Belonging to the Lythraceae family, Lythrum salicaria
L. (or Purple loosestrife) is an herbaceous perennial plant that is quite easily recognizable with its flowering parts displaying small purple flowers. Lythri herba
is widely distributed in Europe, North America, and Asia, and grows in wet places. Lythrum salicaria
L. is known as a medicinal plant that was used in the ancient times. Naturalists and pharmacologists of the Greco-Roman period already reported its medicinal properties [1
], and nowadays the plant is registered in European Pharmacopoeia. It has traditionally been employed as a strong astringent and haemostatic agent to treat gastrointestinal tract ailments, such as dysentery or diarrhea [2
]. In terms of composition, studies reported polyphenols and heteropolysaccharides as principal phytochemical families [1
]. Lythrum salicaria
L. is a rich source of polyphenols and mainly of ellagitannins, a class of hydrolysable tannins [3
]. In this way, the total tannin content is a means of characterization of this herb according to its pharmacopoeial monograph. Polyphenols are natural active compounds that are able to display anti-aging effects as they have strong antioxidant activity [4
]. More generally, botanicals possess various effects and display several activities simultaneously [5
]. Thus, any of them could be considered in their capacity to cope with many different health issues. Moreover, since ancient times, botanical ingredients have been used as remedies and care products and still play an important role in contemporary cosmetics by being highly efficient [4
]. Purple loosestrife—as a native plant in France, widespread and perennial—constituted a sustainable material to initiate a new and modern exploration of its potential. Based on previous knowledge notably about its composition, which suggested the potential of Lythri herba
, the objective of our work was to innovate by searching a new application of a Lythrum salicaria
extract, and more specifically by looking for a beneficial effect for skin physiology.
Skin is a complex system organized as a stratified cellular epidermis lying on a dermal connective tissue (dermis and hypodermis). Epidermis is composed of layers of keratinocytes that are constantly renewed thanks to proliferation and differentiation with an increasing differentiation state from the basal layer (with the less differentiated cells) to the stratum corneum
(top of the epidermis, the most differentiated state) [6
]. In each layer, specific proteins are expressed and characterize differentiated epidermis, such as cytokeratins (keratins 5, 14, 1, 10, etc.), late differentiation markers (transglutaminase M1 (TGK), filaggrin), and proteins of the cornified envelope (involucrin, small proline rich proteins). These cells migrate and ultimately differentiate into corneocytes that form the cornified layer at the surface of the skin. Before becoming corneocytes, the keratinocytes produce proteins and lipids to form a water-impermeable barrier, the stratum corneum
]. Skin needs to be stable, while at the same time retaining dynamics to allow for tissue regeneration and response to cutaneous injuries [6
]. One of the essential functions of skin is to constitute a protective barrier to avoid the loss of inner fluids and to prevent the entry of external aggressive agents [11
]. Indeed, skin has to face daily environmental aggressions, such as ultraviolet radiation from the sun [12
], air pollution or irritations, and wounds. At the same time, a protective barrier but a fragile organ (not thicker than a few millimeters), skin has the faculty to renew itself constantly. Regeneration allows for repairing damages and replacing old cells. Nevertheless, skin cannot escape aging [12
]. Aging involves dramatical changes in the structure, functioning, and appearance of the skin, such as thinning of the epidermis, degradation of the extracellular matrix (ECM), loss of capacity of regeneration or wound healing, elastosis, and the formation of wrinkles [4
]. This protective function of the skin is essentially performed by the stratum corneum
One way to maintain epidermal integrity and more generally to prevent or at least limit the inevitable phenomenon of skin-aging is the use of topical treatments based on natural active ingredients [14
]. Therefore, the goal of the present work was to take another look at Lythrum salicaria
L. by investigating again its metabolite content and its biological activities to highlight its potential interest in skin homeostasis and in the dermo-cosmetic field.
3.1. Extraction, Isolation, Structure Determination and Quantification of Major Compounds
A dry hydro-ethanolic extract was obtained by maceration. In the following text, this extract will be cited as Lythrum salicaria extract (Lyth. s extract) or purple loosestrife extract.
The two major compounds of the hydro-ethanolic extract of Lythrum salicaria L. presented a UV maximum at 235 nm. They were isolated with a level of purity of 97% minimum and had an appearance of off-white amorphous powders.
