Journal of Applied Botany and Food Quality 92, 73 - 80 (2019), DOI:10.5073/JABFQ.2019.092.010 1Department of Chemistry, University of Central Punjab, Lahore, Pakistan 2 Department of Chemistry, University of Agriculture Faisalabad, Pakistan 3 Department of Chemistry, University of Sargodha, Sargodha, Pakistan 4 Department of Forestry & Range Management, Bahauddin Zakariya University, Multan, Pakistan 5Centre for the Research and Technology for Agro-Environment and Biological Sciences, University of Trás-os-Montes e Alto Douro (CITAB/UTAD), Vila Real, Portugal 6Centro de Edafología y Biología Aplicada del Segura, National Council for Scientific Research (CEBAS-CSIC) Univeristy Campus of Espinardo, Espinardo, Spain HPLC-PDA-ESI-MSn profiling of polyphenolics in different parts of Capparis spinosa and Capparis decidua as function of harvesting seasons Tehseen Gull1, 2, Bushra Sultana2, Farooq Anwar3*, Wasif Nouman4*, Eduardo Rosa5, Raúl Domínguez-Perles5, 6 (Submitted: January 17, 2018; Accepted: June 6, 2018) * Corresponding author Summary HPLC-PDA-ESI-MSn analysis of different parts such as stem bark, shoot, flower, fruit and root of Capparis spinosa (C. spinosa) and Capparis decidua (C. decidua), collected in rainy and dry seasons from the Cholistan desert of Pakistan, depicted the occurrence of a wide array of phenolics with quercetin, apigenin and kaempferol derivatives along with dicaffeoylquinic acid, caffeoylquinic acid and feruloylquinic acid as the main compounds. Kaempferol-3-glucoside (28.02-167.21 µg g-1dw) was found to be the principal component in all tested parts of both species while dicaffeoylquinic acid was detected only in the flowers and roots. The roots exhibited maximum contents of flavonoids and hydroxycinnamic acid derivatives. The harvesting period significantly (p<0.05) affected the concentration of phenolics wherein the samples collected in rainy season offered greater levels of phenolics than their counterpart. The roots and fruits of both species were found to be rich sources of phenolics. The findings of this research suggest the harvesting of the selected wild Capparis species in rainy season to maximize their antioxidant and nutraceutical benefits. Keywords: Capparaceae; Flavonoids; Hydroxycinamic acids; Poly- phenols Introduction Plants are valued as a rich source of a wide array of secondary metabolites. Among secondary metabolites, phenolics are one of those bioactive compounds which are widely distributed in the plant kingdom (Muhammad et al., 2015; Sahib et al., 2013; Shahidi, 1997). It is widely accepted that nutraceutical and anti- oxidant attributes of plant foods are mainly associated with their phenolics (Shahidi, 1997). In this regard, consumption of selected vegetables and fruits as well as several other plant foods, is strongly linked with health benefits and/or reduced incidence of degenerative diseases such as aging, inflammation and certain cancers (Lodovici et al., 2001; Oomah, 2001; RobbinS, 2003). Capparis is one of the important genera with known 250 species dis- tributed world wide. Of the Capparis species, Capparis spinosa and Capparis decidua are native to Pakistan (Gull et al., 2015a). These plants have been investigated for their anti-atherosclerosis, anti- hypertensive, anti-inflammatory, analgesic, anti-asthmatic, anti-hyper- lipidemic, hepatoprotective, antibacterial, and antifungal activities (Chahlia, 2009; Duman et al., 2013; Hundiwale et al., 2005; Mali et al., 2005). Besides, stem bark, shoot, root, flower and fruits of both of these species have been reported as good sources of valuable nutrients such as minerals, crude fiber and protein (Gull et al., 2015b; Gull et al., 2015c; Gull et al., 2015d). Different parts of these species are also being employed in the traditional medicine systems for the treatment of several diseases (Gull et al., 2015a). For example, the root extract of C. spinosa has been reportedly used to prepare liver protecting drugs named Liv-52 and CM-52 which are used to cure hepatitis B and liver cirrhosis (EddoukS et al., 2005; RajeSh et al., 2009). The hepatoprotective efficacy of C. spinosa might be attributed to the potential antioxidant activities of its bio- actives following the restoration of liver cell membrane and its per- meability, while the exact mechanism of action needs to be explored. Moreover, C. decidua extracts have also been reported for hepato- protective effects via decreasing the levels of the enzymes SGPT (serum glutamate pyruvate transaminase), SGOT (serum glutamic oxaloacetic transaminase), and ALP (alkaline phosphatase) (Jhajharia et al., 2010). Different parts of Capparis species are also used to cure/prevent diabetes, cardiac diseases, toothache and intermittent fever (Marwat et al., 2011; Özcan, 2005; Sharma and Kumar, 2008). It has been reported in various studies that plant phenolics com- position is affected by geographical zones, climatic conditions and seasonal variability (Nouman et al., 2016; OlSon et al., 2016). Re- searchers are focusing on profiling of the phenolics in vascular plants but there are few studies available revealing the variation in these compounds under seasonal fluctuations (Sampaio et al., 2016) while no study is available explaining the variations in concentration of phenolic acids in different parts of C. spinosa and C. decidua as affected by harvesting seasons. The concentration and availability of phenolic bioactive compounds vary within plants of the same species and their parts. Variation in expression and profiling of these bioactive compounds also vary with respect to cultivar, geogra- phical zones, climatic conditions and seasonal fluctuations (Cartea et al., 2008; CiSka et al., 2000; García-SalaS et al., 2014; Ray et al., 2013). For example, variation in biological activities of dif- ferent Brassica vegetables under seasonal fluctuations such as tem- perature and precipitation might affect the composition and concen- tration of bioactive compounds resulting in affecting antioxidant activities of Brassica vegetables (AireS et al., 2011). Likewise, varia- tion in bioactive compounds, enzymatic antioxidants and antiradical activities of Moringa oleifera leaves as affected by tree age, climatic factors and soil condition has been reported (Nouman et al., 2016; Vázquez-León et al., 2017). Keeping in view the possible variations among bioactive compounds of various plant species and genera, it can be hypothesized that dif- ferent parts of C. spinosa and C. decidua can yield varying concen- tration and profile of bioactive compounds under seasonal effects. The present study was aimed to investigate whether or not the two 74 T. Gull, B. Sultana, F. Anwar, W. Nouman, E. Rosa, R. Domínguez-Perles different seasons such as rainy and dry affect the profile of phenolics in different parts of wild C. spinosa and C. decidua harvested in their natural habitat. Materials and methods Reagents All LC-MS grade solvents were obtained from J.T. Baker (Phillips- burg, NJ). Formic acid was purchased from Panreac (Barcelona, Spain). The standards (-)-epigallocatechin, quercetin-3-O-glucoside, 5-O-caffeoylquinic acid were from Sigma Aldrich (Steinheim, Ger- many). Ultrapure water was produced using a Millipore water puri- fication system. Collection of Plant Samples and Pretreatment As a representative of dry and rainy seasons, two months including April and September were selected for plant sampling based on low/high temperature and rainfall intensities, respectively. Rainfall and temperature data were obtained from Pakistan Council of Re- search in Water Resources (PCRWR), Bahawalpur, Pakistan. Fig. 1 depicts an average weather data for the sampling site while in par- ticular sampling months (April and September), mean temperature (27.9 and 31.1 °C) and average rainfall (7 and 41 mm) was noted, respectively. Different parts including stem bark, shoot, fruits, flow- ers and roots of C. spinosa and C. decidua were collected from Cho- listan desert area of Bahawalpur, Punjab, Pakistan (desert region, latitude 28-15° N; longitude 70-45° ' E and altitude 89 m above mean sea level) in April and September 2013. The samples of selected parts were harvested from ten different plants of each of the mentioned species and then these were pooled in three replications for further analyses. The specimen were further identified and authenticated by Dr. Mansoor Hameed, Taxonomist, Department of Botany, Univer- sity of Agriculture Faisalabad, Pakistan. The collected samples were dried at room temperature for 24 hours followed by oven drying at 45 °C for further studies (nouman et al., 2016). Sample preparation and analysis Samples of the selected parts including stem bark, shoot, flower, fruit and root of C. Spinosa and C. decidua were prepared for the analysis of phenolic compounds using HPLC-PDA-ESI-MSn and HPLC-PAD following the protocol as described by our research group (Nouman et al., 2016). Briefly, samples