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Determination of lawsone in henna powders by


high performance thin layer chromatography

Article in Journal of Separation Science · December 2007


DOI: 10.1002/jssc.200700223 · Source: PubMed

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Nagwa Al-shaer Jihan Badr


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Maha A Aboul-Ela Yousry Mahmoud Gohar


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J. Sep. Sci. 2007, 30, 3311 – 3315 N. S. El-Shaer et al. 3311

Nagwa S. El-Shaer1 Original Paper


Jihan M. Badr2
Maha A. Aboul-Ela1
Yousry M. Gohar3 Determination of lawsone in henna powders by high
1
Department of Pharmacognosy,
performance thin layer chromatography
Faculty of Pharmacy, University
of Alexandria, Alexandria, Egypt The lawsone content has been evaluated quantitatively in eight commercial henna
2
Department of Pharmacognosy, powders and two collected henna leaves. The phenolic, chloroform-soluble fraction
Faculty of Pharmacy, University of the majority of the examined samples showed the presence of lawsone and two
of Suez Canal, Ismailia, Egypt other pigments. Here we aimed to optimize high performance thin layer chroma-
3
Faculty of Science, University of tography for the determination of lawsone. Upon using the optimized method the
Alexandria, Alexandria, Egypt
examined samples showed considerable variation in lawsone concentration ranging
from 0.004 up to 0.608 wt%, indicating that some samples were almost devoid of
lawsone. Some of these products were subjected to preliminary in vivo toxicity stud-
ies.
Keywords: Henna powders / HPTLC / In vivo toxicity studies / Lawsone / Lawsonia alba /
Received: May 22, 2007; revised: August 1, 2007; accepted: August 1, 2007
DOI 10.1002/jssc.200700223

1 Introduction
The use of hair dyes has dramatically increased in indus-
trialized countries during recent decades. Henna leaves
(Lawsonia alba, Family Lythraceae) are widely used as a cos-
metic dye for both hair and skin. One study claimed that
application of henna can induce a severe hemolytic ane-
mia and this could contribute to unexplained neonatal
hyperbilirubinemia in countries where the ceremonial Figure 1. Lawsone.
use of henna is widespread [1]. Lawsone, 2-hydroxy-1,4-
naphthoquinone (Fig. 1), the main active ingredient of
henna [2], was the subject of several studies that attrib- Recent attempts at accurate determination of lawsone
uted the hemolytic action to its redox effect [3 – 6]. used HPLC [10 – 12]. Here we aim to optimize the use of
Although certain studies reported some cases of renal the high performance thin layer chromatography
damage and bladder cancer, however other prospective (HPTLC) as a very simple and accurate technique for
investigations on large populations found no or negative quantitative determination of lawsone in henna pow-
correlations for cancer of the bladder or other cancers, ders. Moreover, representative examples of the tested
especially for consumers or professional exposure to law- henna products have been subjected to an in vivo study to
sone [7, 8]. Nowadays, the increase in number of henna monitor their toxicities.
powders on the market and the wide variation of their
quality necessitate clear documentation which can guar-
antee the content of the main active ingredient. A pre- 2 Experimental
vious method used for estimation of lawsone was based
on colorimetry after its recovery from TLC plates [9].
2.1 Instrumentation

Correspondence: Professor Nagwa S. EL-Shaer, Department of A Camag (Wilmington, NC) Linomat IV sample applicator
Pharmacognosy, Faculty of Pharmacy, University of Alexandria, was used to dispense aliquots of the standard stock solu-
Alexandria 21521, Egypt tion and the prepared samples. The plates were saturated
E-mail: gihan96@hotmail.com in a twin trough chamber; slit dimension settings of
Fax: +20-3-4873273
length 6 and width 0.1 mm, a monochromator band
Abbreviations: ALT, alanine aminotransferase; AST, aspartate width of 20 nm, and a scanning rate of 10 mm/s were
aminotransferase; HPTLC, high performance thin layer chroma- used. Zones were quantified by using a Camag TLC Scan-
tography; TDA, toluenediamine ner III densitometer controlled by CATS version 4.X soft-

i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com


3312 N. S. El-Shaer et al. J. Sep. Sci. 2007, 30, 3311 – 3315

ware in absorption mode using a deuterium source and a


filter with a wavelength of 334 nm.

2.2 Chemicals
Lawsone standard was obtained from Aldrich (Sigma –
Aldrich, Germany). Pre-coated silica gel plates (Merck 60
F254 0.25 mm) were used in HPTLC determinations. All
the solvents used were of chromatographic grade (BDH,
Analar, UK).

