Introduction

Ultraviolet (UV) radiation can cause cancer, premature aging, sunburns, and wrinkles. Skin cancer is a malignant tumor disease common in fair-skinned, light-eyed people, with blond hair (Ferlay et al. 2019). There is a permanent need for protection from UV radiation and prevention from its side effects. Currently, there is a strong tendency to use biodegradable and safe natural products in formulations that could prevent skin cancer. Herbal preparations have a high potential due to their antioxidant activity. Their phenolic compounds can scavenge reactive oxygen species, reduce skin alterations caused by UV exposure, and prevent aging (Estrella-Parra et al. 2019).

Ipomoea genus comprises ca. 700 species distributed in tropical and subtropical regions (Meira et al. 2012). Ipomoea horsfalliae Hook., Convolvulaceae, called “morning glory,” has simple and dark green alternate leaves; its inflorescences have deep fuchsia color and grow on the terminal part of the branches (Delgado et al. 2014). Ipomoea spp. have applications in medicine, due to their hypotensive, antimicrobial, anticancer (Bieber et al. 1986), and antidiabetic properties (Kusano and Abe 2000). The compounds responsible for the biological activity of these species include flavonoids, coumarins, isocoumarins, benzenoids, anthocyanins, glycolipids, and lignans (Truong et al. 2007; Meira et al. 2012; Batiga et al. 2019).

There is a high incidence of skin cancer in Colombia, and melanoma cancer (MC) is the most common type. In 2018, MC caused 518 deaths and 1907 new cases were registered (The Global Cancer Observatory 2019). Twenty-one species of Ipomoea genus are recognized as medicinal plants in Colombia (Bernal et al. 2011); their potential as primary sources of chemicals with protective effect against UV has not been studied yet. This study evaluated the potential of I. horsfalliae flower extract (IHFE) as a starting point for research programs that led to the development of natural skin care products. Chemical characterization, in vitro cytotoxic and sensitizing effects, and in vivo photoprotective effect of IHFE were evaluated.

Materials and Methods

Chemicals

Cyanidin-3-rutinoside, delphinidin-3-glucoside, and kaempferol-3-glucoside were purchased from PhytoLab (Vestenbergsgreuth, Germany). Kaempferol, chlorogenic acid, MTT 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, 2-aminophenol, p-formaldehyde, and eosin were purchased from Sigma-Aldrich (St. Louis, USA). Parsol® was purchased from Alfadelta S.A. of C.V. (Naucalpan de Juárez, Mexico).

Plant Material

Ipomoea horsfalliae Hook., Convolvulaceae, flowers were collected from an experimental plot at CENIVAM (N 07° 08,422′ W 073° 06,960′) in March 2017. Voucher specimen (COL 587134) was deposited at the Colombian National Herbarium, National University of Colombia. Undamaged, fully developed flowers were dried in a VirTis AdVantage Plus tray lyophilizer (Gardiner, USA).

Solvent Extraction

Dried flowers (1 g) were mixed with an ethanol solution (20 ml, 0.5% HCl, 1:1 v/v) and deposited for 5 min in an ultrasound bath (Elmasonic S15H, Singen, Germany). The mixture was filtered, and the residue was extracted twice more. IHFE was rotoevaporated and then dried in a VirTis AdVantage Plus tray lyophilizer.