were air-dried in an oven at 45 °C for 72 h and were ground to a fine powder and stored at -20 ºC for fur- ther analysis. Each sample (40 mg) was extracted with 1.5 mL of 70% methanol for 30 min at 70 ºC, vortexed every 5 min to improve extraction efficacy and then centrifuged (10000×g for 20 min at 4 ºC) (model Sigma 1-13, B Braun Biotech International, Osterode, Ger- many). Supernatants were collected and methanol was removed un- der reduced pressure using a rotary evaporator (EYELA, N-N Series, Rikakikai Co., Tokyo, Japan). The dried residue was reconstituted in ultrapure water (1 mL) and filtered through a 0.22 μm polypropy- lene membrane filter (ANOTOP 10 plus; Whatman, Maidstone, UK). Each sample (20 μL) was analyzed in a LC-PAD-ESI/MSn (Thermo- Scientific, Loughborough, UK) and HPLC-PAD (Gilson Inc., Mett- menstetten, Switzerland) for phenolics identification/authentication and quantification, respectively. The method described by Nouman et al. (2016) was used for these analyses as mentioned below. Analysis of phenolic compounds by HPLC-PDA-ESI-MSn and HPLC-PDA The phenolics profiling, using HPLC-PDA-ESI-MSn chromatograph- ic analysis, was conducted on a Luna C18 column (150 × 2.1 mm, 2.6 μm particle size; Thermo-Scientific, Loughborough, UK). The mobile phase was a mixture of distilled water/formic acid (99:1, v/v) (solvent A) and acetonitrile/formic acid (99:1, v/v) (solvent B). The flow rate was of 0.6 mL/min in a linear gradient following the scheme (t in min; %B): (0; 0%), (5; 20%), (30; 50%), (45, 100%), and (55; 0%). The chromatograms were recorded at 320 and 520 nm. The equip- ment consisted of a LC pump (SRVYR-LPUMP), an autosampler (SRVYR-AS), and a photodiode array detector (SRVYR-PDA5). The HPLC-PDA-ESI-MSn analysis was performed in a mass detector in series, which was an ion trap spectrometer (model LCQ-Advantage- Max) equipped with an electrospray ionization interface controlled by Tune Plus 1.3 SR1 software (Fisher Scientific, Lisboa, Portugal) and operated in negative mode. The ionization conditions were ad- justed at 250 ºC and 4.0 kV for capillary temperature and voltage, respectively. The nebulizer pressure and flow rate of nitrogen were 2.0 bar and 8.0 L/min, respectively. The full scan mass covered the range from m/z 100 up to m/z 1500. Collision-induced fragmentation experiments were performed in the ion trap using helium as the colli- sion gas, with voltage ramping cycles from 0.3 up to 2.0 V. 16 360 361 Fig. 1: Mean annual temperature and rainfall data of Cholistan desert, District Bahawalpur 362 363 0 5 10 15 20 25 30 35 40 45 50 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Average Rainfall (mm) Mean Temerature (°C) Fig. 1: Mean annual temperature and rainfall data of Cholistan desert, District Bahawalpur Profiling of polyphenolics in Capparis species 75 Quantitative analysis was carried out by a HPLC-PDA system (Gil- son Inc., Mettmenstetten, Switzerland) using the same chromato- graphic conditions as described for identification. The equipment consisted of a LC pump (Gilson, 305/306-PUMP), a dynamic mixer chamber (Gilson, 811C), an autosampler (Gilson, Autoinjector-234), and a photodiode array detector (PDA-Plus detector Finnigan Su- riveyor, Thermo-Scientific). Cinnamic acids derivatives were quanti- fied as 5-O-caffeoylquinic acid at 320 nm and flavonols as quercetin- 3-O-glucoside at 360 nm. Statistical Analysis Data were processed using MSTAT-C Statistic Software Package (Michigan State University, Michigan, US). A multi factorial analy- sis of variance (ANOVA) and multiple range test (Tukey’s test) were carried out to evaluate statistical differences. The level of signifi- cance was set at p < 0.05. Results In the present study, on the basis of retention time (RT), molecular masses, and fragmentation patterns, twelve flavonols and five hy- droxycinnamic acids were detected and reported for the first time in the selected Capparis species from Pakistan. A significant (p < 0.05) variation in the qualitative and quantitative composition of flavonoids and hydroxycinnamic acids was recorded among parts of both species as well as function of two seasons. Overall, maxi- mum concentration of total flavonoids was observed in C. spinosa in rainy season (554.53 μg g-1 dw) in comparison with dry months (488.07 μg g-1 dw) which were 11% lower (Tab. 1-5). A similar trend was recorded for hydroxycinnamic acid derivatives as well. Over- all, in comparison with C. spinose, C. decidua