Bioactive Metabolites of the Stem Bark of Strychnos aff . darienensis and Evaluation of Their Antioxidant and UV Protection Activity in Human Skin Cell Cultures

The genus Strychnos (Loganiaceae) is well-known as a rich source of various bioactive metabolites. In continuation of our phytochemical studies on plants from Amazonia, we examined Strychnos aff. darienensis, collected in Peru. This species has been traditionally used in South America and is still presently used as a drug by the Yanesha tribe in Peru. Phytochemical investigation of this plant led to the isolation and structure elucidation by NuclearMagnetic Resonance and High Resolution Mass Spectroscopy of 14 compounds that belong to the categories of phenolic acids [p-hydroxybenzoic acid (1) and vanillic acid (2)], flavonoids [luteolin, (3),3-O-methyl quercetin (4), strychnobiflavone (5), minaxin (6) and 3’,4’,7-trihydroxy-flavone (7)], lignans [syringaresinol-β-D-glucoside (8), balanophonin (9) and ficusal (10)] and alkaloids [venoterpine (11), 11-methoxyhenningsamine (12), diaboline (13) and 11-methoxy diaboline (14)]. The isolated flavonoids—a class known for its anti-aging activities—were further evaluated for their biological activities on normal human skin fibroblasts. Among them, only (6), and to a lesser extent (7), exhibited cytotoxicity at 100 μg/ml. All five flavonoids suppressed intracellularreactive oxygen species (ROS) levels, either basal or following stimulation with hydrogen peroxide or both. Moreover, luteolin and strychnobiflavone protected skin fibroblasts against ultraviolet (UV)-irradiation-induced cell death. The isolated flavonoids could prove useful bioactive ingredients in the cosmetic industry.


Introduction
The flora of the Amazonian forests has proven to be an important source of bioactive ingredients, with a high potential as pharmaceutical or protective agents.The rich ethnobotany of South and Central America is still under thorough investigation for the discovery of skin care products, based on the traditional uses of raw plants or preparations by the indigenous residents [1].The genus Strychnos, although known as the source of highly toxic compounds, still remains a promising source of bioactive ingredients.
Species Strychnos darienensis, closely affiliated to the species studied herein, was initially identified by the botanist Seeman Berthold during an expedition in the Isthmus of Panama (or previously known as Isthmus of Darien), in 1845.Genus Strychnos is a rich source of secondary metabolites, although it is characterized by the presence of terpene indole alkaloids [2].The highly toxic strychnine is without a doubt the most well-known Strychnos alkaloid, although the genus has provided more than 400 different indole alkaloids since its discovery.The presence of those compounds, as well as the traditional use of Strychnos in the preparation of the deadly curare, has led the researchers to focus mainly on the toxicity and less on the medicinal properties of the plant, although the Yanesha use is quite original and definitely medicinal.
The stem bark of Strychnos aff.darienensis has been used for centuries for the preparation of curare from the South American Indian hunters [3] and from the tribe Yanesha as medicine for respiratory and intestinal problems.The medicinal preparation to treat possible infection from worms or parasites involves the consumption of the decoction for three days, with extreme caution [4].
It is important to notice that not all compounds isolated from the genus Strychnos correspond to alkaloids.Among them, phenolic acids, lignans, terpenes, iridoids, and flavonoids have been isolated [5] with various medicinal properties.
Interestingly, other plants of the genus Strychnos have been used for the treatment of various skin disorders [6]; hence, in continuation of our interest inthe plants of Amazonia [7,8], we examine here for the first time the phytochemical composition of Strychnos aff.darienensis under the scope of the antioxidant and photoprotective activities of its components.

General
Fast Centrifugal Partition Chromatography (FCPC) was carried out on a Kromaton FCPC instrument equipped with a column of 1000 mL, adjustable rotation of 200-2000 rpm and a preparative Laboratory Alliance pump with a pressure safety limit of 50 bar.A manual sample injection valve was used to introduce the samples into the column.NMR spectra were recorded at 400 and 600 MHz (Bruker Advance III 600 MHz and Bruker DRX 400, Bruker, Karlsruhe, Germany) in MeOD.2D-NMR experiments, including COSY, HSQC, and HMBC were performed using standard Bruckermicroprograms.The ESI-MS experiments were performed on an LTQ-Orbitrap XL (Thermo-Scientific, Brehmen, Germany).Analytical Thin Layer Chromatography (TLC) was performed on Merck Kieselgel 60 F 254 or RP-8 F 254 plates (Merck, Darmstadt, Germany).Spots were visualized by UV light (254 and 365 nm, Philips, Eindhoven, Netherlands) or by spraying with sulfuric vanillin.The plates were then heated for 5 min at 110 • C. Size exclusion chromatography was performed using Sephadex LH-20 (GE Healthcare, Uppsala, Sweden).