2.3 Plant material


Two samples of henna leaves were collected in June 2005 Figure 2. HPTLC densitogram of 0.3 (lg/spot) injection of
from El-Hannauville, west of Alexandria (sample 1), and lawsone at k 334 nm.
from Aswan in southern Egypt (sample 2). The two sam-
ples were dried and finely powdered. The identity of the
2.5 Sample preparation
plant was confirmed by comparison with a voucher sam-
ple kept in the Faculty of Science, University of Alexan- Each sample of powdered leaf of henna (1 g) was mixed
dria, Egypt. Analysis was performed on these two pow- with 50 mL 5% Na2CO3 aqueous solution and left for one
ders in addition to eight commercial brands of henna hour. Each mixture was filtered into a 100-mL volumetric
powders (samples 3 – 10) which were obtained from local flask; the original flask and the residue were washed
markets (pharmacies and authorized herbalists). Sam- with successive portions of distilled water (3610 mL)
ples 6 and 7 were selected as containing other additives until extraction was complete. The filtrate and washings
together with henna powders, including barium perox- were adjusted to volume with distilled water. An aliquot
ide, citric acid, and toluenediamine (TDA) (sample 6) and of filtrate (20 mL) was transferred quantitatively into a
p-phenylenediamine (sample 7), with the aim of verifying separatory funnel (100 mL), HCl was added to neutrality.
the efficiency of the method for determination of law- The colored phenolic compounds were extracted with
sone in the presence of other components. successive portions of CHCl3 (3610 mL) and the CHCl3
solutions were collected in a distilling flask. The CHCl3
was distilled off under reduced pressure and the residue
2.4 Standard solution and calibration
was dissolved in MeOH and transferred quantitatively to
Stock solution of lawsone was prepared by accurately a volumetric flask (10 mL), then the solution was made
weighing 100 mg of lawsone, dissolving it in MeOH, up to volume with MeOH (sample solution). A 3-lL ali-
quantitatively transferring the solution into a 100-mL quot of each sample and of the standard stock solution
volumetric flask, and making up the volume with MeOH. (0.3 lg/spot) was spotted in triplicate on the plates, and
The solution was kept away from light in a tightly stop- then developed using CHCl3:MeOH (8.5:1.5) under the
pered volumetric flask in a refrigerator. The calibration same conditions as previously described in Section 2.4.
curve was constructed according to the requirements of Zones were scanned at k 334 nm, this wavelength corre-
the International Committee on Harmonization (ICH) sponding to maximum sensitivity among the different
guidelines [13, 14]. Six different concentrations of the wavelengths selected for trials (ranging from 250 to
standard solutions were used, covering the range of 0.1 – 510 nm); moreover, k 334 nm was reported as the kmax of
1 lg/mL. A 1-lL volume of each concentration was lawsone [2]. The peak areas of lawsone were recorded
applied in triplicate (6 mm band length) to a plate (Rf = 0.4).
(2067 cm); the distance from the side edge of the plate
was 1.5 cm and from the bottom of the plate was 1.2 cm.
2.6 Animals and experimental design in toxicity
The application rate was 15 lL/s, and the bands were
studies
developed to a distance of 6 cm using CHCl3 – MeOH
(8.5:1.5). The development time was 10 min, the plates The animals used were 30 adult male albino rats having
were air-dried for 15 min and zones were scanned at initial weights of 100 – 120 g, which were purchased
k 334 nm. Peak areas were recorded for the tracks and a from the breeding unit of the Egyptian Organization for
calibration curve was established for lawsone by plotting Biological Products and Vaccines (Cairo). The samples
the peak area (y axis) against the amount of lawsone in which showed the lowest amounts of lawsone (6, 7, and
lg (x axis). Figure 2 illustrates the chromatogram 10) and a reference (sample 2) were subjected to a toxicity
recorded for a 0.3-lg/mL solution of lawsone at k 334 nm. study. A 10-g portion of each of the four tested henna

