Wound Healing Activity and Phytochemical Screening of Purified Fractions of Sempervivum Tectorum L. Leaves on HCT 116

Received: 31 January 2019 Revised: 12 March 2019 Accepted: 17 April 2019 DOI: 10.1002/pca.2844

SPECIAL ISSUE ARTICLE

Wound healing activity and phytochemical screening of purified fractions of Sempervivum tectorum L. leaves on HCT 116

1 Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi Abstract di Napoli Federico II, Naples, Italy Introduction: Sempervivum tectorum L. (Crassulaceae), is a succulent perennial plant 2 Dipartimento di Farmacia, Università degli widespread in Mediterranean countries and commonly used in traditional medicine Studi di Napoli “Federico II”, Naples, Italy 3 Dipartimento di Bioscienze e Territorio, for ear inflammation, ulcers and skin rashes as a refrigerant and astringent. Università degli Studi del Molise, Pesche, Objective: To demonstrate the therapeutic effects of the plant, various fractions (Isernia), Italy were purified and characterised. The potential wound healing activity, proliferation Correspondence rate and intracellular signalling cascades were investigated by using human epithelial Maria Iorizzi, Dipartimento di Bioscienze e Territorio, Università degli Studi del Molise, colorectal carcinoma (HCT 116) cells. Contrada Fonte Lappone, I–86090 Pesche Methodology: An extraction method without organic solvents was applied for the (Isernia), Italy. Email: [email protected] first time. The purification was carried out by droplet counter current chromatography (DCCC) coupled with high‐performance liquid chromatography (HPLC) and electrospray ionisation mass spectrometry (ESI‐MS) data. By nuclear magnetic resonance (NMR) [1H, 13C and two‐dimensional (2D) experiments] pure components were identified. Wound healing and cell proliferation assays were utilised to determine the role of the isolated S. tectorum (SVT) fraction on cellular migration and proliferation. The signalling pathways elicited from the SVT fractions, were analysed by Western blot analysis. Results: In this study two rare natural components were identified, namely monosaccharide sedoheptulose and polyalcohol 2‐C‐methyl‐D‐erythritol, along with known organic acids and flavonoids. The fractions with high level of sedoheptulose enhance the proliferation and the cellular migration of epithelial HCT 116 cells. The intracellular signalling cascades elicited from the purified fractions induce the c‐Src‐mediated transactivation of EGFR and the activation of the STAT3 pathway which, in turn, are crucially involved in the cellular proliferation and migration. Conclusions: Our study demonstrates the efficacy of purified fractions of S. tectorum L. in enhancing cellular proliferation and migration, suggesting their potential role as topical therapeutic treatments for wound healing.

KEYWORDS

2‐C‐Methyl‐D‐erythritol, NMR spectroscopy, sedoheptulose, Sempervivum tectorum L., wound healing activity