offered 19.23 and 21.97% higher flavonoids and hydroxycinnamic acids, respectively (Tab. 1-5). Stem bark of both species showed different compounds with vary- ing concentration in both seasons. The stem bark of C. spinosa con- tained lower amount of kaempferol-3-glucoside (114.80 μg g-1 dw, on average) than that of C. decidua (144.78 μg g-1 dw, on average). The stem bark of C. decidua exhibited a reasonable level ofapige- nin-7-C-glucoside (isorhoifolin) and quercetin-3-glucoside; however, these compounds were present only in traces in the stem bark of C. spinosa. C. decidua stem bark showed higher contents of the above mentioned phenolics in rainy season while C. spinosa stem bark presented more concentration in dry season (Tab. 1). Beside, 3-p- coumaroylquinic acid and feruloylquinic acid were also detected in C. spinosa stem bark while C. decidua stem bark showed the pre- sence of only feruloylquinic acid (Tab. 1). Shoot of C. spinosa and C. decidua showed lower concentration of these phenolics in comparison with other parts. Apigenin-8-C-glu- coside (isovitexin) (3.15 μg g-1 dw, on average), quercetin-3-glucoside (5.23 μg g-1 dw, on average) and kaempferol-3-glucoside (45.93 μg g-1 dw, on average) were recorded in C. spinosa shoot while quercetin- 3-rhamanoside was recorded in lower quantity. On the other hand, C. decidua shoot contained quercetin-3-glucoside (2.87 μg g-1 dw, on average), apigenin-7-C-glucoside (isorhoifolin) (5.48 μg g-1 dw, on average), kaempferol-3,7-diglucoside (4.34 μg g-1 dw, on average) and kaempferol-3-glucoside (51.71 μg g-1 dw, on average) in both seasons. In case of hydroxycinnamic acids, only feruloylquinic acid was re- corded in both species with its concentration higher in C. spinosa shoot samples collected in rainy season than C. decidua shoot sam- ples of both seasons (Tab. 2). The other flavonoid and hydroxycinna- mic acids were detected in traces. Fruits of both species were noted to be a good source of flavonoids and hydroxycinnamic acids compared to other parts with C. decidua Tab. 1: Identification and quantification of phenolic acids and flavonoids (μg g-1 dw) in stem bark of C. spinosa and Capparis decidua Compounds Capparis spinosa Capparis decidua Dry season Rainy season Dry season Rainy season Flavonoids Quercetin-3,7-diglucoside 0.08±0.01 e 0.06±0.10 e Traces Traces Quercetin-3,7-diglucoside (isomer) Traces Traces Traces Traces Apigenin-8-C-glucoside (isovitexin) Traces Traces Traces Traces Quercetin-3-glucoside Traces Traces 22±1.27 cd 13±0.43 d Apigenin-7-C-glucoside (isorhoifolin) Traces Traces 39±0.94 c 44±1.41 c Kaempferol-3,7-diglucoside Traces Traces 8±1.03 c 8±1.13 c Apigenin-7-rutinoside Traces Traces 6±0.87-d 8±0.54 d Quercetin-3-rhamanoside 0.46±0.09 e 0.42±0.07 e 0.34±0.02 0.39±0.05 Quercetin-3-sophoroside 0.24±0.05 e 0.12±0.02 e Traces 0.49±0.08 Quercetin-3-acetyl-glucoside Traces Traces 0.11±0.03 Traces Kaempferol-3-glucoside 122±3.59 a 107±2.98 ab 122±1.80 b 167±5.96 a Kaempferol-7-glucoside Traces Traces Traces Traces Total 123 A 108 B 197 B 242 A Hydroxycinamic acids Dicaffeoylquinic acid Traces Traces Traces Traces 5-Caffeoylquinic acid Traces Traces Traces Traces 3-Caffeoylquinic acid Traces Traces 0.07±0.01 Traces 3-p-coumaroylquinic acid 31±1.11 c 52±1.73 b Traces Traces Feruloylquinic acid 16±1.38 d 35±1.12 c 22±1.27 cd 35±1.18 c Total 48 B 87 A 22 BC 35 B Values (means ±SD) are average of three samples of each part, analyzed individually in triplicate (p< 0.05). 76 T. Gull, B. Sultana, F. Anwar, W. Nouman, E. Rosa, R. Domínguez-Perles Tab. 2: Identification and quantification of phenolic acids and flavonoids (μg g-1 dw) in shoot of C. spinosa and Capparis decidua Compounds Capparis spinosa Capparis decidua Dry season Rainy season Dry season Rainy season Flavonoids Quercetin-3,7-diglucoside Traces Traces Traces Traces Quercetin-3,7-diglucoside (isomer) Traces Traces Traces Traces Apigenin-8-C-glucoside (isovitexin) 2.3±0.07 c 4.0±0.05 bc Traces Traces Quercetin-3-glucoside 4.4±0.83 bc 6.0±0.19 b 2.4±0.22 d 3.4±0.32 d Apigenin-7-C-glucoside (isorhoifolin) Traces Traces 7.75±1.23 c 3.22±0.71 d Kaempferol-3,7-diglucoside Traces Traces 4.0±0.47 d 4.7±0.38 d Apigenin-7-rutinoside Traces Traces Traces Traces Quercetin-3-rhamanoside 0.73±0.11 d 0.29±0.08 d Traces Traces Quercetin-3-sophoroside Traces Traces Traces Traces Quercetin-3-acetyl-glucoside Traces Traces Traces Traces Kaempferol-3-glucoside 43±0.57 a 48±1.89 a 42±1.40 b 61±2.08 a Kaempferol-7-glucoside