
either the C-3 or the C-5 hydroxyl group of flavones and flavonols. In
addition, aluminum chloride forms acid labile complexes with the orthodihydroxyl groups in the A or B ring of flavonoids. The analytical methods
were used either to determine the glycosylated or the nonglycosylated
flavonoids [8, 9].
Quercetin
Quercetin-3-rutinoside
Quercetin-3-rhamnose (Quercitrin)
Figure 1
The free aglycone and the glycosylated form of quercetin
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The performed hydrolysis in order to produce aglycones, served
multiple purposes: it reduced the number of compounds and made
chromatographic separation easier to achieve; it permitted quantification of
flavonoids because standards for a large number of the glycosylated
flavonoids are not available; and it provided consistent data with the earlier
view that flavonoids were absorbed only in the intestine as aglycones.
Unfortunately, hydrolysis also leads to degradation of the aglycones, thus
giving more importance to those methods based on the glycosylated
flavonoid [9].
Literature survey revealed the presence of two classes of
flavonoids in the extracts of Hibiscus sabdariffa: flavonols (gossypetin), and
the anthocyanins [10,11].
The present study reports some analytical methods used to
determine the flavonol content in different Hibiscus sabdariffa extracts and
the selection of the best procedure to be followed in the quantitative analysis
of flavonoids in the mentioned plant.
MATERIALS AND METHODS
All the experimental procedures were performed to establish the
content of flavonoids in different Hibiscus sabdariffa extracts, calculated as
quercetin equivalent.
Plant Material
The plant samples were provided from the University of Medicine
and Pharmacy “Carol Davila”, Faculty of Pharmacy, Department of
Pharmacognosy, Romania.
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.
Reagents and Solutions
Analytical
substances:
acetone,
hydrochloric
acid,
hexamethylenetetramine, ethyl acetate, aluminium chloride hexahydrate,
sodium citrate, potassium acetate, methanol, ethanol, acetic acid, provided
by Fluka-Chemika.
Standard substance: Quercetin (Sigma, Germany).
Preparation of Plant Extracts
Plant extracts were prepared using three different extracting
solvents: E1-extracted with methanol, E2-extracted with water and E3extracted with 40% ethanol containing 1%HCl. The powdered plant material
was extracted with each of the above solvents using reflux apparatus at 50 0C
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for 1 hour, and then filtrates were completed to 25 ml by the same extracting
solvent.
Analysis of the flavonoid content in extracts.
Determination of flavonoids according to the Christ-Mullers
method [12-14].
This method is applied to the aglycone part; therefore the content
of total flavonoid is calculated as quercetin type.
Procedure: 20 mL of acetone, 2 mL of 25% HCl and 1 mL of 0.5%
hexamethylenetetramine were added to 25 ml extract (corresponding to 2g
of plant material) and refluxed at 560C for 30 minutes. The extract was
filtered and re-extracted twice with 20 ml acetone for 10 min. After cooling
and filtration, the extract was made up to 100.0 mL with acetone (basic
sample solution, BSS). 20 mL of BSS was mixed with 20 mL of water and
then extracted with ethyl acetate (first with 15 mL and then three times with
10 mL). Ethyl acetate extracts were rinsed twice with water then filtered and
made up to 50.0 mL with ethyl acetate (S1). In 10 mL of S1 0.5 mL of 0.5%
solution of sodium citrate and 2 mL of AlCl3 (prepared by dissolving 2 g of
AlCl3 in 100 mL of 5% acetic acid in methanol) were added and then made
up to 25.0 mL with 5% methanolic solution of acetic acid (sample solution,
SS). The same procedure was performed with blank sample solution but
without AlCl3. After 45 minutes, yellow solutions were filtered and
absorbance at 425 nm was measured. The content of total flavonoids was
evaluated upon three independent analyses. The yield was calculated as
quercetin percent using the following expression:
g % = A × 0.772 / b,
where A is absorbance and b represents the mass of dry herbal material in
grams.