1 | INTRODUCTION a method developed in our group. Herein we analyse the effects on cellular migration and proliferation as well as the putative intracellular Herbs and plants have been used in traditional medicine for hundreds of signalling cascades triggered by treatment with S. tectorum in human years for therapeutic purposes since whole herbs contain complex mix- epithelial colorectal carcinoma (HCT 116) cells. tures of organic components that can work together to produce a ben- eficial effect. In recent years there has been growing tendency to use herbal preparations and a renewed interest of chemists in the identifica- 2 | EXPERIMENTAL tion of natural compounds for pharmaceutical or cosmetic applications.1 Sempervivum tectorum L. known as houseleek, is a wild‐growing 2.1 | Plant material succulent perennial plant with thick leaf rosettes, belonging to the family of Crassulaceae, widespread in arid climates such as the Medi- Selected rosettes of wild growing S. tectorum L. (Crassulaceae) were terranean countries from Spain to Asia Minor. The genus Sempervivum collected at Colli al Volturno, a mountainous area of Isernia (central comprises approximately 50 species frequently employed in traditional Italy) in June 2016 and identified by Dr Paola Fortini. A voucher spec- medicine in Serbia, Bosnia and Turkey to cure common ailments, par- imen was deposited (SVT‐129‐2016) at the Herbarium of the ticularly earache,2 ear inflammations and dermatophyte infections.3,4 Dipartimento di Bioscienze e Territorio, University of Molise (Pesche, Among botanicals, S. tectorum leaves, fresh juice and bruised leaves Isernia). The rosettes were immediately analysed. from S. tectorum, are used in folk medicine in Europe almost exclusively for external purposes such as for the treatment of warts, ulcers, 2.2 | General experimental procedures skin rashes and corns, as a refrigerant and astringent.5 In Italy the juice and leaves have been applied for insect bites while the beaten aerial Electrospray ionisation mass spectrometry (ESI‐MS) experiments were part placed on the brow has been reported to reduce the severity of performed on an Applied Biosystem API 2000 triple‐quadrupole mass headaches.6 It has also been found that a S. tectorum extract has spectrometer. Optical rotations were determined on a Jasko P‐2000 lipid‐lowering activities in rats,7 significant antinociceptive activities5 polarimeter. Nuclear magnetic resonance (NMR) spectra were and liver protecting with membrane stabilising effects.8 Antioxidant obtained on a Varian Inova 500 NMR spectrometer (1H at 500 MHz activities have been evidenced in vitro9-12 and in vivo13 and have been and 13C at 125 MHz) equipped with a Sun hardware, δ (ppm), J in attributed to the presence of the plant's phenolic compounds. Drink- Hz, using a solvent signal for the calibration (13CD OD at δ 49.0 ing tea prepared from the leaves is recommended in folk medicine 3 C and residual CD HOD at δ = 3.31). The spectra in D O (deuterated for ulcer treatment.14 Several papers have reported the polyphenol 2 H 2 water) contained TSP‐d [3‐(trimethylsilyl)propionic‐2,2,3,3‐d acid composition5,15 and the flavonoid content of S. tectorum from differ- 4 4 sodium salt] as the internal standard. The heteronuclear single‐ ent geographical areas.16 quantum coherence (HSQC) spectra were optimised for an average Sempervivum tectorum as a succulent plant, has a crassulacean acid 1J of 140 Hz; the gradient‐enhanced heteronuclear multiple bond metabolism (CAM), and presents a typical ecophysiological adaptation CH correlation (HMBC) experiments were optimised for a 3J of 8 Hz. to arid conditions due to an efficient water conservation strategy and CH Droplet counter current chromatography (DCCC) fractionation was a specialised photosynthetic mode with a carbon dioxide (CO2) con- performed on a DCC‐A apparatus (Tokyo Rikakikai Co., Tokyo, Japan) centrating mechanism. During the night, carbon dioxide is fixed and equipped with 250 glass‐columns. Sephadex LH‐20 (4 cm × 80 cm; is stored in cell vacuoles in the form of malate. Throughout the follow- 25–100 μm) (Sigma‐Aldrich Chemie GmbH, Steinheim, Germany). ing day, malate is released from the vacuoles and CO2, produced by High‐performance liquid chromatography (HPLC) was performed the malate decarboxylation can enter the Calvin cycle and can be used using a Waters 510 pump equipped with a Rheodyne 7125 injector as a substrate for Rubisco to provide sugars and other carbohydrates and a Waters 401 differential refractometer as the detector on a Luna to the plant. The physiological homeostasis of CAM plants under C column (5 μm, 250 mm × 4.6 mm i.d.) (Phenomenex, Torrance, CA, long‐term drought is due to a significant starch conservation and an 18 USA); Eurospher 100–5NH (250 mm × 4.6 mm i.d.) (Vertex Plus, adaptation of the equilibrium of carbohydrate balances.17 2 Knauer, Berlin, Germany). A number of homeopathic products containing houseleek are marketed in the United Kingdom and the United States (Homeopathic Medicine Boiron; Washington Homeopathic Products) while others 2.3 | Extraction and isolation are used as cosmetics (Ilcsi Natural Cosmetics, Hungary) or in cosmetic formulations.18 Eight fresh rosettes (492 g) of S. tectorum (SVT) were cleaned, washed Although several pharmacological activities have been reported for and homogenated at room temperature without any solvent. The the S. tectorum leaves, a complete characterisation of the extracts is homogenate was then centrifuged to give a supernatant and a precip- still not available, and previous analyses have highlighted only the itate. The supernatant was freeze‐dried to give 4.07 g of lyophilised polyphenolic components. extract (ST1), while the precipitate was extracted overnight with The present study is focused on the determination of the phyto- methanol (MeOH) (500 mL) at room temperature and provided chemical profile by applying an extraction any organic solvents using 5.10 g of MeOH extract (ST2). CATTANEO ET AL. 3

