Fragaria vesca var. nubicola Lindl. ex Hook.f.; commonly known as wild strawberry is synonym of Fragaria nubicola (Lindl. ex Hook.f.) Laciata. It is a perennial herb belonging to family Rosaceae that mostly grows along roadsides and in forests. The family Rosaceae comprises of 85 genera and nearly about 3000 species widely distributed in Europe and North America while represented by 27 genera and about 160 species in Pakistan (Stewart, 1972; Mabberley, 2008).

The vegetative parts of F. vesca var. nubicola has been used as stimulant, diuretic agent, detoxifying agent and for treatment of diarrhea (Neves et al., 2009). The achenes and thalamus parts of F. vesca has been reported to have high phenolic content and showed good antioxidant activity (Cheel et al., 2007). Literature revealed that fruits as well as whole plant of F. vesca var. nubicola has potential analgesic and anti-oxidant activity (Kanodia and Das, 2008; Kanodia et al., 2011). Similarly, the ethanolic extract of F. vesca var. nubicola possesses anti-convulsant activity and is effective for treatment of epilepsy (Patil et al., 2012).

Strawberry fruits reported to have phenolic compounds like ellagic acid, ellagic acid-glycoside, coumaryl glycoside along with various anthocyanidin as their glycosides. The fruits are also reported to have anti-oxidant, anticancer, anti-inflammatory and anti-neurodegenerative properties (Hannum, 2004; Seeram et al., 2006). As previously reported the phytochemical investigation showed agrimoniin; an anti-tumor and antidiarrheal agent as major ellagitannins in F. vesca (Miyamoto et al., 1987; Vrhovsek et al., 2012). The roots of F. vesca var. nubicola are rich in proanthocyanidin (Vennat et al., 1986). Depression is one of serious disease prevailing all over the world affecting 13–20% population (Licinio and Wong, 1999). The discovery of new antidepressants is of utmost importance as significant proportion of patients develops resistance against medicines already available in market. Plants could be an effective approach for discovery of new antidepressant agents that can act via different mechanisms (Zhang, 2004).

Owing to its multiple traditional uses and strong phytochemical background of F. vesca var. nubicola, the current study was designed for isolation and characterization of bioactive secondary metabolites followed by their evaluation for antidepressant potential in various animal models.

Two new benzyl derivatives have been isolated from ethyl acetate fraction of F. vesca var. nubicola through successive column chromatography, while characterization was made through various spectroscopic techniques and comparison with literature. The isolated compounds 1 and 2 exhibited significant antidepressant like effects in tail suspension and forced swim tests.

Compound 1 was isolated as colorless oil with molecular formula C₁₉H₂₀O₅ as suggested by HR-EI-MS with highest molecular ion peak at m/z 328.1305 [M⁺] and m/z values obtained were 310, 296, 292, 264, 260, 228, 150, and 120. The UV spectrum showed absorption band at λ_max 222 (2.9), 252 (3.6), 298 (3.9), 324 (4.6) and presence of various groups like hydroxyl group, alkane, alkene, aromatic moiety, and C-O were suggested by absorption bands in IR spectrum at υ_max 3372, 2920, 1640, 1610, and 1230, respectively.

The ¹H-NMR spectrum revealed the presence of five aromatic protons at chemical shift values of δ_H 6.54 (1H, d = 2.6 Hz, H-10), 6.40 (1H, d = 2.6 Hz, H-12), 6.68 (1H, d = 2.4 Hz, H-2), 6.89 (1H, d = 8.1 Hz, H-5), and 6.72 (1H, dd = 8.1, 2.4 Hz, H-6). Similarly two methylene proton signals appeared at δ_H 2.76 (2H, m, H-7) and 2.94 (2H, m, H-8), while a methine proton resonated at δ_H 6.30 (1H, s, H-15) along with two oxidized protons at δ_H 4.28 and 3.80 showing germinal coupling with each other (1H, d = 15.6 Hz each) as given in Table 1. The ¹H-NMR spectrum also suggested the presence of two methoxy groups at δ_H 3.74 (3H, s, -OCH₃) and δ_H 3.65 (3H, s, OCH₃).

The ¹³C-NMR and DEPT spectrum revealed the presence of nineteen 19 carbon atoms including two methyl, three methylene, six methine, and eight quaternary carbon atoms. Two methoxy carbons resonated at δ_C 56.4 (C-11) and 58.4 (C-3) while three methylene carbons were observed at δ_C 40.2 (C-7), 38.9 (C-8), and 64.3 (C-17). ¹³C-NMR showed five aromatic methine carbon atoms at δ_C 102.3 (C-12), 107.9 (C-10), 119.2 (C-2), 115.7 (C-5), and 124.8 (C-6) with another methine carbon at δ_C 104.4 (C-15) as given in Table 1. The quaternary carbons resonated at δ_C 149.9 (C-3), 148.3 (C-4), 137.2 (C-1), 145.8 (C-9), 162.1 (C-11), 160.6 (C-13), 109.7 (C-14), and 150.6 (C-16).

