SPME-GC-MS analysis of commercial henna samples (Lawsonia inermis L.)

The aim of this work was to provide a characterisation of volatile constituents from different commercial batches of henna (Lawsonia inermis) leaves of different geographic origin. Headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography–mass spectrometry (GC–MS) was used for the purpose. A total of 72 components were identified by GC–MS in the headspace of different henna samples which proved to differ considerably from each other, because they were characterised by different classes of components, mainly aliphatic compounds (9.0–64.7%), terpenoids (5.8–45.5%) and aromatics (7.9–45.2%), with alkanes (0.9–18.5%), aldehydes (2.1–18.8%) and carboxylic acids (3.1–29.3%), monoterpenes (3.4–30.0%) and sesquiterpenes (0.8–23.7%) and phenyl propanoids (0.6–43.1%), being the most abundant, respectively. Major representatives of these groups were n-hexadecane (0.5–4.7%), (2E)-hexenal (0.5–11.7%) and acetic acid (2.8–24.5%), limonene (0.8–14.7%), carvol (3.8–7.1%), geranyl acetone (1.4–7.9%) and (E)-caryophyllene (3.3–8.4%), and (E)-anethole (0.6–35.0%), respectively. We assume that factors such as the manufacturing process, the storage conditions and the different geographic origin of the samples may contribute to such variability.


Introduction
Derived from cut and dried leaves of Lawsonia inermis L. (Lythraceae), henna has been used for centuries for body art, especially in Arabic and Hindu cultures (De Groot 2013). Today, henna is used as a hair dye and for temporary tattoos on the skin, frequently for self-use and easy domestic application, because of its strong colouring properties from the active component lawsone (2-hydroxy-1,4-naphthoquinone) and because people perceive henna as a natural product without the unfavourable effects of chemical colouring agents. Henna use has increased recently because of the fashion of temporary tattoos in both Europe and the USA, even if its use in the USA is not approved by the food and drug administration (FDA), which does not classify lawsone as a colourant permitted for direct application on the skin (FDA 2012). On the contrary, henna is authorised as a colouring agent for hair, and is widely available in India, the Middle East, Europe, Australia, Canada and the USA.
Studies focused on qualitative analysis of volatile components in henna as a finished product, that is, sold in the market as a hair dye, however, are lacking. Thus, the aim of our work was to provide a characterisation of volatile profiles of henna samples. Starting from different commercial henna hair, hand and tattoo batches found in the retail trade, we performed a headspace solid-phase microextraction (HS-SPME) coupled to gas chromatography -mass spectrometry (GC -MS) analysis to characterise the volatile constituents occurring in different commercial samples of different geographic origin. We used SPME technique because this is a non-destructive and non-invasive method to evaluate aroma compounds in different kinds of matrices (Demyttenaere et al. 2001;Hamm et al. 2003;Demyttenaere, Dagher, et al. 2003a, Demyttenaere, Sánchez Martinez, et al. 2003bDemyttenaere, Moriña, et al. 2004a, Demyttenaere, Vanoverschelde, et al. 2004bVan Lancker et al. 2008). It has been largely applied to aroma analysis in combination with GC and GC -MS, offering solvent-free and rapid sampling with low cost and ease of operation; moreover, it is sensitive, selective and also compatible with low detection limits (Pawliszyn 1997).
Interestingly, acetic acid is a marker of fermentation, which probably occurs during manufacturing of henna. Being a weak acid, acetic acid slightly lowers pH of henna solution (about 5.5), allowing to extend the duration of the colour (Leung & Foster 2003). Therefore, samples with high levels of this compound may be considered of higher stability. Other components present in noteworthy levels were the sesquiterpenes ar-curcumene (7.9%) and b-sesquiphellandrene (7.3%) in the Zarqa sample (Pakistan) and the lactone dihydroactinidiolide (2.3 -6.8%) in the Mogano (Iran), Mohini (India), Minaridi (India) and Sahara (Morocco) samples.
Multivariate analysis showed that the variability of data was generated mostly by the content of acetic acid, (E)-anethole and (E)-caryophyllene, as shown in the loading plot (Figure 1(b)). They were correlated with henna samples taking place in the same position of the score plot (Figure 1(a)). Sample 6 from Morocco was correlated with (E)-anethole, while samples 2 and 5 from India and Morocco, respectively, were mainly characterised by high levels of acetic acid. Finally, samples 1, 3 and 4, from Iran and Pakistan, respectively, were mostly correlated with sesquiterpenes such as (E)-caryophyllene.

Conclusions
Our study found significant variability in the headspace volatile components of the different henna products analysed, confirmed by the principal component analysis. We assume that factors such as the manufacturing process, the storage conditions and the different geographic origin of the samples may contribute to such variability. Results obtained by SPME analysis showed different chromatographic profiles for the six henna samples, leading to the conclusion that the volatile components in the samples of henna are heterogeneous, depending on the different origin and manufacturing process of the samples. Since there are no studies in the literature that offer qualitative and semi-quantitative analysis and comparison of data between different samples of commercial henna to highlight their variability, the results of this project fill a gap in knowledge about this plant derivative that is widely used for cosmetic purposes, but still poorly understood.

Supplementary material
Supplementary material relating to this paper are available online at http://dx.doi.org/10.1080/ 14786419.2015.1055491, alongside Table S1 and Figure S1.