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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-
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).
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 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.
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.
tested samples caused both hepatic and renal disorders [4] Munday, R., Smith, B. L., Fowke, E. A., J. Appl. Toxicol. 1991, 11,
in varying degrees upon oral administration, while tri- 85 – 90.
glycerides values were not significantly altered in all [5] Kok, A. N., Ertekin, M. V., Avci, B., Int. J. Clinl. Pract. 2004, 58, 530 –
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.
2004, 82, 647 – 655.
ples 6 and 7 recorded a mortality of 6 and 5 (out of 6)
rats, respectively. Although previous studies stated that [7] Gerhard, J. N., Rolf, F., Florence, B., Herve, T., Food Chem. Toxicol.
2004, 42, 517 – 523.
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,
ing from G6PD deficiency [19]. Accordingly, the fatal tox- 76 – 78.
icity noted for some animals may be attributable to the
[10] Scarpi, C., Ninci, F., Centini, M., Anselmi, C., J. Chromatogr. A
chemical additives mentioned in samples 6 and 7. Pre- 1998, 796, 319 – 325.
vious studies reported that TDA, one of the components [11] Lobstein, A., Brenne, X., Feist, E., Metz, N., Weniger, B., Anton, R.,
of sample 6, causes ataxia, tachycardia, nausea, irrita- Phytochem. Anal. 2001, 12, 202 – 205.
tion, vomiting, convulsions, and respiratory depression [12] Babula, P., Mikelova, R., Potesil, D., Adam, V., Kizek, R., Havel, L.,
[20]. Moreover, when administered by subcutaneous Sladky, Z., Biomed. Papers 2005, 149, 25 – 28.
injection, TDA caused death of rats on day 18, which was [13] Zhao, R., Liu, S., Zhou, L., Chromatographia 2005, 61, 311 – 314.
accompanied by significant teratogenic effects [21]. The [14] Abou-Donia, A. H., Toaima, S. M., Hammoda, H. M., Shawky, E.,
high dose induced a statistically significant increase in Chromatographia 2006, 64, 109 – 112.
the incidence of hepatic neoplasia in male rats and it [15] Reitman, S., Frankel, S., Am. J. Clin. Path. 1957, 28, 56 – 63.
induced a significant dose-related positive trend in the [16] Rartels, H., Bohmer, M., Clin. Chim. Acta 1971, 32, 81 – 85.
incidence of liver neoplasms in both sexes [22]. p-Phenyl- [17] Barham, D., Trinder, P., Analyst 1972, 97, 142 – 145.
enediamine, added to sample 7, was reported to induce [18] Fossati, P., Prencipe, L., Berti, G., Clin. Chem. 1980, 26, 227 – 231.
allergy [23, 24]; it also caused dermal toxicity and patho- [19] Raupp, P., Hassan, J. A., Arughese, M. V., Kristiansson, B., Arch.
morphological lesions in guinea-pigs [25]. A recent study Dis. Child. 2001, 85, 411 – 412.
revealed that p-phenylenediamine induced DNA damage [20] Gosselin, R. E., Clinical Toxicology of Commercial Products, The Wil-
liams and Wilkins Co, Baltimore 1976, pp. 141 – 145.
in SV-40 immortalized human uroepithelial cells [26]
and p53-mediated apoptosis in Mardin-Darby canine kid- [21] Marks, T. A., Gupta, B. N., Ledoux, T. A., Staples, R. E., Teratology
1981, 24, 253 – 265.
ney cells [27]. These previous toxicological studies may
[22] Cardy, R. H., J. Natl. Cancer. Inst. 1979, 62, 1107 – 1116.
explain the death of the rats treated with henna products
[23] White, J. M., Kullavanijaya, P., Duangdeeden, I., Zazzeroni, R.,
to which TDA and p-phenylenediamine have been added. Gilmour, N. J., Basketter, D. A., Mc Fadden, J. P., Clin. Exp. Allergy
However, these hazards should be taken in consideration 2006, 36, 1289 – 1293.
even when henna products are used topically, due to the [24] Ho, S. G., White, I. R., Rycroft, R. J., Mc Fadden J. P., Contact Derma-
probable absorption of these harmful compounds espe- titis 2004, 51, 213 – 214.
cially through the skin of hands or feet. [25] Viswanathan, P. N., Gupta, V., Misra, V., Int. J. Cosmet. Sci. 1985, 7,
213 – 218.
[26] Huang, Y. C., Hung, W. C., Kang, W. Y., Chen, W. T., Chai, C. Y.,
Toxicol. Lett. 2007, 170, 116 – 123.
4 References
[27] Chen, S. C., Chen, C. H., Chern, C. L., Hsu, L. S., Huang, Y. C.,
[1] Zinkham, W. H., Oski, F. A., Pediatrics 1996, 97, 707 – 709. Chung, K. T., Chye, S. M., Toxicol. in Vitro 2006, 20, 801 – 807.