Analyses in HRMS were conducted to detect the accurate masses of molecular ions and obtain the molecular formulae. Fragmentation of the compounds was also registered. HRMS and MS2
data are detailed in Table 1
. The two major compounds (1
) provided comparable signals: m
935.078 for [M+H]+
952.105 for [M+NH4
933.063 for [M-H],−
were detected. Also, a slightly unusual form of ion was detected with m/z
477.019 attributed to [M-3H+Na]−
according to the suggested formula. The neutral molecular formula was determined to be C41
corresponding to a monoisotopic mass of 934.0712 g·mol−1
. Based on the literature of the studied plant, this information indicated two isomers of the class of ellagitannins: vescalagin and castalagin [3
]. The structures of these ellagitannins are presented in Figure 1
Compounds 1 and 2 were observed in both negative and positive ionisation modes. In positive mode, singly charged ions were predominant, whereas in negative mode, doubly charged ions were the most intense. Regarding the fragmentation patterns, neutral losses of water and/or carbon dioxide were recovered and were consistent with the hypothesis of structures (presence of hydroxy and ester functions on the ellagitannins). Moreover, characteristic ions for ellagitannins were observed, such as the neutral loss of a hexahydroxyphenoyl (HHDP) group with the observed m/z 633.072 (+)/631.057 (−), or the HHDP group itself with the m/z 303.014 (+)/300.999 (−).
To confirm the hypotheses and to distinguish compound 1 from compound 2, they were analyzed by 13
C-NMR and 1
H-NMR experiments. The assignment of sugar and aromatic protons and carbons was achieved based on one dimensional NMR experiments (see Table 2
Based on HRMS and literature, we supposed that the two compounds were the epimers vescalagin and castalagin. The literature indicated that the chemical shift and the coupling constant of the anomeric proton signal were different between the pair of isomers. Vescalagin has a coupling constant equal or less than 2 Hz because the anomeric proton is in the beta orientation, whereas for castalagin, the coupling constant is larger. In our experiments, for compound 1, a chemical shift of 4.87 ppm and a coupling constant was 2.00 Hz indicated the beta orientation of the hydroxyl group at the C1
of the sugar moiety [18
]. On the contrary, for compound 2, a chemical shift of 5.71 ppm and a coupling constant of the anomeric proton signal larger than the one of compound 1 (coupling constant of 4.40 Hz) for the proton H1
indicated the alpha orientation of the hydroxyl group at the C1
of the sugar moiety [18
]. Moreover, all of the NMR data collected for compounds 1 and 2 were consistent with data presented in literature for vescalagin and castalagin, respectively [3
The two major compounds of the extract of aerial parts of Lythrum salicaria L. are vescalagin (1) and castalagin (2). They were quantified respectively at 4.6% and 2.6% in the dry extract.
Assays to study biological effects of both the extract of Lythrum salicaria L. and vescalagin were performed at non-cytotoxic concentrations (concentrations selected following a MTT (MethylThiazolyldiphenyl-Tetrazolium) assay (data not shown).
3.3. Pro-Differentiation Effect of Lythrum Salicaria Extract
3.3.1. Levels of Expression of Genes Implicated in Differentiation
In order to identify a potential effect on epidermis differentiation, NHEK were incubated with Lythrum salicaria
L. extract and the effect of this extract was evaluated at the gene expression level by RT-qPCR. As shown in Figure 2
, the incubation of the cells with extract induced a strong increase in the mRNA of differentiation markers, mainly TGK, keratin 1 (KRT1) and KRT10, and to a lesser extent, filaggrin, loricrin and involucrin (from 400% to 6000% of the untreated control depending of the marker). Looking at the presence of various proteins, such as involucrin, loricrin, and filaggrin gives indications of fully differentiated epidermal keratinocytes and corneocytes [20
]. These structural proteins help to maintain the cutaneous barrier. Filaggrin helps to stabilize the keratin network, which serves as a crucial scaffold for other components of the cornified layer. Indeed, involucrin and loricrin are cross-linked by TGK1 [6
]. One could also note that for most of the aforementioned markers (except for involucrin), the stimulation induced by Lythrum salicaria
L. extract was stronger than that of the well described keratinocyte differentiating agent CaCl2
]. Within the epidermis, at high concentration, calcium generates the differentiation of keratinocytes rather than their proliferation [13
]. Specific keratins KRT1 and KRT10 were much more expressed in response to Lythrum salicaria
extract. These keratins are markers of supra-basal layers and clearly indicate that epidermal cells are developing towards a terminal differentiation state [20
]. The high levels of filaggrin and loricrin in skins treated with the extract participated to the resistance of the cornified envelope. The level of TGK was increased with the extract. It plays a major role in the assembly of components of the cornified layer.