Traces Traces Traces Traces Total 51 B 59 B 57 B 72 A Hydroxycinamic acids Dicaffeoylquinic acid Traces Traces Traces Traces 5-Caffeoylquinic acid Traces Traces Traces Traces 3-Caffeoylquinic acid Traces Traces Traces Traces 3-p-coumaroylquinic acid Traces Traces Traces Traces Feruloylquinic acid 7.5±0.96 b 17±0.99 b 2.4±0.22 d 3.9±0.47 d Total 7.5 B 17 A 2.4 A 3.9 A Values (means ±SD) are average of three samples of each part, analyzed individually in triplicate (p< 0.05). Tab. 3: Identification and quantification of phenolic acids and flavonoids (μg g-1 dw) in fruit of C. spinosa and Capparis decidua Compounds Capparis spinosa Capparis decidua Dry season Rainy season Dry season Rainy season Flavonoids Quercetin-3,7-diglucoside Traces Traces Traces Traces Quercetin-3,7-diglucoside (isomer) Traces Traces Traces Traces Apigenin-8-C-glucoside (isovitexin) 17±1.03 c 20±0.1 c 2.6±0.42 e 2.9±0.31 e Quercetin-3-glucoside 0.08±0.009 0.12±0.009 25±1.30 c 32±1.08 c Apigenin-7-C-glucoside (isorhoifolin) Traces Traces 0.42±0.15 f Traces Kaempferol-3,7-diglucoside Traces Traces 0.21±0.02 f 1.2±0.14 ef Apigenin-7-rutinoside Traces Traces 7.2±0.49 d 11±0.48 d Quercetin-3-rhamanoside 0.47±0.08 e 0.89±0.12 e 0.41±0.08 1.11±0.18 Quercetin-3-sophoroside 0.12±0.15 e 0.78±0.23 e Traces Traces Quercetin-3-acetyl-glucoside 0.11±0.05 e 0.43±0.09 e 0.19±0.009 f 0.97±0.10 f Kaempferol-3-glucoside 123±1.66 b 147±1.34 a 116±1.54 b 131±5.38 a Kaempferol-7-glucoside Traces Traces Traces Traces Total 140 B 170 A 152 B 180 A Hydroxycinamic acids Dicaffeoylquinic acid Traces Traces Traces Traces 5-Caffeoylquinic acid Traces Traces Traces 0.49±0.08 3-Caffeoylquinic acid Traces Traces 0.09±0.012 f 1.1±0.17 ef 3-p-coumaroylquinic acid 4.1±0.59 de 5.0±0.47 de 2.6±0.42 e 5.0±0.97de Feruloylquinic acid 7.6±0.85 d 8.2±0.37 d 24.9±1.30 c 29.0±1.03 c Total 12 A 13 A 28 B 36 A Values (means ±SD) are average of three samples of each part, analyzed individually in triplicate (p< 0.05). Profiling of polyphenolics in Capparis species 77 Tab. 4: Identification and quantification of phenolic acids and flavonoids (μg g-1 dw) in flower of C. spinosa and Capparis decidua Compounds Capparis spinosa Capparis decidua Dry season Rainy season Dry season Rainy season Flavonoids Quercetin-3,7-diglucoside Traces Traces 1.18±0.56 b 1.16±0.32 b Quercetin-3,7-diglucoside (isomer) 3.6±0.12 de 2.7±0.09 Traces Traces Apigenin-8-C-glucoside (isovitexin) 2.6±0.04 de 5.0±0.20 d Traces Traces Quercetin-3-glucoside 2.8±0.40 de 3.5±0.13 de 1.8±0.09 b 2.2±0.11 b Apigenin-7-C-glucoside (isorhoifolin) Traces Traces 2.2±0.31 b 2.1±0.27 b Kaempferol-3,7-diglucoside Traces Traces Traces Traces Apigenin-7-rutinoside Traces Traces Traces 0.61±0.19 Quercetin-3-rhamanoside 0.82±0.05 e 1.3±0.13 e 0.9±0.03 1.4±0.09 Quercetin-3-sophoroside Traces Traces Traces Traces Quercetin-3-acetyl-glucoside Traces Traces Traces Traces Kaempferol-3-glucoside 41±0.78 b 52±1.77 a 40±1.07 ab 48±1.97 a Kaempferol-7-glucoside Traces Traces 2.1±0.85 3.0±0.78 Total 51 B 65 A 48 B 59 A Hydroxycinamic acids Dicaffeoylquinic acid 17±0.52 c 24±0.88 c Traces Traces 5-Caffeoylquinic acid Traces Traces Traces Traces 3-Caffeoylquinic acid Traces Traces Traces Traces 3-p-coumaroylquinic acid Traces Traces Traces Traces Feruloylquinic acid Traces Traces 1.8±0.59 b 2.3±0.12 b Total 17 B 24 A 1.8 A 2.3 A Values (means ±SD) are average of three samples of each part, analyzed individually in triplicate (p< 0.05). Tab. 5: Identification and quantification of phenolic acids and flavonoids (μg g-1 dw) in root of C. spinosa and Capparis decidua Compounds Capparis spinosa Capparis decidua Dry season Rainy season Dry season Rainy season Flavonoids Quercetin-3,7-diglucoside 0.10±0.01 e 0.32±0.01 e Traces Traces Quercetin-3,7-diglucoside (isomer) Traces Traces Traces Traces Apigenin-8-C-glucoside (isovitexin) 14.±1.5 cd 19.±0.65 c 18±1.25 f 19±0.62 f Quercetin-3-glucoside Traces Traces 55±0.82 c 69±2.21 bc Apigenin-7-C-glucoside (isorhoifolin) Traces Traces 48±0.45 d 59±2.15 Kaempferol-3,7-diglucoside Traces Traces 6.0±0.35 g 8.0±0.37 g Apigenin-7-rutinoside Traces Traces 21±0.77 f 18±1.02 Quercetin-3-rhamanoside 0.48±0.08 e 0.97±0.09 e 51±1.20 d 69±2.07 bc Quercetin-3-sophoroside Traces Traces Traces Traces Quercetin-3-acetyl-glucoside 0.51±0.008 e 1.19±0.10 e 44±0.66 de 57±2.17 c Kaempferol-3-glucoside 107±1.76 b 132±4.34 a 28±1.87 e 44±1.71 de Kaempferol-7-glucoside Traces Traces 20±1.05 25±1.13 ef Total 123 B 154 A 290 B 368 A Hydroxycinamic acids Dicaffeoylquinic acid Traces Traces 29±1.29 e 34±1.19 e 5-Caffeoylquinic acid Traces Traces 3±0.16 g 4±0.19 g 3-Caffeoylquinic acid Traces Traces 78±1.03 b 97±3.21 a 3-p-coumaroylquinic acid 8±1.00 d 11±0.85 cd 18±1.25 f 30±1.13 e Feruloylquinic acid 7.0±0.55 d 22±1.10 c 55±0.82 c 61±2.47 bc Total 15 B 33 A 183 B 226 A Values (means ±SD) are average of three samples of each part, analyzed individually in triplicate (p< 0.05). 