Then, 2.00 g of the ST1 extract was submitted to DCCC with to give kaempferol‐3‐O‐rhamnosyl‐glucoside‐7‐O‐rhamnoside (9.2 mg) n‐BuOH–Me2CO–H2O (3:1:5) in the descending mode (the upper and kaempferol‐3,7‐di‐O‐rhamnoside (2.2 mg). phase was the stationary phase) to give six main fractions (A–F, see Table 1). The obtained fractions were monitored by TLC on silica gel plates with n‐BuOH–HOAc–H2O (12:3:5) and CHCl3–MeOH–H2O 2.4 | Reagents and cell culture (80:18:2). Fraction A (180 mg) was composed of isocitric acid, malic acid and malic acid sodium salt; fraction B (98.3 mg) was a mixture The sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐ of malic acid and free sugars; fractions C (65.7 mg) and D (120.5 mg) PAGE) reagents were provided by Bio‐Rad (Hercules, CA, USA). The contained malic acid, free sugars and sedoheptulose in different anti‐phospho‐EGFR Y845; anti‐phospho‐Src Y416 and anti‐phospho‐ percentages; fraction E (32.7 mg) was exclusively composed of 2‐C‐ STAT3 Y705 were from Cell Signalling Technology (Denvers, MA, methyl‐D‐erytritol, and fraction F (60.7 mg) mainly contained polyphe- USA). The anti‐α‐tubulin, anti‐rabbit and anti‐mouse antibodies were nolic components. purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The fractions A–E were then purified by HPLC on a Eurospher The protein A–horseradish peroxidase was from Amersham Pharmacia 100–5‐NH2 (250 mm × 4.6 mm i.d., flow rate 1.0 mL/min) column Biotech (Little Chalfont, UK). AG1478, PP2, PP3 were from with CH3CN–H2O (65:35) as the eluent to give in this order: 2‐C‐ Calbiochem (La Jolla, CA, USA). methyl‐D‐erythritol (16.3 mg), fructose (10.4 mg), sedoheptulose The HCT 116 human colon cancer cell line was obtained from the (18.0 mg), malic acid (76.5 mg) and isocitric acid (6.7 mg). Fraction American Type Culture Collection (ATCC, Manassas, VA, USA). The F was purified by HPLC on a 5 μ Luna C18 column (250 mm × cells were cultured in Dulbecco's modified Eagle's medium (DMEM) 4.6 mm i.d., flow rate 1.0 mL/min) with CH3CN–H2O (Thermo Fisher Scientific, Monza, Italy) supplemented with 10% foetal [15:85 + 0.1% trifluoroacetic acid (TFA)] as eluent and gave two bovine serum (FBS) (Invitrogen, Carlsbad, CA, USA). The cells were flavonol glycosides in small amount: kaempferol‐3‐O‐α‐ grown until they reached 80% confluence, serum starved for 24 hours, rhamnopyranosyl‐7‐O‐α‐rhamnopyranoside (1.7 mg)5 and and successively stimulated with purified fraction of S. tectorum (SVT) kaempferol‐3‐O‐β‐[α‐rhamnopyranosyl–(1 → 2)glucopyranoside]‐7‐ at different concentrations. In other experiments, serum deprived cells O‐α‐rhamnopyranoside (0.3 mg).19 were preincubated with 10 μM PP2 for 45 min, 10 μM PP3 for 45 min The ST2 extract (2.10 g) was fractionated over Sephadex LH‐20 or 2 μM AG1478 for 1 h before the stimulation with the SVT with MeOH–H2O (2:1) and afforded five fractions (F1–F5). The central fractions. fraction F2 (620 mg) contained a complex mixture of malic acid, free sugars, sedoheptulose and 2‐C‐methyl‐D‐erythritol while fraction F4, was a mixture rich in polyphenolic components. Fraction F2 was sub- ‐ – – mitted to DCCC separation with n BuOH Me2CO H2O (3:1:5) in the 2.5 | Protein extraction and Western blotting same condition as reported earlier. Four main fractions (A1–A4) were 20 obtained and purified by HPLC on a Eurospher 100–5‐NH2 column Whole lysates were obtained as previously described. HCT 116 with CH3CN–H2O (65:35) as the eluent and led to the isolation of: 2‐ cells were serum‐starved for 24 h and stimulated with different C‐methyl‐D‐erythritol (6.2 mg), fructose (3.0 mg), sedoheptulose concentrations of SVT fractions in the presence or absence of the (2.5 mg) and malic acid (15 mg). Fraction F4 was purified by HPLC on appropriate amount of the specific inhibitors. Briefly, the cells were a5μ Luna C18 column with CH3CN–H2O (15:85 + 0.1% TFA) as eluent rinsed in cold phosphate buffered saline (PBS), and lysed by incubation at 4°C for 45 min with RIPA buffer containing: 50 mM Tris–HCl, pH 7.4, 150 mM sodium chloride (NaCl), 1% NP‐40, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.25% sodium TABLE 1 ST1 fractionation by DCCC and HPLC purification deoxycholate, 1 mM sodium fluoride (NaF), 10 μM sodium