Plant Material
The stem bark of Strychnos aff.darienensis was collected from Peru in November 1997 and identified by Dr. Sydney McDaniel.The collected plant material was dried by freeze-drying, pulverized, and stored in dark glass bottles where oxygen was replaced by nitrogen for atmospheric stability.The pulverized plant material was kept in a cool, dry, and dark room until used.A voucher specimen (IBE12195-B) was deposited in the National Center for Natural Products Research in the University of Mississippi, USA.
The stem bark of Strychnos aff.darienensis (600 g) was initially treated with 400 mL EtOAc/ EtOH/NH 3 (96:3:1) and subsequently, extraction by maceration followed, using EtOAc three times (3 × 3.5 L) and then with MeOH three times (3 × 3.5 L) for 24 h each.Solvents were removed under vacuum and the corresponding dry extracts of EtOAc (3.18 g) and MeOH (27.01 g) were stored at −20 • C until used.
The MeOH extract (27 g) was subjected to absorbent chromatography using the XAD4 resin (650 g) and was eluted with EtOH to afford 13.63 g of extract (yield 51.4%).This extract was diluted in 750 mL EtOAc and was subjected to liquid-liquid extraction with 750 mL (×3 times) of 4% HOAc to give fraction M1 (4.19 g).The remaining acidic (pH 3) solution was extracted by CH 2 Cl 2 (1.2 L × 3 times) to give fraction M2 (241.0 mg), then basified to pH 8 with Na 2 CO 3 and repeatedly extracted with CH 2 CL 2 (1.2 L × 3 times) to give fraction M3 (240.5 mg).The same extraction was made at pH 10 (alkalinization with NH 3 ) and pH 12 to afford fractions M4 (92.5 mg) and M5 (84.9 mg), respectively.The water after evaporation gave a residue of 8.5 g, see Supplementary Material, Figure S1.

Cells and Cell Culture Conditions
A commercially available normal human neonatal foreskin fibroblast strain was used (AG01523c, Coriell Institute for Medical Research, Camden, NJ, USA).Cells were routinely cultured in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 15% fetal bovine serum (FBS), as described previously [9], and subcultured twice a week at a 1:2 split ratio, using a trypsin-citrate solution (0.25-0.3%, respectively).Cell counting after trypsinization was performed using a Z1 Coulter counter (Beckman Coulter International SA, Nyon, Switzerland).Cells were tested periodically and found to be mycoplasma free.The cells were used within 10 passages from their purchase.

Assessment of Cytotoxicity
The cells were plated in 96-well flat-bottomed microplates at a density of 7000 cells/well in DMEM 15% Fetal Bovine Serum (FBS) and were left to adhere for 18 h.Then, the test compounds were added, appropriately diluted with Dimethyl Sulfoxide (DMSO) in serum-free DMEM.After a 72-h incubation, the medium was replaced with methylthiazolyldiphenyl-tetrazolium bromide (MTT; Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) dissolved at a final concentration of 1 mg/mL in serum-free, phenol-red-free DMEM (Biochrom) for a further 4 h of incubation.Then, the MTT formazan was solubilized in 2-propanol and the optical density was measured using an Infinite ® M200 microplate reader (Tecan, Männedorf, Switzerland) at a wavelength of 550 nm (reference wavelength 690 nm).

IntracellularReactive Oxygen Species (ROS) Assay
The cells were plated in 96-well clear-bottomed black microplates at a density of 7000 cells/well in DMEM 15% FBS and were left to adhere for 18 h.Then, the test compounds were added and appropriately diluted with DMSO in serum-free, phenol-red-free DMEM.After a 24 h incubation, 2 ,7 -Dichlorodihydrofluorescein diacetate (DCFH-DA) (Sigma-Aldrich) was added at a 10-µM final concentration for a further 45 min.Then, the medium was replaced by phosphate buffered saline (PBS), and, after a further 15 min, fluorescence emission was determined at 520 nm following excitation at 485 nm in an Infinite ® M200 microplate reader (Tecan).Alternatively, 30 min after DCFH-DA addition, the cell cultures received H 2 O 2 at a final concentration of 500 µM for 15 min, and then fluorescence determination, as described above, followed.

UV-Protection Assay
Photo-protective activity was assessed by a modification of the method used previously [10].Briefly, cells were plated in 96-well flat-bottomed microplates at a density of 7000 cells/well in DMEM 15% FBS and were left to adhere for 18 h.Then, the test compounds were added and appropriately diluted with DMSO in serum-free, phenol-red-free DMEM.After pre-incubation for 24 h, cells were irradiated under aseptic conditions using four Sankyo Denki (Hiratsuka, Kanagawa, Japan) UV-B lamps (energy spectrum 280-360 nm peaking at 306 nm) for 10 min (726 mJ/cm 2 ).Following further incubation for 72 h, viability was assessed by using the MTT assay, as described above.