78 T. Gull, B. Sultana, F. Anwar, W. Nouman, E. Rosa, R. Domínguez-Perles fruits offering higher concentration of these compounds (Tab. 3). The fruits from C. spinosa exhibited maximum concentration of kaemp- ferol-3-glucoside (134.80 μg g-1 dw, on average) followed by api- genin-8-C-glucoside (isovitexin) (18.62 μg g-1 dw, on average) while C. decidua fruit contained quercetin-3-glucoside (28.21 μg g-1 dw, on average) and kaempferol-3-glucoside (123.50 μg g-1 dw, on average). Moreover, 3-p-coumaroylquinic acid and feruloylquinic acid were re- corded in fruits of both species in both seasons while 3-caffeoylquinic acid was recorded only in C. decidua fruit (Tab. 3). Likewise, shoot and flowers of both species exhibited lower quanti- ties of identified flavonoids and phenolic acids. Among flavonoids, only kaempferol-3-glucoside was detected in both species while the other compounds were present in traces. Dicaffeoylquinic acid (20.56 μg g-1 dw, on average) was quantified in flowers of C. spinosa while feruloylquinic acid was present in C. decidua flowers (Tab. 4). It can be guessed from the data of Tab. 5 that roots of C. decidua contained higher amount of flavonoids and hydroxycinnamic acids than its counterpart. Meanwhile, the roots from C. spinosa were found to be a good source of kaempferol-3-glucoside (119.66 μg g-1 dw, on average) while the other flavonoids were found in traces. Re- garding hydroxycinnamic acids, dicaffeoylquinic acid, 5-caffeoyl- quinic acid and 3-caffeoylquinic acid were identified in traces in C. spinosa roots while 3-p-coumaroylquinic and feruloylquinic acids were found in reasonable concentration. On the other hand, C. decidua roots exhibited maximum amount of 3-caffeoylquinic acid (87.58 μg g-1 dw, on average) followed by feruloylquinic acid (58.28 μg g-1 dw, on average), dicaffeoylquinic acid (31.40 μg g-1 dw, on average), 3-p-coumaroylquinic acid (23.69 μg g-1 dw, on average) and 5-caffeoylquinic acid (3.28 μg g-1 dw, on average) (Tab. 5). Discussion HPLC-PDA-ESI-MSn analysis of aqueous methanolic extracts of different parts of C. spinosa and C. decidua demonstrated a vari- able composition of phenolic compounds in dry and rainy seasons. Among these compounds, flavonols were found as the main consti- tuents as shown in Tab. 1-5. Beside these, hydroxycinnamic acids were also found in the tested parts of these species with varying concentrations. Previously, few studies have shown the presence of rutin, kaempferol, kaempferol rutinoside and quercetin rutinoside (ArGentieri et al., 2012; Inocencio et al., 2000; RodriGo et al., 1992) while the others were detected first time in the present study. The fruits of C. spinosa presented highest amount of total flavonoids in rainy seasons (169.50 μg g-1 dw) followed by roots (153.61 μg g-1 dw) and fruits of dry season (140.35 μg g-1 dw). Total flavonoids, ex- pressed in dry season by stem bark and roots of C. spinosa, were 27% lower than fruits (Tab. 1-5). Kaempferol-3-glucoside was noted as main flavonoid in C. spinosa fruits (134.80 μg g-1 dw, on average) followed by apigenin-7-C-glucoside (isorhoifolin) (Tab. 3). Apigenin and kaempferol have previously been reported in C. spinosa fruits collected from Xinjiang province of China (Yu et al., 2006; Zhou et al., 2010; Zhou et al., 2011) but these compounds were identified for the first time in capparis fruit samples collected from Pakistan. Beside this, a few other derivatives of kaempferol were reported in C. spinosa shoots which were confirmed in the present investigation (SiracuSa et al., 2011). It was noted that C. spinosa shoot is a rich source of kaempferol derivative. Such variation in flavonoids distri- bution within plants of different origins may be linked to varying mode of extraction employed, and genetic and agro-climatic factors prevailing in different sites (HaShempour et al., 2010; ISlam et al., 2003). The flavonoid compounds detected in the present analysis of Cap- paris species were consisted of twelve compounds including