DCCC HPLC separation: orthovanadate (Na3VO4), 1 mM phenylmethylsulfonylfluoride, fractions Mixture of components column, eluent. 10 μg/mL aprotinin, 10 μg/mL pepstatin, 10 μg/mL leupeptin.21

A Isocitric acid, malic acid, Eurospher‐100‐5‐NH2 The protein concentration was determined by a Bio‐Rad protein – malic acid sodium salt CH3CN H2O (65:35) assay. Then 50 μg of the whole lysates were resolved on 10% B Malic acid and free sugars SDS‐PAGE, followed by blotting to polyvinylidene fluoride (PVDF)

C Malic acid (30%), free sugars Eurospher‐100‐5‐NH2 membranes. The membranes were probed with specific primary – (30%), Sedoheptulose (30%) CH3CN H2O (65:35) antibodies and then incubated with horseradish peroxidase‐

D Malic acid (30%), Eurospher‐100‐5‐NH2 conjugated secondary antibodies. The protein bands were detected ‐ ‐ – sedoheptulose (5%), 2 C CH3CN H2O (65:35) by autoradiography.22 The bands densitometry was estimated using methyl‐D‐erythritol (40%) a Discover Pharmacia scanner equipped with a sun spark classic ‐ ‐ ‐ ‐ E2C Methyl D erythritol densitometric workstation. To determine the equal amount of μ‐ ‐ – F Mixture of polyphenols 5 Luna C18 CH3CN H2O loaded protein the same filters were reprobed with an anti‐α‐tubulin (15:85 + 0.1% TFA) antibody. 4 CATTANEO ET AL.

2.6 | Wound healing assay image was taken of the same area of the plates. Time‐lapse images were taken by using the Leica AF6000 Modular System and processed Wound healing assay was performed as previously described.23HCT by using the Leica LAS AF lite software. The covered surface was 116 cells were cultured in DMEM supplemented with 10% FBS at quantified with Image J software. 37°C until confluences. Subsequently the cell monolayer was μ scratched with an 80 m diameter sterile tip and the plates were 2.7 | Cell proliferation assay washed in PBS to remove the detached cells. Every 24 h for 72 h an

Here, 104 cells were seeded in each well of a 24‐well plate and cultured in DMEM supplemented with 10% FBS for 24, 48 and 72 h at 24 1 13 37°C, 5% CO2, as previously described. The number of trypan TABLE 2 H‐ and C‐NMR in CD3OD and D2O + TSP‐d4 internal blue‐positive and negative cells was determined by direct counting standard (500 and 125 MHz) data for sedoheptulose and fructose. at different time points using Burker's chamber. Sedoheptulose β‐ β‐D‐ β‐D‐ Furanose form Fructofuranose Fructopyranose

a a 2.8 | Statistical analysis Position δH δC δH δC δH δC

CD OD 3 All the presented data are expressed as means ± standard deviation b – 1 64.0 3.60 64.1 3.47 3.62 65.9 (SD) and are representative of three or more independent experi- 2 — 103.3 103.2 99.2 ments. The statistical analyses were performed in accordance with 3 4.04 77.8 4.05b 77.5 4.02b 69.4 the Student's t test for paired data and the results were considered 4 4.26 t (7.2) 77.4 4.05b 77.1 4.20 t (6.9) 71.8 significant at p < 0.05. 5 3.73 83.2 3.90b 83.1 3.63b 71.3 6 3.69 73.7 3.61b 64.0 3.70b 65.9 3 | RESULTS AND DISCUSSION 7 3.46 3.61 64.2