i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com


J. Sep. Sci. 2007, 30, 3311 – 3315 Other Techniques 3313

powders was extracted with 90 mL of polyethylene gly-


col. Each group was given an oral dose of 1 mL of one of
the four tested samples. Oral administration was contin-
ued for 21 days for all groups, in addition to a control
group where the rats were treated only with the solvent
polyethylene glycol. After 21 days, blood samples were
obtained from the orbital sinus of the animals and cen-
trifuged to obtain their sera which were kept at – 808C
prior to performing the assays. Investigations of the liver
and kidney functions as well as determination of the tri-
glycerides concentrations were performed as follows:
aspartate aminotransferase (AST) and alanine amino-
transferase (ALT) were determined using a Diamond
Diagnostics colorimetric kit on serum samples [15], crea- Figure 3. HPTLC densitogram showing the separation of
tinine was determined using a Human Creatinine Liqui- lawsone from other phenolic, chloroform soluble components
of Lawsonia alba at k 334 nm.
Color kit applying the Jaffe reaction [16], while uric acid
was estimated using a Uric acid – LS Uricase PAP kit [17,
18]. Values of the assays were expressed as mean l S.E. Table 1. Precision of HPTLC method used for estimation of
and statistically analyzed using Student’s t-test. lawsone.

Experiment Concentration Mean l SD RSD


number (lg/spot) (%)
3 Results and discussion
1 0.1067 0.1069 l 0.001882 1.76
3.1 Validation of the method 0.1041
0.1100
3.1.1 Linearity 2 0.2047 0.2062 l 0.003107 1.50
The presented method exhibited strict linearity between 0.2098
the area under the peak and concentration over the 0.2042
3 0.3010 0.3021 l 0.006143 2.03
range from 0.1 – 1 lg/spot with a correlation coefficient 0.3087
(r) of 0.99974. The linear regression equation was found 0.2966
to be y = 400.82 + 10915.9428 x, where y is the spot area 4 0.3938 0.4004 l 0.005801 1.44
and x is the concentration in lg/spot. 0.4023
0.4050
3.1.2 Selectivity 5 0.4955 0.5032 l 0.008426 1.67
0.5122
After sample preparation according to Section 2.5, selec- 0.5019
tive baseline separation was obtained and lawsone found 6 0.5958 0.6011 l 0.007232 1.20
to be well separated from the other two pigments. Hence 0.5981
0.6093
lawsone could be easily estimated without any interfer-
ence. The scan densitogram obtained from a representa-
tive sample is presented in Fig. 3.
of standard lawsone solution (0.3 mg/mL ) to give mix-
3.1.3 Precision (repeatability) ture 1 and another 5 mL of the same sample was mixed
The precision of the method was assessed by determina- with 5 mL of standard lawsone solution (0.6 mg/mL) to
tion of six different concentrations of lawsone, each give mixture 2. The original sample and the two fortified
applied as 1 lL in triplicate (Table 1). Repeated analysis ones were analyzed on the same plate by application of
of a homogeneous sample (0.109 mg/mL) was performed 1 lL, each in triplicate (Table 2). The low RSD values indi-
using the same equipment, analytical procedure, and cated suitability of the method for lawsone analysis in
plate. The % RSD was found to be 1.72, indicating the pre- henna powder.
cision of the method. Accordingly, the suggested technique represents a sim-
ple, accurate, and precise method for the determination
3.1.4 Accuracy of lawsone without any interference from other compo-
The accuracy was validated using the standard addition nents of the examined products. Considerable variation
analysis method by application of the proposed method of lawsone content was observed between the two refer-
to three sample solutions, one of them being unfortified ence henna leaves and the eight commercial henna pow-
and the other two fortified. A 5-mL volume of a sample of ders (Table 3, Fig. 4). The reference sample collected from
known concentration 0.109 mg/mL was mixed with 5 mL Aswan (sample 2) showed much higher content (0.545%)

i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com


3314 N. S. El-Shaer et al. J. Sep. Sci. 2007, 30, 3311 – 3315

Table 2. Results of the standard addition experiment by the


proposed method.

Experiment Concentration (lg/spot)


number
Expected Found l SD Recovery RSD%
(%)

1 (sample) 0.1090 0.1077 l 0.001204 98.8 1.12


2 (mixture 1) 0.2045 0.1924 l 0.001501 94.1 0.78
3 (mixture 2) 0.3545 0.3503 l 0.005231 98.8 1.49

Table 3. Concentration of lawsone in different henna sam-


ples.
Figure 4. Variation of concentration of lawsone in the ten
Sample numbera) Concentration of lawsoneb) examined samples (using HPTLC).
(wt% of sample)c) l SD