quer- cetin-3,7-diglucoside, quercetin-3,7-diglucoside (isomer), apigenin- 8-C-glucoside, quercetin-3-glucoside, apigenin-7-C-glucoside (iso- rhoifolin), kaempferol-3,7-diglucoside, apigenin-7-rutinoside, quer- cetin-3-rhamnoside, quercetin-3-O-sophoroside, quercetin-3-acetyl- glucoside, kaempferol-3-glucoside, and kaempferol-7-glucoside with their composition significantly differed among different parts of both species in either of the seasons. As stated earlier, fruits and roots of C. spinosa presented maximum content of flavonoids while a few such as apigenin-7-C-glucoside (isorhoifolin), kaempferol-3,7- diglucoside, apigenin-7-rutinoside and kaempferol-7-glucoside were detected only in traces amount in C. spinosa. In case on hydroxy- cinnamic acids, stem bark and fruits of C. spinosa exhibited maxi- mum amount of these acids in either of the dry and rainy seasons, respectively while 5-caffeoylquinic acid and 3-caffeoylquinic acid were recorded in traces (Tab. 1-5). Kaempferol-3-glucoside was the most abundant constituent (437.0 and 486.9 μg g-1 dw) and repre- sented 89.0 and 87.8% of flavonoids detected in in dry and rainy sea- sons, respectively. Apigenin-8-C-glucoside (isovitexin) was found as the second most abundant flavonoid in C. spinosa with contribution 36.09 and 48.49 μg g-1 dw in dry and rainy seasons, respectively. Apigenin-8-C-glucoside (isovitexin) was mainly present in the fruits representing 43.3% of total apigenin-8-C-glucoside (isovitexin) con- tents of C. spinosa while in stem bark, it was not detected. The least amounts of flavonoids were quantified in C. spinosa shoot. Dicaffeoylquinic acid, 5-caffeoylquinic acid, 3-caffeoylquinic acid, 3-p-coumaroylquinic acid, and feruloylquinic acid were identified in C. spinosa (Tab. 1-5). As a whole, C. spinosa presented 99.10 and 173.80 μg g-1 dw of hydroxycinnamic acids in dry and rainy months, respectively (Tab. 1-5). Stem bark and roots exhibited maximum amount of these acids (47.85 and 33.06 μg g-1 dw, respectively) while the least count was recorded in shoots. Of the studied hydroxycin- namic acids, 3-p-Coumaroylquinic acid presented maximum amount in dry month (43.33 μg g-1 dw) while in rainy month feruloylquinic acid gave maximum quantity (81.44 μg g-1 dw). Both caffeoylquinic acid derivatives were found in traces while dicaffeoylquinic acid was as high as 17.38 and 23.74 μg g-1 dw (Tab. 1-5). The availability of these phenolic acids supports the traditional health benefits of cap- paris. The researchers reported that roots, fruits and flowers of these species are a good for curing infectious diseases (Iwu et al., 1999). Such functions of capparis tissues might be attributed to the phe- nolic acids (Sharma and Kumar, 2008). The researchers extracted kaempferol, apigenin and cinnamic acids from fruit extracts of C. spinosa and identified these as anti-inflammatory agents against in- duced rat paw edema (Zhou et al., 2010). So, the findings of the pre- sent investigation support that capparis tissues, especially roots can be used in pharmaceutical industries. C. decidua presented more amounts of flavonoids and hydroxycin- namic acids. In dry and rainy months, C. decidua exhibited 65.5 and 52.9% more flavonoids than C. spinosa, respectively. These data also showed that C. decidua provided more flavonoids in rainy months rather than dry ones (920.38 and 734.49 μg g-1 dw, respectively) as was recorded in case of C. spinosa. Among different parts of C. decidua, roots ranked higher in flavonoids followed by stem bark and fruits. The least amount of flavonoids was recorded in shoots (64.42 μg g-1 dw, on average) which was 80.6% lower than roots and 70.7% lower than stem bark. Among all studied flavonoids, Querce- tin-3,7-diglucoside (isomer) was found in traces while all others were also detected except quercetin-3-sophoroside and Quercetin-3,7-di- glucoside (0.49 and 1.17 μg g-1 dw on average, respectively). Kaemp- ferol-3-glucoside ranked highest among all detected flavonoids (400.10 μg g-1 dw, on average) followed by quercetin-3-glucoside and apigenin-7-C-glucoside (isorhoifolin). These three compounds re- presented 74.00% of total flavonoids. Interestingly, a few flavonoids like apigenin-7-rutinoside and quercetin-3-sophoroside were not de- tected in dry months but these were found in the samples (flowers and stem bark) harvested/plucked during rainy month. Previously, various compounds like apigenin, kaempferol, kaempferol-3-O- Profiling of polyphenolics in