D2O + TSP‐d4 Sempervivum tectorum leaf samples were centrifuged and the superna- 1 3.60b 62.4 3.60b 62.3 3.58‐3.74b 63.8 tant, which was solvent free, allowed a wide range of metabolites 2 — 101.6 101.4 97.9 (Supporting Information, Figure S1). Their structures were elucidated ‐ 3 4.13 75.7 4.13b 75.3 3.82 d 67.5 using spectroscopic methods including one dimensional (1D) and two‐dimensional (2D) NMR experiments (COSY, HSQC and HMBC) 4 4.32 t (7.5) 75.4 4.12b 74.4 3.91 dd 69.6 and spectrometric techniques. 5 3.79b 80.1 3.84b 80.6 4.03 m 69.1 In agreement with the physiological behaviour in CAM plants, 6 3.87b 72.7 3.69b 62.6 4.06–3.72b 63.3 characterised by the accumulation of malic acid, the major organic acid – 7 3.81 3.58 62.3 identified was malic acid followed by isocitric acid. However, two rare aCoupling constants (J) are in parentheses and given in hertz. components were identified: the rare sugar sedoheptulose and the bOverlapped with other signals; data extracted from COSY and HSQC unusual polyalcohol 2‐C‐methyl‐D‐erythritol. The free fructose was experiments. abundant in the extract while the glucose was evidenced as a minor

1 13 TABLE 3 H‐ and C‐NMR data (CD3OD and D2O, 500 and 125 MHz) of isocitric acid, malic acid sodium salt and 2‐C‐methyl‐D‐erythritol.

Isocitric acid Malic acid sodium salt 2‐C‐Methyl‐D‐erythritol

a a a b Position δH δC δH δC δH δC δH

1 — 182.9 175.5 3.42 d (11.5) 3.53 67.2 3.47 d,(11.7) d (11.5) 3.57 d (11.7) 2 4.46 brs 73.0 4.23 dd (7.6, 4.2) 68.3 — 73.8 3 3.30 m 48.0 2.35 dd (15.8, 4.2) 40.0 3.61 m overlap 74.9 3.69 dd, 2.75 dd (15.8, 7.6) 4 — 38.0 — 173.1 3.81 brd 62.6 3.83 dd, 3.57 d overlap 3.60 dd 5 2.70 dd overlap 183.0 1.11 s 18.5 1.15 s 6 — 183.0 aCoupling constants are in parentheses and given in hertz. b1 H‐NMR was recorded in D2O. CATTANEO ET AL. 5