The data of HMBC spectrum was quite supportive for accurate placement of substituents in compound 1. The HMBC correlation of methoxy protons at δ_H 3.74 showed strong correlation with C-3, C-4, and C-2 confirming its position at C-3 similarly second methoxy group (δ_H 3.65) was placed at position C-11 showing HMBC correlation with C-10, C-11, and C-12. The placement of two hydroxyl groups at C-4 and C-16 were established from HMBC correlations, like H-2 (δ_H 6.68) and H-6 (δ_H 6.72) showed connectivity with C-4 and correlation of H-17 and H-15 with C-16 respectively. The methylene protons at position C-7 showed strong HMBC correlations with C-1, C-2 and C-6, while protons at position C-8 showed HMBC correlations with C-9 and C-10 suggesting presence of bibenzyl group in compound 1 as depicted in Figure 2 (Zhan et al., 2016). The HMBC correlations of methylene protons at C-17 showed strong correlation with C-13, C-15, and C-16 while correlation of H-15 with C-13, C-14, C-16, and C-17 formed an oxygen heterocyclic ring as shown in Figure 1. The structure of compound 1 was suggested as 5-(4-hydroxy-3-methoxyphenethyl)-7-methoxy-2H-chromen-3-ol on the basis of all spectral data and literature comparison (Zhan et al., 2016).

Compound 2 was isolated as colorless oil from ethyl acetate fraction of F. vesca var. nubicola. Its molecular formula was C₂₀H₂₂O₆ as suggested by highest molecular ion peak at m/z 358.1409 [M⁺] while other m/z values were 340, 326, 322, 294, 290, 264, 258, 226, 148, and 118. The absorption bands in UV spectrum were obtained at λ_max 212 (3.6), 240 (2.8), 296 (4.1), 310 (3.9) and IR spectrum showed absorption bands at υ_max 3380, 2960, 1650, 1610, and 1230 suggesting presence of hydroxyl group, alkane, alkene, aromatic moiety, and C-O respectively.

The ¹H-NMR and ¹³C-NMR data of compound 2 is quite identical to compound 1. Compound 2 had one additional methoxy group which was evident from all spectral data. The ¹H-NMR showed chemical shift value for three methoxy groups at δ_H 3.74 (3H, s, δ_C = 57.1) and 3.59 (3H, s, δ_C = 55.1) at position C-3 and C-11 similar to compound 1 with additional methoxy group at position C-15 having chemical shift value of δ_H 3.57 (3H, s, δ_C = 56.9). The ¹H-NMR spectrum showed presence of five aromatic protons at δ_H 6.60 (1H, d = 2.1 Hz, H-2, δ_C = 115.4), 6.90 (1H, d = 8.4 Hz, H-5, δ_C = 117.7), 6.76 (1H, dd = 8.4, 2.1 Hz, H-6, δ_C = 123.8) for ring A along with δ_H 6.50 (1H, d = 2.3 Hz, H-10, δ_C = 111.6) having meta coupling with proton at δ_H 6.45 (1H, d = 2.3 Hz, H-12, δ_C = 99.6) at ring B.

¹³C-NMR spectrum and DEPT showed presence of twenty carbon atoms including three methoxyl group, three methylene, five methine, and nine quaternary carbons given in Table 1. The HMBC correlations of substituents were quite identical to those for compound 1, the only difference observed was a methoxy group at position C-15 showing HMBC connectivity of protons with C-15, C-16 and C-14. On the basis of all spectral data and literature comparison the structure established for compound 2 was 5-(4-hydroxy-3-methoxyphenethyl)-4,7-dimethoxy-2H-chromen-3-ol.

The acute toxicity and behavioral test is mandatory for further detail studies. In our study both compounds were found safe with no behavior changes in the specified time, nor any mobility or mortality.

The TST and FST are reported to be the most commonly used predictive tests of antidepressant effects sensitive to the acute administration of antidepressant substances (Cryan et al., 2005). These behavioural tests are based on induction of immobility in rodents that face an inescapable situation and the antidepressant activity is indicated reduction in immobility time (Porsolt et al., 1977). In this study, compounds 1 and 2 displayed antidepressant like effects in both FST and TST. Although many antidepressant including monoamine oxidase inhibitors, tricyclics, selective serotonin reuptake inhibitors and selective noradrenaline reuptake inhibitors are available in the market for the treatment of various types of depressive disorders but even the first line antidepressant are facing many challenges in terms of unwanted effects (Kennedy, 2013; Johannessen et al., 2016).

The antidepressant like potential of natural isolated compounds is well-documented (Belozertseva et al., 2007; Pervez et al., 2016). Herbal therapies including the famous St. John’s wort have been found effective in the treatment of various depressive disorders. There has been renewed interest recently for novel pharmacotherapy from medicinal plants and compounds isolated from plant extracts. It is assumed that they might follow a different mechanistic pathway and better pharmacokinetic profile and thus better patient compliance. The compounds 1 and 2 may serve as leads for the development of newer antidepressant agents from natural sources. However, further detail studies are needed to ascertain their clinical status.

AK and UF conceived and designed the study. SN performed isolation and NK performed in vivo studies. JH analyzed the data. UF and AK wrote the manuscript with inputs and comments from all co-authors. All authors have read and approved the final version of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.