Capparis species 79 rutinoside and quercetin-3-O-rutinoside have been isolated from C. spinosa fruits (Yu et al., 2006; Zhou et al., 2010; Zhou et al., 2011). Beside these, Capparis leaves and shoots have also been reported as a good source of kaempferol and quercetin. Rutin, kaempferol, 3-O- rutinoside, and isorhamnetin 3-O-rutinoside have also been reported as major flavonoids in C. spinosa (SiracuSa et al., 2011). Hydroxy- cinnamic acids such as dicaffeoylquinic acid, 5-caffeoylquinic acid, 3-caffeoylquinic acid, 3-p-coumaroylquinic acid, and feruloylquinic acid were also detected first time in C. decidua. These were found in all parts of C. decidua in appreciable amount with maximum levels recorded in roots of this species followed by fruits and stem bark. Roots represented 77.00 and 74.72% of total hydrocinnamic acids in dry and rainy months, respectively. Among all hydroxycinnamic acids, feruloylquinic acid ranked highest followed by 3-caffeoylqui- nic acid > 3-p-coumaroylquinic acid > dicaffeoylquinic acid > 5- caffeoylquinic acid. The comparative profile of flavonoids and hydroxycinnamic acids of C. spinosa and C. decidua is first time investigated and reported in the present investigation. It can be noted that C. decidua is an impor- tant source of studied phenolics as it possesses 59.2 and 50.7% more flavonoids and hydroxycinnamic acids than C. spinosa on average, respectively. Both species exhibited higher contents of phenolics in rainy season than dry ones. The reasons for higher concentration of phenolic acids might be rainfall as researchers reported a positive correlation of rainfall with phenolics and flavonoid contents in seve- ral studies (Sarrazin et al., 2015; SouSa et al., 2010). YanG et al., (2006) studied 120 tropical and sub-tropical edible plants for their antioxidant potential and reported a higher antioxidant activity of these plants in hot wet season. Higher antioxidant activity is an in- dicator for the presence of higher phenolic acids (Siddhuraju and Becker, 2003). The varying concentration of bioactive compounds such as rutin has been reported to be linked with varying daylight in different Capparis parts. For example, maximum rutin contents in flowers and leaf samples of C. spinosa were detected at morning and night times while floral buds of C. spinosa gave maximum rutin in the morning. Stem and fruits of this species provided maximum rutin contents in morning at 11:00 and afternoon at 16:00 h (Behnaz et al., 2013). In the present work, variations in the phenolics profile can be mainly linked to varying climatic factors such as temperature, humidity and rainfall fluctuation in both seasons. Conclusion In the present comprehensive study, we for the first time reported that how different parts of C. spinosa and C. decidua exhibit varying le- vels of polyphenolics in dry and rainy seasons. A notable variation in the profile of phenolics was recorded among selected species parts as function of both harvesting seasons. Different parts of both species exhibited impressive range of phenolics but the roots possessed maxi- mum amount of these compounds which were mostly derivatives of quercetin such as kaempferol, apigenin, caffeoylquinic acid and feruloylquinic acid. These findings support the nutra-pharmaceutical potential of C. spinosa and C. decidua due to offering a wide array of polyphenols. 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DOI: 10.1021/jf105017j Addresses of the authors: Tehseen Gull1, 2, Bushra Sultana2, Farooq Anwar3*, Wasif Nouman4*, Eduardo Rosa5, Raúl Domínguez-Perles5, 6 1 Department of Chemistry, University of Central Punjab, Lahore, Pakistan 2 Department of Chemistry, University of Agriculture Faisalabad, Pakistan 3 Department of Chemistry, University of Sargodha, Sargodha, Pakistan 4 Department of Forestry & Range Management, Bahauddin Zakariya University, Multan, Pakistan 5 Centre for the Research and Technology for Agro-Environment and Biological Sciences, University of Trás-os-Montes e Alto Douro (CITAB/ UTAD), 5001-801, Vila Real, Portugal 6 Centro de Edafología y Biología Aplicada del Segura, National Council for Scientific Research (CEBAS-CSIC) Univeristy Campus of Espinardo, Edif. 25. 30100 Espinardo, Spain * Corresponding author’s E-mail: wnouman@gmail.com; fqanwar@yahoo.com © The Author(s) 2019. This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creative- commons.org/licenses/by/4.0/deed.en). mailto:wnouman@gmail.com mailto:fqanwar@yahoo.com