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Supercritical fluid extraction of hernandulcin from Lippia dulcis Trev.

Lippia dulcis Trev. is a medicinal plant of the Verbenaceae family. It is intensely aromatic and the
leaves and flowers have a strong sweet taste [1]. The presence of a sweetener compound
(hernandulcin) in the extract is mentioned in most of the publications on this plant matrix. The
substances present in this plant have been previously investigated and isolated by different methods,
such as hydrodistillation, solvent extraction and microwave-assisted hydrodistillation
(MWHD) [1], [2], [3] and [4], but there are no reports available on the supercritical fluid extraction of
this plant matrix. Compounds identified in extracts of this plant include camphor, limonene, terpineol,
α-pinene, α-copaene, trans-caryophyllene, δ-cadinene, α-bisabolol, (+)-4β-hydroxy-hernandulcin,
(+)-hernandulcin and its stereoisomer (− )-epi-hernandulcin[1] and [4]. In the literature available, the
compound hernandulcin is recommended as a low cariogenic sweetening agent, useful as a food or
beverage additive, or in pharmaceutical formulations and oral hygiene products, either alone or in
combination with other additives and sweeteners [5] and [6].

Regarding the method of extraction, hydrodistillation is commonly applied to obtain essential oils, as
described in the Brazilian Pharmacopeia [7]. However, using this method may cause thermolabile
compounds to degrade, since high temperature is needed. Hernandulcin is sensitive to high
temperature and thus this technique might not be suitable to obtain this product. Moreover, with the
use of organic solvents there is the inconvenience of a further processing stage being required in
order to remove the residual solvent.

In this context, supercritical fluid extraction (SFE) using CO 2 as the solvent was the method of choice
to obtain L. dulcis extracts, not leaving any residue in the final product and with the possibility of
working at mild temperatures, avoiding thermal degradation of compounds. The extraction of
natural compounds is one of the most studied applications of supercritical fluids [8].
Therefore, in this study, a preliminary investigation on the supercritical fluid extraction parameters
of L. dulcis was performed. Also, the composition of the extracts as well as the presence of the
sweetener compound hernandulcin was verified by chromatographic techniques.

Materiales y métodos
L. dulcis was dried immediately after harvest in an oven with air circulation (Marconi, MA035) at 30 °C
to give a moisture content of 9.0 ± 0.5% (wet basis). The temperature was selected in order to avoid
thermal degradation of the compounds present in the plant, and also because it is lower than the
temperature used in the SFE process. To measure the moisture content, plant samples were placed in
an oven at 105 ± 1 °C until a constant weight was achieved, according to the methodology described
in the Brazilian Pharmacopeia [7]. The initial moisture content of the plant was 75 ± 1% (wet basis).
Measurements were performed in triplicate.

After drying, the stems of L. dulcis were manually separated from the leaves and flowers and
discarded. According to the literature [9], the compound hernandulcin is present in the leaves and
flowers of L. dulcis. The dried leaves and flowers were subsequently ground in a knife mill (Marconi,
Brazil), which was coupled to a sieve of mesh 30 (pore size 0.595 mm) to standardize the particle
size. Dried and ground L. dulcis was placed in a hermetically sealed bottle and stored in a freezer, in
order to avoid loss of volatile compounds and material degradation.

A facile synthesis of (6S,1′S)-(+)-hernandulcin and (6S,1′R)-(+)-epihernandulcin

(+)-Hernandulcin (1) was isolated in 1985 by Kinghorn and co-workers1 as colorless oil from Lippia
dulcisTrev. (Verbenaceae), obtained at a marketplace near Mexico City. This plant was described in a
book entitled Natural History of New Spain that was written between 1570 and 1576 by the Spanish
physician Francisco Hernandez. L. dulcis is a medicinal plant used by the Aztec people, known to
them for centuries by the Nahuatl name ‘Tzonpelic xihuitl’, meaning ‘sweet herb’. 2 Initially, (+)-
hernandulcin was isolated as a minor volatile oil constituent with a very low yield (0.004% w/w). Later
the yield was improved to 0.154% (w/w) by isolating from L. dulcis leaves and flowers collected in
Panama, where it was being sold as a remedy for respiratory ailments. 3 It has been shown that (+)-
hernandulcin could be produced from transformed shoot cultures of L. dulcis in a yield of 2.9% dry
weight. 4 (+)-Hernandulcin was judged, on a molar basis, by human taste panel to be more than
three orders of magnitude sweeter than sucrose. Its sweetness, however, was considered somewhat
less pleasant than that of sucrose, and some bitterness, off- and after-taste were perceived as well. 5 (
±)-Hernandulcin showed no acute toxicity to mice at doses up to 2 g per kilogram of body weight
nor mutagenic activity. 3

Entre las plantas oleaginosas no comestibles que se han descrito en el la literatura como fuente de
éster de alquilo (biodiesel), es decir, el NET (Azadirachta indica A. Juss;. Meher et al, 2005), Karanja
(Pongamia pinnata L.;. Modi et al, 2007.; Raheman y Ghadge, 2007; Raheman y Phadatare, 2004;

Sharma y Singh, 2008; Srivastava y Verma 2008), mahua (sp Madhuca;. Puhan et al. 2005b), UNDI
(también conocido asNagchampa o polanga; Calophyllum inophyllum L.; Azam et al., 2005;
Banapurmath et al., 2008; Sahoo et al., 2007), castor bean (Ricinus communis L.; Goodrum y
Geller, 2005; Scholz y da Silva, 2008), simarouba (Simarouba glaucaDC;. Adjaye et al, 1995;. Azam
et al, 2005.), Benoil árbol (Moringa oleifera Lam;.. Rashid et al, 2008), de jojoba (Simmondsia
chinensis (Link) Schneid;. Bouaid et al, 2007;.. Canoira et al, 2006), árbol de caucho(Hevea
brasiliensis (Willd. ex Adr Juss) Muell Arg;..... Ramadhas et al, 2005), árbol de sebo de China
(Sapium sebiferum (L.) Roxb;. Gao et al, 2008). Babasú (Attalea speciosa C. de Marte;. Oliveira et
al, 1999.), Tucumán (Astrocaryum vulgare Mart;. Lima de Oliveira et al, 2008), Zanthoxylum
(Zanthoxylum. bungeanum Max;. Zhang y Jiang, 2008), el mango del mar (Cerbera Odollam Gartn.
Engl. o Cerbera manghas L.; Gaillard et al, 2004)- J.. curcas (Kumar y Sharma, 2008) merece una
atención especial debido al papel principal que se espera que juegue en un futuro próximo.