MATERIALS AND METHODS

Plant materials: Leaves of Bejaria aestuans Mutis ex L. (named BA), Cavendishia pubescens (Kunth) Hemsl. (named CP) and Cavendishia bracteata (Ruiz and Pav. Ex J. St.-Hil) Hoerold, (named CB) were collected at Llanos de Cuivá, Santa Rosa de Osos, Antioquia, Colombia, at 2730 m above sea level (Geographical coordinate: 6°49'50.6" N; 75°29'29.9" W). Botanical identification for BA, CP and CB was performed in the Herbarium of the University of Antioquia (HUA) and a voucher specimen was deposited under registry code HUA190418, HUA190421 and HUA190424, respectively.

Dry and powdered leaves of the B. aestuans, C. pubescens and C. bracteata (c.a. 1.0 g) were mixed with 50.0 mL of acetone/HCl (99.5:0.5 v/v) for 24 h at room temperature and magnetically stirred at 900 rpm. The resultant solution, previously filtered, was concentrated to dryness in a rotary evaporator under reduced pressure at 40°C to get rid of any residual solvent. Then, extracts of BA, CP and CE were redisolved in ethanol and keep at 4°C before analysis.

Phytochemical screening: Dry leaves of BA, CP and CB were powdered and then dissolved in ethanol:water (1:1) for determining the presence of chemical compounds like phenols, flavonoids, anthocyanin and tannins (Dahou et al., 2003).

Total Polyphenol Contents (TPC): The total polyphenol content in BA, CP and CB extracts was determined using the Folin-Ciocalteau colorimetric technique with some modifications (Singleton et al., 1999). Briefly, the BA, CP and CB extract solutions (c.a. 0.5 mL), were added to 0.125 mL of the Folin-Ciocalteau reagent and 1.25 mL of 20% Na₂CO₃. After incubation for 90 min at room temperature, the absorbance was read at 760 nm using an Evolution 60S spectrophotometer (Thermo Fisher Scientific, Inc., Shanghai, China). The results were expressed as Gallic acid equivalents per gram of dry extract (GAE g^–1 dry extract).

Total Anthocyanin Content (TAC): Total anthocyanins were estimated by a pH differential method (Lee et al., 2005) with some modifications. An aliquot (c.a. 1.0 mL) of solution of extract was mixed with 3.0 mL of buffer at pH 1.0 (HCl/KCl; 0.025 M) and another at pH 4.5 (acetic acid/sodium acetate; 0.4 M). After that, absorbance (A) was measured in a Thermo Scientific Evolution 60S UV-Visible spectrophotometer, at 510 and at 700 nm. The anthocyanin pigment concentration was expressed as cyanidin-3-glucoside equivalents using the following equation:

where, A= absorbance = (A₅₂₀-A_{700 nm})_pH1.0-(A₅₂₀-A_{700 nm})_{pH 4.5}.

The MW is the molecular weight for cyanidin-3-glucoside (cyd-3-glu) = 449.2 g mol^–1, DF is the dilution factor established, l is the path length of the cell in cm; ε is the molar extinction coefficient of cyd-3-glu = 26,900 L mol^–1 cm^–1 and 10³ is the conversion factor.

In vitro DPPH radical assay: The radical scavenging capacity for each fraction in different concentrations was estimated according to the method described previously (Brand-Williams et al., 1995) with some modifications; the effective relative concentration (EC₅₀) at which 50% of DPPH has been removed was expressed as mg of extract μmol^–1 of DPPH radical, based on the following equation:

The exact initial DPPH concentration (92.18 μmol L^–1) in the reaction system was calculated according to a calibration curve (y = 1.145 E-2× -4.192 E-3, r = 0.9999, where y = absorbance and x = concentration of DPPH) at 514 nm). All experiments were performed in triplicate. All spectrophotometric data were obtained using a Thermo Scientific Evolution 60S UV-Visible spectrophotometer. Disposable cuvettes (1×1×4.5 cm) were used for visible absorbance measurements for analyses.

UVA-UVB absorption capacity: Briefly, an aliquot (c.a. 50 μL) of crude extract was added to 2000 mL of methanol in a quartz cuvette and its absorption spectra (wavelengths 200-400 nm) were acquired using a Thermo Scientific Evolution 60S UV-Visible spectrophotometer against a blank containing methanol. The UVA-UVB absorption capacity was determined using the absorption coefficient (absorbance DM mg^–1 mL^–1) measured at 290, 310, 340 and 380 nm. Benzophenone 3 (B3), butyl methoxydibenzoylmethane (BMDM) and ethylhexyl methoxycinnamate (EHMC) were used as conventional sunscreen standards.

Statistical analysis: Results were expressed as Mean±Standard Deviation (at least three replicates). Analysis of variance and significant differences among means and correlation analysis were performed with one-way ANOVA. The experimental data was analyzed using SPSS V.21.0 software.

RESULTS AND DISCUSSION

The UV radiation accelerates skin aging leading to loss of skin elasticity and decreasing the capacity of the skin to hold water, which are implicated in the formation of the most obvious sign of aging, skin wrinkling. Some promising topical treatments of skin aging include herbal extracts, vitamin and antioxidant supplements, which have been widely accepted to scavenge free radicals from skin cells and to restore skin elasticity.