Biological Evaluation of The Isolated Flavonoids
For the biological evaluation, the well-studied class of flavonoids-known to possess limited cytotoxicity and significant anti-aging activity [26,27]-was selected to be evaluated for their antioxidant and UV-protection activity.Alkaloids were excluded for any further evaluation due to their high toxicity [3,28], while phenolic acids and lignans were less promising as antioxidants than flavonoids since they had fewerfree hydroxyl groups [29,30].The first step was the evaluation of the cytotoxicity of the isolated compounds that was performed on human skin fibroblasts using the MTT-method.The compounds tested were luteolin (3), 3-O-methyl-quercetin (4), strychnobiflavone (5), minaxin (6), and 3',4',7-trihydroxyflavone (7).As shown in Figure 3, minaxin (6) was the most toxic compound for human fibroblasts, suppressing their viability to 56% (±1) of the control at the highest concentration tested (100 µg/ml), while at 20 µg/mL, viability was 75% (±3) of the control.3',4',7-trihydroxyflavone (7) was less cytotoxic since it suppressed viability to 68% (±16) of the control only at the highest concentration (100 µg/mL).Hence, this concentration (i.e., 100 µg/mL) was excluded during the assessment of the antioxidant and UV-protection capacities of 6 and 7.The remaining three compounds werecompletely non-cytotoxic and actually enhanced cell viability to 140-148% of the control at the highest concentration of 100 µg/ml, see Figure 3.In total, fourteen (1-14) compounds have been isolated and identified using spectroscopic methods.1D and 2D NMR experiments, see Supplementary Material, Tables S2-S15, as well as, high-resolution MS led to unambiguous structure elucidation of the isolated compounds.All isolated compounds had identical spectroscopic data tothose previously described.

Biological Evaluation of The Isolated Flavonoids
For the biological evaluation, the well-studied class of flavonoids-known to possess limited cytotoxicity and significant anti-aging activity [26,27]-was selected to be evaluated for their antioxidant and UV-protection activity.Alkaloids were excluded for any further evaluation due to their high toxicity [3,28], while phenolic acids and lignans were less promising as antioxidants than flavonoids since they had fewerfree hydroxyl groups [29,30].The first step was the evaluation of the cytotoxicity of the isolated compounds that was performed on human skin fibroblasts using the MTT-method.The compounds tested were luteolin (3), 3-O-methyl-quercetin (4), strychnobiflavone (5), minaxin (6), and 3',4',7-trihydroxyflavone (7).As shown in Figure 3, minaxin (6) was the most toxic compound for human fibroblasts, suppressing their viability to 56% (±1) of the control at the highest concentration tested (100 µg/ml), while at 20 µg/mL, viability was 75% (±3) of the control.3',4',7-trihydroxyflavone (7) was less cytotoxic since it suppressed viability to 68% (±16) of the control only at the highest concentration (100 µg/mL).Hence, this concentration (i.e., 100 µg/mL) was excluded during the assessment of the antioxidant and UV-protection capacities of 6 and 7.The remaining three compounds werecompletely non-cytotoxic and actually enhanced cell viability to 140-148% of the control at the highest concentration of 100 µg/ml, see Figure 3.
Compounds 3, 4, 5, and 6 were found to inhibit the basal levels of intracellular reactive oxygen species (ROS) dose-dependently, see Figure 4A, with the most efficient being luteolin (3).In fact, 3 at 100 µg/mL was more efficient than Trolox, which was used as a positive control (38% vs. 50%, 0.0002).On the other hand, 3',4',7-trihydroxyflavone (7) was not attenuating the basal levels of intracellular ROS, as shown in Figure 4A.All compounds, with the exception of strychnobiflavone (5), were also observed to inhibit the hydrogen peroxide-induced ROS stimulation, see Figure 4B; although, none of them was as potent as the positive control (Trolox).Our results agree with literature reports from epithelial cells regarding the antioxidant activity of 3 [31] and 4 [32], while this is the first study to show the capacity of 5, 6, and 7 to suppress ROS in human skin fibroblasts.Compounds 3, 4, 5, and 6 were found to inhibit the basal levels of intracellular reactive oxygen species (ROS) dose-dependently, see Figure 4A, with the most efficient being luteolin (3).In fact, 3 at 100 µg/mLwas more efficient than Trolox, which was used as a positive control (38% vs. 50%, 0.0002).On the other hand, 3',4',7-trihydroxyflavone (7) was not attenuating the basal levels of intracellular ROS, as shown inFigure 4A.All compounds, with the exception of strychnobiflavone (5), were also observed to inhibit the hydrogen peroxide-induced ROS stimulation, seeFigure 4B; although, none of them was as potent as the positive control (Trolox).Our results agree with literature reports from epithelial cells regarding the antioxidant activity of 3 [31] and 4 [32], while this is the first study to show the capacity of 5, 6, and 7 to suppress ROS in human skin fibroblasts.Concerning the capacity of the compounds to protect from UV-B irradiation, the most active was luteolin (3), which at both concentrations of 20 and 100 µg/mL fully protected human skin Concerning the capacity of the compounds to protect from UV-B irradiation, the most active was luteolin (3), which at both concentrations of 20 and 100 µg/mL fully protected human skin fibroblasts from UV-B, as shown in Figure 5, in agreement with previous observations [33].Strychnobiflavone (5) was also capable to reverse UV-B-induced lethality in a dose-dependent manner, while compounds 7 and 4 exhibited a minor protective effect at the concentration of 20 µg/mL, see Figure 5. Interestingly, strychnobiflavone has been identified as one of the metabolites from the Brazilian plant Strychnos pseudoquina possibly related with its anti-inflammatory and wound healing properties [34][35][36].Concerning the capacity of the compounds to protect from UV-B irradiation, the most active was luteolin (3), which at both concentrations of 20 and 100 µg/mL fully protected human skin fibroblasts from UV-B, as shown in Figure 5, in agreement with previous observations [33].In conclusion, the flavonoids isolated from Strychnos aff.darienensis in the present study are capable of suppressing intracellular ROS levels, while two of them, luteolin (3) and strychnobiflavone (5), act as protective agents against UV-irradiation, properties that imply that they may be used as bioactive ingredients in the food and cosmetic industries.