UHPLC-ESI+-Orbitrap-MS Analysis

Flower extracts were analyzed on an UHPLC Dionex™ UltiMate™ 3000 (Thermo Fisher Scientific TFS, Bremen, Germany), coupled to an Orbitrap™ mass detector (Exactive Plus, TFS, Bremen, Germany), using a heated-electrospray interface (HESI-II), operated in positive ion mode (350 °C). Separation used a Hypersil GOLD™ aQ column (TFS, Sunnyvale, USA), of 100 mm × 2.1 mm id, × 1.9 μm of particle size, at 30 °C. The mobile phase was as follows: A: water (0.2% formic acid) and B: acetonitrile (0.2% formic acid). Analysis started with 100% A and changed linearly up to 100% B in 8 min, remained for 4 min, then returned to 100% A in 1 min, where it remained in equilibrium for 3 min. Flow was 0.3 ml/min and the injection volume 1 μl. Capillary voltage (3.5 kV, 320 °C) and higher energy collisional dissociation (HCD) were used. Mass range in all experiments was set at m/z 80–1000. The data obtained was processed with the Thermo XCalibur™ Roadmap software, version 3.1.66.10. Compound identification was based on the extracted ion current (EIC), the exact masses of the protonated target compounds, and by comparison with certified standards.

Cytotoxicity Assay

MTT assay (Mosmann 1983) was used to evaluate the potential of IHFE to reduce the viability of human cells. Six cell lines were selected (Table S1, Supplementary material), which represented cells from hepatocellular carcinoma (HepG-2), leukemia (THP-1), normal kidney (HEK-293), normal lung (MCR-5), and xeroderma pigmentosum (XP4PA and XP12RO-SV). Confluent monolayers of cells in 96-well cell culture microplate were treated with IHFE at concentrations in the range of 50 to 500 μg/ml for 3 days at 37 °C in a 5% CO2 humidified atmosphere. Non-treated cells were run in parallel. MTT (5 mg/ml) was added to each well (20 μl), and the microplate was kept at 37 °C for 4 h. Dimethylsulfoxide (100 μl) was added, and absorbances were measured at λ = 580 nm using a plate reader. A dose-response curve was plotted, and the half-maximal cytotoxic concentration (CC50) was determined from the plot.

In Vitro Test to Screen Skin Sensitizers in IHFE

The production of interleukin (IL)-8 by stimulated THP-1 cells is used as a biomarker of sensitizing effect (Takahashi et al. 2011); in the present study, the protocol described by Parise et al. (2015) was followed. THP-1 cells were seeded at a density of 2.5 × 105 cells/ml in 24-well plates and were incubated in culture medium with or without IHFE at a non-cytotoxic concentration of 80 μg/ml for 24 h at 37 °C, 5% CO2. Cells incubated in culture medium with 50 μM of 2-aminophenol were run in parallel. 2-Aminophenol is recognized as a strong sensitizer (Parise et al. 2015). The supernatants from cell cultures were collected for the determination of IL-8 by using an ELISA Kit (Invitrogen™). Three independent experiments in duplicate were carried out.

Protective Effect Against Skin Damage Induced by UV-B

Photoprotective activity was measured, according to Estrella-Parra et al. (2019), with some modifications. The procedure was performed with 20 BALB/c mice, from 4 to 6 weeks of age, and a weight of 16 ± 2 g. The mice were depilated with Nair™ sensitive skin cream (Church and Dwight, Princeton, USA) 24 h before starting the experiments.

UV-B exposure acute effect on mouse skin was studied in A, B, C, and D groups of five individuals. Groups A and B were treated with ethanol applied topically (100 μl, 70% v/v). Groups A and C were not irradiated. In groups C and D, the dorsal area of each mouse was divided into two and Parsol® (100 μl, 30 mg/ml) and IHFE (100 μl, 30 mg/ml) were applied on the left and right zones, respectively.

Groups B and D were irradiated for 3 min with a Spectroline EB-280C UV-B lamp (λ = 312 nm), located 15 cm away, with an irradiation dose of 6 mJ/cm2. This procedure was repeated three times, every 24 h. All mice were sacrificed by asphyxia in a CO2 chamber, 48 h after receiving their last radiation dose.

Histological Analysis

Histological analysis was performed according to Estrella-Parra et al. (2019). Samples were placed on cassettes for histology and suspended in a p-formaldehyde solution (500 ml, 2% w/v in phosphate buffer, 0.1 M, pH 7.2) for 24 h. They were washed with water (3 h), dehydrated in ethanol solutions (500 ml, 70, 80, 96, or 100% v/v) for 2 h, and included in paraffin. Samples of 5 μm thickness were cut in a Leica® RM2125RT rotation microtome (Leica Biosystems, Wetzlar, Germany). Histological sections were stained with hematoxylin and eosin (H&E) and were observed in a Leica® DM500 optical microscope.