1 0.173 l 0.008301
2 0.545 l 0.005186
between 35 and 458C. The plant does not thrive where
3 0.306 l 0.004771 the minimum temperatures are below 118C, and temper-
4 0.608 l 0.001307 atures below 58C will kill the henna plant [9]. The com-
5 0.607 l 0.005612 mercial samples also showed varying concentrations of
6 0.039 l 0.006546 lawsone. Samples 4, 5, and 8 showed higher concentra-
7 0.019 l 0.007312
8 0.598 l 0.005781 tions than the reference samples (0.608, 0.607, and
9 0.449 l 0.005772 0.598 wt% respectively). Concentrations of lawsone in
10 0.004 l 0.004802 samples 3 and 9 were 0.306 and 0.449, respectively,
a)
whereas samples 6 and 7 showed much lower contents of
Samples 1 and 2: reference henna powders. Samples 3 –
lawsone (0.039 and 0.019 wt%, respectively). The lowest
10: commercial henna powders.
b) content of lawsone was found in sample 10 (0.004%),
Results are average of three readings.
c)
wt%: g of lawsone/100 g of dry henna powder. which could be attributed to collection of the sample at
an inappropriate time or place, as previously mentioned.
Surprisingly, samples 6 and 7 showed the darkest color
than that collected from North-West Egypt (sample 1), during extraction, but most of this color was retained in
which contained 0.173% of lawsone. This is in agreement the aqueous solution rather than being extracted into
with a previous study which reported that the lawsone the CHCl3, indicating that these henna products are
content varies according to the time and area of collec- almost devoid of the main coloring ingredient of henna
tion where it is assumed to attain its highest concentra- leaves and, instead, synthetic chemical additives impart
tion in henna grown in tropical regions at temperatures the color of the product

Table 4. Effect of daily oral administration of sample 2 (extract of henna leaves), samples 6, 7, 10 (commercial henna powders
showing low concentration of lawsone) and control (polyethylene glycol) on liver function, kidney function and triglyceride concen-
tration in rats, expressed as mean l SE.

Group Liver function Kidney function Triglyceride Number of


concentration dead rats
(mg/dL) (out of 6)
n ALT (l/L) AST (l/L) Creatinine Uric acid
(mg/dL) (mg/dL)

Group 1 6 26.0 l 0.9 22.5 l 1.2 1.0 l 0.2 5.5 l 0.5 40.58 l 0.8 0
Control
Group 2 6 84.8 l 2.3a) 76.6 l 2.3a) 6.4 l 0.40a) 17.5 l 0.5a) 44.8 l 3.0 0
Sample 2
Group 3 6 102.0 l 3.5a) 79.5 l 4.0a) 6.0 l 0.4a) 9.3 l 0.60a) 78.0 l 1.8a) 6
Sample 6
Group 4 6 35.7 l 3.0a) 80.0 l 2.8a) 4.7 l 0.2a) 12.5 l 0.80a) 43.2 l 2.9 5
Sample 7
a) a) a) a)
Group 5 6 50.2 l 3.3 80.6 l 2.6 8.8 l 0.3 13.3 l 0.60 43.5 l 1.3 1
Sample 10
a)
Statistically significant from the control group at p a0.05.

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J. Sep. Sci. 2007, 30, 3311 – 3315 Other Techniques 3315

3.2 Toxicity study [2] Thomson, R. H., Naturally Occurring Quinones, Academic Press, Lon-
don, New York 1971.
Results of toxicity studies demonstrated that all the four [3] Osman, A. M., J. Appl. Toxicol. 2003, 23, 209 – 212.
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samples (Table 4). It was found that death occurred in 532.
only 1 rat out of the 6 in the case of sample 10, while sam- [6] Millan, D. C., Sarvate, S. D., Oatis, J. E., Jollow, D. J., Toxicol. Sci.
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rats, respectively. Although previous studies stated that [7] Gerhard, J. N., Rolf, F., Florence, B., Herve, T., Food Chem. Toxicol.
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lawsone may cause hemolytic oxidation when taken
[8] David, K., Daniel, M., Mut. Res. 2003, 537, 183 – 199.
orally, yet this could be fatal only in case of people suffer-
[9] Karawya, M. S., Abdel Wahab, S. M., Zaki, A. Y., Lloydia 1969, 32,
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[10] Scarpi, C., Ninci, F., Centini, M., Anselmi, C., J. Chromatogr. A
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[23] White, J. M., Kullavanijaya, P., Duangdeeden, I., Zazzeroni, R.,
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[26] Huang, Y. C., Hung, W. C., Kang, W. Y., Chen, W. T., Chai, C. Y.,
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[27] Chen, S. C., Chen, C. H., Chern, C. L., Hsu, L. S., Huang, Y. C.,
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