FIGURE 1 SVT purified fractions improve wound healing and proliferative activity. (A), Representative images (top) and bar graphs quantification (bottom) of HCT 116 cells migration in presence or absence of SVT fractions containing high levels of sedoheptulose and malic acid at the final concentration of 50 μg/mL or 100 μg/mL, after 0, 24, 48 and 72 h of SVT treatment (n = 5 independent experiments; *P < 0.05 vs. untreated cell; bar = 200 μm). (B), Proliferation rate of HCT 116 cells was measured at 24, 48 and 72 h after plating (for all groups, 104 cells/well) in presence or absence of SVT fractions containing high levels of sedoheptulose and malic acid at the final concentration of 50 μg/mL or 100 μg/ mL. Each experiment was performed in triplicate (*P < 0.05 vs. untreated cell) 6 CATTANEO ET AL. component. Sempervivum tectorum also contained several flavonoid However the HPLC purification was always a compromise glycosides that have previously been identified. between the separation of fructose and the yields of sedoheptulose, since, after multiple purifications, the complete separation was not possible and, indeed in the positive ESI‐MS, quasimolecular ion peaks at m/z 203 [M + Na]+ and m/z 219 [M + K]+ were observed due to the 3.1 | Metabolite identification presence of the fructose unit as a minor component. 1 ‐ 3.1.1 | Sedoheptulose The H NMR spectrum was rather complex due to the overlap- ping signals of sedoheptulose in its β‐furanose form, associated with In higher plants glucose, fructose and other free sugars are present signals of the fructose unit in equilibrium with its two main tauto- ubiquitously and play important roles in many metabolic and develop- mers: the β‐fructopyranose and β‐fructofuranose forms. Among the mental processes. Sedoheptulose (D‐altro‐2‐heptulose) is one of the signals, sedoheptulose was identified by means of its diagnostic sig- δ δ ‐ ‐ few heptoses found in nature and it is a relatively unknown metabolite nals at H 4.32 and H 4.13 (H 4 and H 3 respectively in D2O) 13 compared with its phosphorylated form, sedoheptulose 7‐phosphate (Table 1 and Figure S2). As expected, the C‐NMR experiment (S7P), which is recognised as an intermediate molecule of the primary showed three anomeric carbons: 101.6 ppm (C‐2oftheβ‐furanose glucose metabolism.25 form of sedoheptulose), 101.4 ppm (C‐2ofβ‐fructofuranose) and 97.9 ppm (C‐2ofβ‐fructopyranose) (see Figure S3). Assignments Sedoheptulose has a molecular formula C7H14O7 and a molecular weight of 210.18 Da. The positive ESI‐MS showed quasimolecular for sedoheptulose were possible using a combination of COSY, ion peaks at m/z 233 [M + Na]+ and m/z 249 [M + K]+ respectively HSQC and HMBC experiments (Table 2) as well as the existing liter- 26,28 for sedoheptulose. According to Ceusters et al.,26 the sedoheptulose ature. The COSY experiment revealed the spin sequence from H‐3toH‐7, HSQC provided correlations between protons and the unit, in a D2O solution, is present in three conformations: α‐furanose (16%), β‐furanose (64%), and α‐pyranose (20%), forms. The chemical corresponding carbons (Figures S4 and S5 expanded portion) and ‐ ‐ shift assignments of the main β‐furanose form are listed in Table 2 HMBC added long term correlations with cross peaks between H2 and the NMR data can be clearly differentiated from those obtained 1 (3.60) and C‐2 (101.6); and H‐7 (3.81) and C‐5 (80.1). Based on for mannoheptulose.27 these data, the signals were attributable to the β‐furanose form of

FIGURE 2 SVT promotes EGFR trans‐phosphorylation and c‐Src activation. Whole lysates were purified from 24 h serum‐starved HCT 116 cells stimulated for 5 min with different concentration of 100 μg/mL SVT fractions containing high levels of sedoheptulose and malic acid. Proteins were resolved on 10% SDS‐PAGE and hybridised with phospho‐specific anti‐p‐EGFR Y845 (A, C, D) or anti‐p‐Src Y416 (B, C, D) antibodies. Cells were also pre‐incubated with 10 μM PP2 or PP3 (C) or 2 μM AG1478 (D) before SVT stimulation. Densitometric analysis was performed with ImageJ program. An anti‐Tubulin antibody served as a control for loaded protein. All blots are representative of at least three independent experiments (*P < 0.05 vs. serum starved cell; §P < 0.05 vs. SVT 100 μg/mL stimulated cell) CATTANEO ET AL. 7

sedoheptulose (D‐altro‐2‐heptulose) (Table 2 and Figures S3, S4, S5 experiments performed in D2O showed the presence of two and S6). preponderant tautomers, the β‐fructopyranose and β‐fructofuranose In the literature free sedoheptulose was first reported in Sedum forms (Table 2). The free glucose was evidenced in a minor amount 29 1 spectabile Bor. (Crassulaceae) and, in lower concentrations, in some and in the H‐NMR spectra (D2O; Figure S1) the anomeric protons edible vegetables, such as the carrot (Daucus carota L.), in which it is considered a minor carbohydrate,30 and in the avocado (Persea americana Mill.).31 In plant tissues the free heptose sugars might emerge from the Calvin–Benson cycle or by the oxidative pentose phosphate pathway (ox PPP) in which S7P is an intermediary reagent and could be involved in the cyclic regeneration of D‐ribulose for the