Data Analysis

R software for Windows (version 3.5.2, http://www.R-project.org) was used. The dose-response curve was plotted, and the half-maximal cytotoxic concentration (CC50) was calculated. Results were expressed as the means ± standard error of the mean (SEM) from duplicate assays of independent experiments. Levels of significance were calculated by one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc and Student’s t tests.

Results

UHPLC-ESI+-Orbitrap-MS Analysis of IHFE

The extracted ion currents of the exact masses of [M]+ or [M + H]+ of compounds present in IHFE were obtained from the total ion current (TIC) (Fig. 1). In Table 1, the experimental exact masses of M+ and [M + H]+ identified in IHFE are shown together with the criteria used for compound identification, and with the main ion-fragments obtained at the HCD under different collisional energies. In Fig. 2, mass spectra of kaempferol-diglucoside and pelargonidin-sophoroside-glucoside are shown. The fragmentation pattern of these compounds is characterized by the consecutive loss of sugar moieties from [M + H]+ ions.

Fig. 1
figure 1

Ipomea horsfalliae flower extract chromatogram (extracted ion chromatogram, EIC), obtained by UHPLC-ESI+-Orbitrap-MS. Peak identification appears in Table 1

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Table 1 Characteristic ions exact masses of compounds, identified by UHPLC-ESI+-Orbitrap-MS in Ipomea horsfalliae flower extract
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Fig. 2
figure 2

Mass spectra of a kaempferol-diglucoside (HCD, 10 eV) and b pelargonidin-sophoroside-glucoside (HCD, 30 eV), obtained by UHPLC-ESI+-Orbitrap-MS

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Table 2 presents the linearity and sensitivity of the method used to quantify phenolic compounds by UHPLC-ESI+-Orbitrap-MS. Cyanidin-3-rutinoside, delphinidin-3-glucoside, kaempferol, kaempferol-3-glucoside, and chlorogenic acid were used as external standards. The analytical method had low detection (LOD = 0.1–0.3 μg/ml) and quantification (LOQ = 0.2–1 μg/ml) limits. Coefficients of determination (R2 = 0.9896–0.9990) demonstrated good method linearity in the range of concentrations evaluated (2–10 μg/ml).

Table 2 Linearity and sensitivity determined by UHPLC-ESI+-Orbitrap-MS for some phenolic compounds present in Ipomea horsfalliae flower extract
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Cytotoxicity

IHFE did not exhibit relevant cytotoxicity for both cancerous and normal cells according to dose-response curves (Fig. S1, Supplementary material) and the World Health Organization parameter (WHO 2019). CC50 values were higher than 200 μg/ml for cells derived from hepatocellular carcinoma (HepG-2, 270 ± 80 μg/ml), monocytic leukemia (THP-1 > 500 μg/ml), normal kidney (HEK-293, 340 ± 110 μg/ml), and normal lung (MRC-5, 250 ± 50 μg/ml). For XP cells, CC50 values were lower compared with normal cells (XP4PA, 110 ± 20 μg/ml; XP12RO-SV, 63 ± 5 μg/ml), which was expected since the cells were deficient in a key DNA repair protein, and therefore, they were more sensitive to treatment with IHFE (Maher et al. 1977).

Sensitizing Effect

Allergic contact dermatitis results from T cell–mediated immune responses induced by compounds called sensitizers. When THP-1 cells were stimulated with the representative sensitizer, 2-aminophenol, the level of IL-8 in the culture medium increased (from 1.079 ± 44 to 247 ± 77 pg/ml in non-stimulated cells; p < 0.01, ANOVA). In contrast, THP-1 cells stimulated with IHFE at a concentration of 80 μg/ml did not increase the IL-8 production (204 ± 54 pg/ml) which suggests the lack of potential sensitizing effect at the concentration tested.