We observed significant stimulations of the expression of keratinocyte genes that are involved in differentiation and structuration of the complexity of the epidermis thanks to the treatment by Lythrum salicaria extract. At this stage, the extract turned out to be a pro-differentiating agent.
3.3.2. Expressions of Characteristic Proteins in Differentiated Keratinocytes
We then confirmed the effect of Lythrum salicaria
extract at the protein level. Expression of TGK, KRT10, and filaggrin proteins was evaluated in NHEK by immunofluorescent staining of these proteins after 72 h of treatment (see Figure 3
). The positive control condition was a treatment with CaCl2
at 1.5 mM. The negative control consisted in untreated cells. By comparison with these conditions, the observation of the impact of a treatment by Lythrum salicaria
extract at 20 µg·mL−1
In cells without any treatment, the proteins were weakly expressed. The expression of these proteins showed the same trend as previously observed at the mRNA level. Lythrum salicaria extract had a pro-differentiating effect with the same range of effect as that of calcium. Again, the Lythrum salicaria extract demonstrated its capability to promote the production of proteins, allowing for proper and efficient epidermis functions and the differentiation of its constitutive cells.
3.3.3. Histological Characterization of Reconstructed Human Epidermis (RHE)
In order to get deeper in the characterization of the positive effect of Lythrum salicaria
extract on epidermis differentiation, we then turned on a three dimensional model of epidermis, consisting in reconstructed human epidermis (RHE). In this model, epidermis differentiation can be enhanced by the presence of vitamin C in the culture medium. Therefore, in order to identify a proper effect of Lythrum salicaria
extract, it was added in the culture medium in the absence of vitamin C. Then, after seven days of treatment, the expression of three differentiation markers KRT10, TGK, and filaggrin was analyzed by immunohistochemistry. Histological cross-sections of RHE are shown in Figure 4
In our study, RHE in presence of vitamin C expressed large amounts of KRT10, filaggrin, and TGK. On the contrary, the absence of vitamin C resulted in RHE with a weaker expression of the last three markers.
We noticed strong similarities of appearance between a treatment by Lythrum salicaria extract and vitamin C. RHE treated with the extract expressed high levels of markers, as it is showed by the intensity of the red coloration. These treated RHE were no more comparable with those that were obtained in absence of vitamin C. We could conclude that the extract had a positive effect on RHE development since it favored the differentiation of keratinocytes.
3.4. Reinforcement of the Skin
3.4.1. Skin Barrier Function Enhancement on Full Thickness Reconstructed Skin Models
Aging is characterized by a progressive loss of cohesion and architecture of the skin that is associated with an increase of a basal inflammation level, the so-called inflammaging [22
]. The results that were obtained in RHE suggested that Lythrum salicaria
extract could potentially help to reinforce aged or damaged skin. Therefore, the effect of this extract and of one of its constituents, vescalagin, was evaluated in a model of full thickness reconstructed skin (FTSK) cultivated in a depleted medium to induce an aged phenotype. Histological cross-sections of reconstructed skins are presented in Figure 5
First, we checked the global appearance of control FTSK cultivated in a complete medium. These “normal” FTSK were characterized by a dermal compartment (dermal fibroblasts embedded in a collagen matrix), and an epidermal area that was composed of characteristic epidermal cell layers: a basal layer, a spinous cell layer, a granular layer, and the cornified layer (or stratum corneum).
In comparison with control FTSK skins described just above, aged reconstructed skins (used as control of a depleted medium of culture) presented a disorganization that was characterized by the loss of the basal layer, a narrowing of the granular layer with decreased numbers of keratohyalin granules, a thicker stratum corneum and the loss of the cohesion at the dermo-epidermal junction.