CO2 fixation in plant photosynthesis. The physiological role of free sedoheptulose in plants remains unclear. It has been suggested that it could act as an alternative carbon store under elevated CO2 conditions, as reported in Kalanchoe pinnata,26 contributing to carbon and phosphorus homeostasis in CAM plants. Pitkanen et al.32 first evidenced the existence of sedoheptulose in human urine and recently it has also been found in blood spots33 and within mouse cells.34 In recent years, sedoheptulose has been patented for the treatment of inflammation35 and as nutritional supplement.36

3.1.2 | 2‐C‐Methyl‐D‐erythritol (2S,3R)

The 2‐C‐Methyl‐D‐erythritol is a soluble carbohydrate and is one of the four stereoisomers of 2‐methyl‐1,2,3,4‐butanetetrol. These four components rarely exist in plants. However, they are often recognised as secondary organic aereosols in the atmosphere above the Amazo- nian rainforest and Chinese and European forests, thus suggesting that they may be emitted from living plants.37 The polyalcohol moiety was identified as 2‐methyl‐1,2,3,4 butanetetrol by 1H‐ and 13C‐NMR signals and by the correlation between the HMBC and HSQC spectra (Table 3). In the positive ESI‐MS a quasimolecular ion peak was observed at m/z 159 [M + Na]+. The absolute configuration was determined as 2S,3R by comparing its NMR data and optical rotation with those reported in the literature.38 In the natural compound from S. tectorum we found [α] 20 D = +6.42 (c=0.5 MeOH). In the last few years a non‐mevalonate pathway has been discov- ered in bacteria and higher plants39 and 2‐C‐methyl‐D‐erythritol has been considered as a putative C5 intermediate in the alternative mevalonate‐independent pathway for terpenoids biosynthesis. The 2‐C‐methyl‐D‐erythritol is believed to derive from the condensation of pyruvate and glyceraldehyde‐3‐phosphate40 and this unit has previously been isolated only from Convolvolus glomerulatus,41 Liriodendron tulipiferga,42 Ferula sinaica43 and Phlox sublata L.44 FIGURE 3 Src‐mediated EGFR trans‐phosphorylation triggers STAT3 pathway. Serum starved HCT cells were stimulated with different 3.1.3 | Free monosaccharides concentration of SVT fractions containing high levels of sedoheptulose and malic acid (A), or were pre‐incubated with 2 μM AG1478 (B), or Free D‐fructose was the most abundant monosaccharide identified in 10 μM PP2 or 10 μM PP3 (C), before the stimulation with 100 μg/mL ‐ the extract of S. tectorum. As a free sugar, the fructose has a complex SVT. Proteins were resolved on 10% SDS PAGE and STAT3 Y705 phosphorylation was detected with a phospho‐specific antibody. 1H‐NMR spectrum as a result of its existence in at least five tautomers Phosphorylated proteins levels were measured by densitometry. An in different solutions. In water at 20°C the distribution of β‐pyranose, anti‐Tubulin antibody served as control for loaded protein. All blots are β‐furanose, α‐furanose, α‐pyranose and the keto tautomers was found representative of at least three independent experiments (*P < 0.05 vs. to be 68.23%, 22.35%, 6.24%, 2.47% and 0.50%, respectively.45 The serum starved cell; §P < 0.05 vs. SVT 100 μg/mL stimulated cells) 8 CATTANEO ET AL.