Determination of flavonoids by Chang et al. method [15,16)]
Flavonols in Hibiscus sabdariffa extracts were expressed as
quercetin equivalent. Quercetin (Sigma, Germany) was used to perform the
calibration curve (standard solutions of 6.25, 12.5, 25.0, 50.0, 80.0 and
100.0 µg mL-1 in 80 % ethanol (V/V). Sample extracts (1g plant material in
25 ml extract) were all evaporated to dryness and re-dissolved in 80 %
ethanol to be ready for the analytical test.
1 mL of a sample (ethanolic solutions Hibiscus) was mixed with 3
mL 95 % ethanol (V/V), 0.2 mL 10 % aluminum chloride (m/V), 0.2 mL of 1
mol L–1 potassium acetate and 5.6 mL water. A volume of 10 % (m/V)
aluminum chloride was substituted by the same volume of distilled water in
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703
blank. After incubation at room temperature for 30 minutes, the absorbance
of the reaction mixture was measured at 415 nm.
Chang et al. modified methods
Several modifications were made to the Chang et al. method in
order to clarify the accuracy of this method. These modifications were as
follows:
Modified method A
The modification is applied to the sample preparation using the
same standard quercetin curve. Each extract was hydrolyzed with 2 N HCl
(1:1) for 8 hours, then the flavonoids were extracted with three quantities of
ethyl acetate (each of 15 mL), after which the ethyl acetate layer was
evaporated to dryness under reduced pressure, then the residue was redissolved using 80 % ethanol to a volume of 25 mL. This solution was used
for the analytical procedure. The flavonoid content was expressed as
quercetin equivalent.
Modified method B
We performem the same modification as in method A except that
the hydrolysis of flavonoids is carried out using 4 N HCl conc. for 30
minutes. The flavonoid content was also expressed as quercetin equivalent.
Verification of the Chang et al method
Procedure: 0.1g of quercetin was dissolved in 80% ethanol to a
volume of 100 mL in a volumetric flask (this is sol. A). 0.1 g of quercetin
were dissolved in 5 mL of concentrated ethanol in a 100 mL volumetric flask,
then we added 2.5 g of Magnesium strips and 16 mL of concentrated HCl,
each of which was added step wise. The color changed from yellow to dark
violet-red color. The volume was then completed to 100 mL using 80%
ethanol (this is sol. B). The verification experiment was performed using the
original Chang et al, method (without modifications) using the same standard
curve equation. In three series of 10 mL volumetric flasks we put 0.75,1, and
1.25 mL of soln A in the first series; while in the second series we put a
mixing quantities of (1mL soln A + 1mL soln B),( 0.75 soln A + 1.25 mL
soln B), and finally (1.25 soln A + 0.75 mL soln B) , the third series contained
0.75, 1, and 1.25 mL of soln B. After incubation at room temperature for 30
minutes, the absorbance of the reaction mixture was measured at 415 nm.
Aluminum chloride was excluded from the blank series. (Note that the
volume of the sample + 95 % ethanol should be collectively 4 mL).
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Statistical analysis
The results of the spectrophotometric analysis were expressed as
the Mean ± SD (SDOM) upon three independent analyses.
RESULTS AND DISCUSSION
To perform the calculations of total flavonols content in the studied
plant using the Chang et al method, a standard curve is needed which is
obtained from a series of different quercetin concentrations.
0 .1 0 7 1 2
0 .1
Abs
0 .0 5
0
0
0 .5
1
1 . 51 . 6 3 7 5
C o n c .[ % ]
Figure 1
Quercetin standard curve
Table I
Quercetin calibration curve
Calibration curve: Linear
Expression: Abs = A + B* Conc.( Factors: A= 0.0007; B = 0.0603
Coefficient r 2 = 0.9997)
Standard blank = 0.0460
Sample No.