Protective Effect of IHFE Against Skin Damage Induced by UV-B

Figure 3 illustrates the extent of histological skin lesions of BALB/c mice from group B, treated with ethanol (70% v/v) and UV-B. The number of lesions with an incidence of 100% was higher than that in group A, treated with ethanol and no UV-B exposure. Fusiform nuclei with focal extension were evidenced in the stratum corneum. Cells of burn, hyperplasia, hypertrophy, atrophy, and pleomorphism were manifested in the epidermis of all organisms (Fig. 4). Skin, exposed to UV radiation, accelerated mitotic activity, and the number of layers in the epidermis increased. A higher number of congested blood vessels, extravasation of erythrocytes, and polymorphonuclear cells were observed in the dermis.

Fig. 3
figure 3

Extension of lesions evaluated in the skin of the BALB/c mice treated with ethanol (70%, v/v), Parsol®, or Ipomea horsfalliae flower extract (3 mg/ml), exposed to UV-B. The extension is focal (0 to 20%), multifocal (20 to 50%), or diffuse (50 to 100%). Pk, parakeratosis; Hk, hyperkeratosis; Sp, spongiosis; BC, burn cells; Hp, hyperplasia; Ht, hypertrophy; At, atrophy; Pl, pleomorphism; Ed, edema; Hg, hemorrhage; IF, inflammatory infiltrates; CG, congestion

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Fig. 4
figure 4

BALB/c mouse skin histological sections of the group treated with ethanol (70% v/v, a and c) or Ipomea horsfalliae flower extract (3 mg/ml) and UV-B (d and e). H&E stain. SC, stratum corneum; E, epidermis; BC, burn cells; Ht, hypertrophy; At, atrophy; Pl, pleomorphism; D, dermis; SG, sebaceous gland; HF, hair follicle; RF, reactive fibroblasts; BV, blood vessel; M, muscle; IF, inflammatory infiltrates; H, hypodermis

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The extent of the histological lesions in the individuals treated with Parsol® or IHFE was lower than that in the group without protection (Fig. 3). The group with IHFE or Parsol® showed significant differences, according to Student’s t test, in comparison with the group treated with ethanol and UV-B. Stratum corneum presented normal appearance, contained flattened cells that lacked nuclei and organelles. In the epidermis, round burn cells were detected, with contracted nuclei and eosinophilic cytoplasm; the extension of these cells was multifocal and smaller than that of the group without protective substance. IHFE caused changes in cell growth in the epithelium (Fig.4); multifocal atrophy (30%), hypertrophy (5%), and focal hyperplasia (11%) were observed. Parsol® changed the size and shape of epithelial cells, which caused focal pleomorphism (8%) and multifocal atrophy (24%). There was no edema in the dermis of individuals covered with IHFE or Parsol®. In connective tissue, congestion of blood vessels (50%), erythrocyte extravasation (30%), and polymorphonuclear number (54%) were higher in mice treated with Parsol® than in those treated with IHFE.

Discussion

Evaluation of the cytotoxic effect for cultured cells is a preliminary approach to predict organ-specific toxicity, and the first step of the discovery process ending with a new natural anticancer product (Zhang et al. 2007). A standardized reference value of CC50 is not available to estimate the cytotoxic potential of plant extracts. It has been proposed that extracts with CC50 > 90 μg/ml could be classified as non-cytotoxic, while extracts with CC50 < 90 μg/ml as moderately or highly cytotoxic according to the WHO (2019). The US National Cancer Program suggests a cut-off for the effective dose 50 of 30 μg/ml in cell-based assays used to select plant extracts with potential anticancer effect (WHO 2019). IHFE seems not to have the potential to kill the cancerous cells tested in this study since it showed CC50 values > 250 μg/ml. Nevertheless, the result does not exclude the potential effect on other cancer cells. In addition, IHFE did not exhibit a relevant cytotoxic effect on cells derived from both normal kidney and lung, which are target cell models used in predictive toxicology testing. Consequently, IHFE might be a good candidate for research on natural products for human health. We recognize that our preliminary cytotoxic analysis of IHFE is limited; further studies with accurate and relevant tests should be carried out.