The treatment of the aged reconstructed skins (cultivated in depleted medium) with the extract and vescalagin led to a global improvement of their morphology. As shown in Figure 5
, they were almost similar to FTSK grown in a complete medium. Overall proportions of the three areas (dermis, epidermis, stratum corneum
) were similar to those of normal skins. Within the epidermis, the basal and granular layers were well defined. The cohesion between the dermis and the epidermis was totally recovered with the extract, but only partially with vescalagin.
These results indicated that the molecule alone had the same properties as the extract. Indeed, when tested at the same concentrations (30 µg·mL−1), their activity seemed to be equivalent. A histological study of the effect of the extract or the molecule that was isolated from it showed that they were able to reinforce the skin. FTSK cultivated in depleted medium containing Lythrum salicaria extract or vescalagin presented an aspect that was close to that of normal skins rather than an aspect of aged skins.
3.4.2. Beneficial Effects on Extracellular Matrix
Inflammation and aging are characterized by an increased release of enzymes degrading the extracellular matrix (ECM) and a decreased content of ECM components, such as collagen I. Therefore, the levels of some metalloproteinases (MMP) in the subnatants of the FTSK were monitored by ELISA. Results are illustrated in the following graphs (see Figure 6
, Figure 7
and Figure 8
). Matrix metalloproteinases (MMP) are enzymes that participate to the remodeling of the extracellular matrix. MMP-1 (Matrix metalloproteinase 1) is the predominant collagenase that degrades structural collagens, whereas MMP-3 (Matrix metalloproteinase 3) degrades collagens, proteoglycans, and matrix glycoproteins [23
]. Furthermore, procollagen I, one of the major constituents of ECM, was also monitored in parallel.
As expected, in FTSK cultivated in depleted conditions, there was an increased release of MMP-1 and MMP-3 that was associated with a decreased content in procollagen I. Variations for each marker were larger than 10 fold.
The presence of both the extract and vescalagin exerted a very large decrease of expression of products that were responsible for the degradation of ECM, namely MMP-1 and MMP-3. The effect of the extract was dose-dependent (Figure 6
and Figure 7
). In parallel, an increase of procollagen I was observed, but only in response to the total extract (see Figure 8
These elements were strong indicators of the fact that purple loosestrife extract reinforced the dermis. Treated skins were no more comparable to skins reconstructed in depleted media, but returned to a normal state.
This last experiment indicated that the products obtained from purple loosestrife acted in a favorable manner towards the dermis, which is the basement of the epidermis.
In this study, the main goal was to evaluate the potential of an extract of Lythrum salicaria L. for beneficial effects on the skin. We focused on a hydro-alcoholic extract of aerial parts of Lythrum salicaria L., which was rich in ellagitannins and contained vescalagin at a rate of 4.6%. This tannin is the chemo-marker of the extract.
The biological activities of the natural products tested are associated with dermo-cosmetic applications as we tested effects towards constitutive cells of the skin through topical treatments. The in vitro biological tests allowed for demonstrating the effect of the skin treatment with purple loosestrife extract or one of its molecules at different scales. We monitored the effects from gene expression to skin morphology using normal human epidermal keratinocytes, reconstructed human epidermis, and full thickness reconstructed skin.
In the presence of the extract, the expression of markers of differentiation within monolayers of keratinocytes, and RHE was important. These components participate to the structuration of the cornified layer, which is essential for the skin to be protected and to maintain homeostasis. The more differentiated the epidermis is, the better defined the stratum corneum
. This specific and outermost layer of the skin forms a skin barrier function. Not only is skin the largest organ of the human body [13
], but it also has to maintain its protecting function against external aggressions. The extract was able to counteract the starving effect of a depleted medium during the development of FTSK. Skins that were treated with the extract were well structured and their morphologies were comparable to normal skins.
Vescalagin was able to generate the same effects as the extract. This molecule was able to strongly impact the morphology of FTSK, as well as to lower the quantity of MMP in aged reconstructed skins. Thus, the chemo-marker also constitutes a biomarker of the extract.
We demonstrated that the Lythrum salicaria extract could be used as an active ingredient with beneficial effects for the skin homeostasis with a pro-differentiating effect and a global protecting or reinforcement action. This effect was characterized by the generation of well-structured epidermis and by maintaining the skin protective functions for the body.
Lythrum salicaria L. may now be considered as a new great source of active ingredients with protecting and anti-aging effects for skin.