Conc. (% mg /100 mL)
Absorbances
1
0.1250
0.0080
2
0.2500
0.0158
3
0.5000
0.0315
4
0.7500
0.0447
5
1.0000
0.0618
6
1.2500
0.0765
7
1.5000
0.0906
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The final test sample concentration using the Chang et al method is
then obtained using the following formula:
(R * DF * V *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.
The comparison of the results obtained from the methods discussed
above is made on gram bases i.e. the flavonoid content in the extracted
solutions is calculated as g % (g Quercetin per100g of dried plant material),
as represented in the table II.
Table II
Flavonoid percent content analyzed by different
spectrophotometric methods in three extracting solvents
Ext.
No.
Measurement by
Chang et al
method
0.068 g % ± 0.02
0.049g % ± .065
Modified method
A
Modified method
B
E-1
E-2
Measurement
by
Christ-Mullers
method
0.029 g % ± 0.034
0.024 g % ± 0.034
0.03 g % ±0.023
0.018 g % ±0.012
0.05 g % ±0.020
0.023 g % ±0.016
E-3
0.02 g % ± 0.035
0.059 g % ± 0.03
0.024 g % ±0.015
0.035 g % ±0.019
The results in table I ensure the accuracy, less degradation, and the
highest percent flavonoid value as determined by Chang et al. method.
Indeed this method is the sum of two individual colorimetric methods:
method with AlCl3 (for the determination of flavones and flavonols) and
method with 2,4-dinitrophenylhydrazine (for the determination of
flavanones calculated as naringenin), but as the literature survey revealed
the absence of the flavanones, the second part of this method was omitted.
The flavonoid content of the extracts was calculated in terms of quercetin
equivalent using the standard curve equation (y = 0.0603 x + 0.0007, r2 =
0.9997).This equation was also applied for the modified methods A and B.
The results shown above proved that the hydrolysis of flavonoid glycosides
using method (B) is better for obtaining higher aglycone percent and
reducing the degradation and loss during hydrolysis. On the other hand and
as a result of the presence of anthocyanins in the studied plant, a verification
step to the Chang et al. method was performed on standard solutions of each
of the flavonoids represented by quercetin (sol. A), and the anthocyanins
represented by cyanidin (sol. B). The results of each of the absorbance
values and the calculated concentrations are shown in table II, calculated
(directly from the equation) as quercetin mg per 100 mL solution.
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Table II
The verification procedure results
Test solution T.S
Absorbance
Abs.
T.S (1.25 mL sol. A)
T.S(1.25 mL
sol.A+0.75 mL sol. B)
Differences of the
two T.S
T.S (1 mL sol. A)
T.S(1mL sol.A+1 mL
sol. B)
Differences of the
two T.S
T.S (0.75 mL sol. A)
T.S (0.75mL sol. A+
1.25 mL sol. B)
Differences of the
two T.S
T.S (for all the third
series of sol. B)
The mean of the
differences of series 1
and 2 T.S
0.012
0.010
0.009
0.008
0.005
0.004
(-) negative
Concentration
Conc.
mg/100ml
solution
0.18739
0.15422
Abs.
Differences
Conc.
Differences
0.002
0.033
0.001
0.016
0.001
0.016
-------
-------
0.001
0.021
0.13764
0.12106
0.07131
0.05472
(-) negative
As noted from the above table, the other constituents as
anthocyanins present in the plant extract have a little interference on the
actual concentration of flavonoids that could be considered as a negligible
effect. In addition, no color interference was observed.
CONCLUSION
The present study revealed that the flavonoid content determined
by the method reported by Chang et al is a simple, rapid, and economic
procedure, which gave the best results among all other method cited in this
article. In addition the presence of other constituents in the plant extracts as
anthocyanins do not greatly interfere with the obtained results. More over it
was found that the hydrolysis using 4N HCl for 30 minutes is the ideal
method for the aglycone release.
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707
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