Many compounds in plant extracts can function as sensitizers because of their immunomodulatory effect. In three independent experiments, the levels of IL-8 in IHFE-stimulated and non-stimulated THP-1 cells were similar, which suggests the absence of a sensitizing effect. We could speculate that phenolic acids in IHFE could be responsible for the absence of IL-8 production. These phytochemicals displayed anti-inflammatory properties downregulating the NF-κB pathway signaling involved in the IL-8 gene transcription (Liu et al. 2018). A standardized reference value of effective dose 50 in the THP-1/IL-8 assay is not available to estimate the sensitizing effect of plant extracts. Consequently, further analysis with accurate and relevant tests such as the mouse local lymph node assay (LLNA) and human the cell activation test (h-CLAT) are required to investigate the presence of chemical sensitizers in IHFE.

Some compounds isolated from plants are used as sunscreens because they are antioxidants and absorb UV radiation (Radice et al. 2016). In IHFE, several anthocyanins were detected; cyanidin, pelargonidin, and their glycosylated derivatives have been recognized as antioxidants and photoprotective agents (Afaq et al. 2005). Glycosylated compounds of cyanidin, pelargonidin, and delphinidin were found in pomegranate fruits and showed a protective effect against skin changes in SKH-1 mice, induced by UV-B, and acted as modulators of photocarcinogenesis biomarkers (Afaq et al. 2005; Afaq et al. 2010).

Dicaffeoylquinic acid, a major component of IHFE, is an ester of caffeic and quinic acids (Truong et al. 2007). Caffeic acid protects the cells from the cytotoxic effect caused by UV-C; its photoprotective activity was evidenced in fibroblasts and epidermoid carcinoma cells. Proliferation of these cells exposed to UV-C was higher in the presence of caffeic acid (56 or 167 μM) compared with the control (Neradil et al. 2003). Caffeic acid esters act as sunscreens; they are stable against UV-A or UV-B radiation and have a sun protection factor of ca. 93% (Rivelli et al. 2010).

Scopoletin, found in IHFE, has been recognized for its anti-inflammatory (Moon et al. 2007), antioxidant (Parra et al. 2018), and vasodilator (Kwon et al. 2002) activities. Coumarin derivatives reduced the embryotoxic effects of UV-B radiation in sea urchin gametes and have been considered as possible photoprotectors (de Araujo Leite et al. 2015).

Results from this study suggest that the topical application of IHFE can prevent acute inflammatory response, reducing the number of burn cells, inflammatory infiltrates, and can prevent also intercellular edema. Some studies have shown the protective effect of phenolic compounds detected in IHFE, on the skin carcinogenesis induced by UV. Caffeoylquinic acid, quercetin-rhamnoside, and glycosylated compounds of kaempferol, present in Prunus persica (L.) Batsch flower extract, inhibited the increase in the number of layers of the epidermis and prevented oxidative deterioration by enzymatic modulation of superoxide dismutase and glutathione peroxidase (Kwak et al. 2018).

Conclusion

IHFE could be considered a good starting point for research programs which seek to develop natural skin care products. Topical application of IHFE on mouse skin reduced acute inflammation caused by exposure to UV-B. Phenolic acids, coumarins, flavonols, and anthocyanins could be responsible for the photoprotective effect of IHFE. Some limitations of this study need to be considered. IHFE toxicity and skin sensitization potentials need to be investigated using accurate and relevant tests, and the mechanistic explanation of the photoprotective effect should be provided. Despite these limitations, the present study gives valuable information on the potential use of I. horsfalliae flower extracts.