In this study, the phytochemical study showed the presence of phenols, flavonoids, anthocyanins, tannins and quinones in all three extracts (Table 1). Data from Ericaceae species is difficult to compare due to its environmental conditions. However, the results of phytochemical analysis obtained are in good agreement with data reported by some authors (Akkol et al., 2008; Harnafi et al., 2007), which have detected the presence of flavonoids, tannins, proanthocyanidins, coumarins on Ericaceae species.

A suitable mix of conventional sunscreens must absorb in UVA-UVB range and provide a broad-spectrum UVB/UVA protection. Extracts of B. aestuans, C. bracteata and C. pubescence showed an interesting broad-spectrum UVB/UVA absorption. However, B. aestuans and C. pubescence extracts displayed a higher ultraviolet radiation absorption in the range of UVB than C. bracteata extracts. Nevertheless, all of them showed a broad-spectrum range similar to the standards (Fig. 1). This result has been correlated with the presence of phenolic compounds in the extracts that have demonstrated good absorption of UV radiation (Stevanato et al., 2014). On the other hand, the absorption coefficients of the extracts was calculated in order to obtain comparative data at wavelength 290, 310, 340 and 380 nm, typical regions for UVA and UVB solar spectrum (Fig. 2). Here, we can observe that all of them have broad-spectrum coverage, but this was much higher in the UVB range.

Table 1:	Phytochemical screening of Bejaria aestuans, Cavendishia pubescence and Cavendishia bracteata leaf extracts
+: Detected, According to these results, the UV absorption spectra of the BA, CP and CB extracts and three common UV filters, Benzophenone-3 (B3), EHMC: Ethylhexyl methoxycinnamate and BMDM Butyl methoxydibenzoylmethane were recorded (Fig. 2)

Fig. 1:	UVA-UVB absorption coefficient assays (absorbance DM mg –1 mL–1) at 290, 310, 340 and 380 nm

Fig. 2:	UV spectrum of Bejaria aestuans, Cavendishia pubescence and Cavendishia bracteata leaf extracts (at 0.015 mg mL–1, all of them) and a mixed sample of benzophenone 3, butyl methoxydibenzoylmethane, ethylhexyl methoxycinnamate (at 0.017 mg mL–1, all of them)

Table 2:	In vitro antioxidant capacity, total polyphenol and anthocyanin contents
*Total phenolic content. **Effective concentration at 50%. ***Total anthocyanin content, Means in the same column followed by the same letter do not differ significantly (p<0.05)

This increase could be attributed to the inherent presence of polyphenols and anthocyanins in the extracts (Table 2), which have demonstrated the ability to act as broad-spectrum sunscreens, covering the UVA and UVB range (Velasco et al., 2008).

Polyphenolic compounds from plants exert their beneficial health effects mainly through their antioxidant capacity due to their property of scavenging free radicals, chelating metal catalysts, activating antioxidant enzymes and so on. Total Phenolic Content (expressed in GAE g^–1 DS) of the antioxidant extracts from selected plants are presented in Table 2. The highest concentration of TPC (27.35 mg GAE g^–1 DS) was obtained from C. pubescens, followed by B. aestuans and C. bractaeata. Difference analysis between the three cultivars of Ericaceae species showed significant differences (p<0.05) in TPC. The average TPC value (c.a 21.48 mg GAE g^–1 DS) of all selected Andean Colombian plants was higher than that reported for common cultivars, like strawberries (0.844 mg GAE g^–1) (Kajdzanoska et al., 2011), common legumes (0.57-9.60 mg GAE g^–1) (Xu et al., 2007) and Arbutusandrachne L. (Fam. Ericaceae) fruits from Turkey (2.4-4.1 mg GAE g^–1) ( Serce et al., 2010). Concerning the anthocyanin contents, the results were similar to those obtained with TPC and ranged from 0.36-3.31 mg C3GE g^–1 DS, with the highest content in C. pubescens extract, followed by B. aestuans and C. bractaeata (Table 2).

The antiradical activities of Ericacea plant extract were expressed as EC₅₀ value. The EC₅₀ values found were 2.64 E-04, 1.96 E-04 and 3.75 E-04 mg μmol^–1 DPPH of reaction medium in the reduction of DPPH. These results are closely to those reported by Guendouze-Bouchefa et al. (2015) on methanolic extracts of three Ericaceae species (Guendouze-Bouchefa et al., 2015). Therefore, the extracts showed an interesting antiradical capacity that could be linked to defense mechanisms against oxidative stress in plants of high altitude and could be useful as cell protectors in our own bodies.

Present study confirms that Ericaceae extracts should be useful asa potential source of natural photoprotection and antioxidants compounds because of their capability to absorb UV radiation and scavenge free radicals. In addition, a chemical study of these plants could be performed in order to identify novel compounds that are responsible of these biological properties.

CONCLUSION

The in vitro test showed that these matrices in association with conventional organic and inorganic sunscreens could be useful as potential ingredients on dermatological formulations with health benefits and will be used as an additive for photoprotection and free radical scavenging.

ACKNOWLEDGMENTS

Authors acknowledges CODI-University of Antioquia (Project No. IN632CE) for financial support.