Figure 3 .
Figure 3. Cytotoxicity of the isolated compounds.The cytotoxicity of the isolated compounds was evaluated in human skin fibroblasts using the MTT-method, as described in the Materials and Methods.Results are expressed as a percentage of the control and represent the mean of three independent experiments performed in quadruplicate.

Figure 3 .
Figure 3. Cytotoxicity of the isolated compounds.The cytotoxicity of the isolated compounds was evaluated in human skin fibroblasts using the MTT-method, as described in the Materials and Methods.Results are expressed as a percentage of the control and represent the mean of three independent experiments performed in quadruplicate.

Figure 4 .
Figure 4. Intracellular antioxidant activity of the isolated compounds.The ability of the isolated compounds to attenuate basal (a) or hydrogen-peroxide-stimulated (b) (ROS) in human skin fibroblasts was evaluated using DCFH-DA, as described in the Materials and Methods.Vehicle (DMSO) was used as negative control and Trolox as positive one.Results are expressed as percentages of the value of the respective control concentration, and they represent the mean of three independent experiments performed in quadruplicate (* 0.01 < p≤ 0.05; ** p ≤ 0.01).

Figure 5 .
Figure 5. UV-protective capacity of the isolated compounds.The capacity of the isolated compounds to protect human skin fibroblasts from the lethal effects of UV-B-irradiation was assessed as described in the Materials and Methods.Results are expressed as absorbance values at 550 nm (reference wavelength 690 nm) and represent the mean of three independent experiments performed in quadruplicate.

Figure 4 .
Figure 4. Intracellular antioxidant activity of the isolated compounds.The ability of the isolated compounds to attenuate basal (a) or hydrogen-peroxide-stimulated (b) (ROS) in human skin fibroblasts was evaluated using DCFH-DA, as described in the Materials and Methods.Vehicle (DMSO) was used as negative control and Trolox as positive one.Results are expressed as percentages of the value of the respective control concentration, and they represent the mean of three independent experiments performed in quadruplicate (* 0.01 < p ≤ 0.05; ** p ≤ 0.01).

Figure 4 .
Figure 4. Intracellular antioxidant activity of the isolated compounds.The ability of the isolated compounds to attenuate basal (a) or hydrogen-peroxide-stimulated (b) (ROS) in human skin fibroblasts was evaluated using DCFH-DA, as described in the Materials and Methods.Vehicle (DMSO) was used as negative control and Trolox as positive one.Results are expressed as percentages of the value of the respective control concentration, and they represent the mean of three independent experiments performed in quadruplicate (* 0.01 < p ≤ 0.05; ** p ≤ 0.01).

Figure 5 .
Figure 5. UV-protective capacity of the isolated compounds.The capacity of the isolated compounds to protect human skin fibroblasts from the lethal effects of UV-B-irradiation was assessed as described in the Materials and Methods.Results are expressed as absorbance values at 550 nm (reference wavelength 690 nm) and represent the mean of three independent experiments performed in quadruplicate.

Figure 5 .
Figure 5. UV-protective capacity of the isolated compounds.The capacity of the isolated compounds to protect human skin fibroblasts from the lethal effects of UV-B-irradiation was assessed as described in the Materials and Methods.Results are expressed as absorbance values at 550 nm (reference wavelength 690 nm) and represent the mean of three independent experiments performed in quadruplicate.