FIGURE 4 Natural components from Sempervivum tectorum L. leaves were sufficiently resolved for both anomers of α‐ and β‐glucopyranose According to the previous studies on S. tectorum L., only the flavo- (5.24 ppm H‐1ofα‐D‐glucopyranose; 4.65 ppm H‐1of nol glycoside compounds with the kaempferol skeleton were isolated β‐D‐glucopyranose). in the ST2 extract while in the ST1 extract, they were found in small amounts. Their structures were determined by correlating the spectral data (1H‐, 13C‐NMR, 2D‐NMR experiments, ESI‐MS) with literature values.5,19 | 3.1.4 Organic acids and flavonol glycosides Two major flavonoids were identified as kaempferol‐3‐O‐β‐[α‐ rhamnopyranosyl‐(1 → 2)glucopyranoside]‐7‐O‐α‐rhamnopyranoside5 Simple organic acids are typical products of the CAM. In our extracts and kaempferol‐3‐O‐α‐rhamnopyranosyl‐7‐O‐α‐rhamnopyranoside the most abundant was malic acid, also detected as sodium salt, which showed respectively in the ESI‐MS (negative ion mode) followed by isocitric acid. Their 1H‐ and 13C‐NMR signals are reported quasimolecular ion peaks at m/z 739 (M‐H)− and m/z 577 (M‐H)−. in Table 3. The malic acid showed in the negative ESI‐MS a quasimolecular ion peak at m/z133 (M‐H)− while the isocitric acid − evidenced a quasimolecular ion peak at m/z191 (M‐H) in the 3.2 | Wound healing activity ESI‐MS (negative ion mode). The malic acid sodium salt showed in the ESI‐MS (positive ion mode) a quasimolecular ion peak at m/z 157 Purified fractions of S. tectorum containing high levels of sedoheptulose + α 20 − [MNa +H]and an optical rotation [ ]D = 2.64 (c=0.7 MeOH). and malic acid enhance cell proliferation and migration. CATTANEO ET AL. 9

The process of repair that follows injury to the skin and other soft We observed a significant increase of the phosphorylation level of tissues is also known as wound healing. The skin wound healing and the Y705 residue in cells incubated for 5 min with the ST1 fractions repairing effect is promoted by several plant products and has been (Figure 3A). We also observed that pre‐incubation with 2 μM AG1478 recently demonstrated for some traditionally used herbs, such as (Figure 3B) or 10 μM PP2 (Figure 3C) prevents the phosphorylation of Malva silvestris46 and Opuntia ficus indica,47 and tropical plants, such the Y705 residue of STAT3. as Aloe vera.48 Taken together these data suggest that stimulation with the ST1 Cellular proliferation and migration are crucially involved in tissues fractions, at the final concentration of 100 μg/mL, enhances prolifera- repair and in many physiopathological processes. However, the effects tion and cellular migration through a molecular mechanism which of the intracellular signalling cascades involved in these processes are requires the c‐Src‐mediated transactivation of EGFR. EGFR Y845 little known. Therefore, we tested the effects of purified fractions of S. phosphorylated residue triggers, in turn, the activation of the STAT3 tectorum (Table 1) on cellular proliferation and migration in epithelial pathways and the nuclear translocation of this transcriptional factor, HCT 116 cells. which could be crucially involved in the transcription of genes associ- We incubated cells with different purified fractions of S. ated with cellular proliferation and migration. Further studies are in tectorum, at different concentrations, for up to 72 h, and we progress to analyse the molecular mechanism responsible for the c‐ analysed the cellular migration and proliferation. We observed that Src activation mediated by the ST1 fractions. the ST1 fraction containing high levels of sedoheptulose and All natural compounds have been reported in Figure 4. malic acid (fractions C and D, Table 1) at the final concentration of 100 μg/mL, significantly improves the cellular migration ACKNOWLEDGEMENTS (Figure 1A) and ameliorates proliferative activity (Figure 1B) of This research was supported by the University of Naples Federico II, HCT 116 cells. Project “Basi molecolari del morbo di Alzheimer”, 2019. NMR spectra The epithelial wound repair involves both migration and prolifera- were provided by Centro di Servizio Interdipartimentale di Analisi 49 tion and in these processes the tyrosine kinase receptors (RTK) are Strumentale (CSIAS), Università di Napoli “Federico II”, Napoli, Italy. crucially involved.50 EGFR is a transmembrane RTK that triggers many signalling pathways, which are crucially involved in several processes, ORCID such as angiogenesis, inhibition of apoptosis, cell migration and prolif- Fabio Cattaneo https://orcid.org/0000-0002-5833-8333 eration. We observed that incubation for 5 min with the fractions C Simona De Marino https://orcid.org/0000-0002-0300-5048 and D of SVT, at a final concentration of 100 μg/mL, induces a signif- Melania Parisi https://orcid.org/0000-0003-4064-6634 icant phosphorylation of EGFR on its cytosolic tyrosine residue in Carmen Festa https://orcid.org/0000-0001-8879-4332 position 845 (Y845) (Figure 2A). 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