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Phenolic constituents from apple tree leaves and their in vitro biological activity
Industrial Crops and Products 90 (2016) 118–125
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Phenolic constituents from apple tree leaves and their in vitro biological activity Shalika Rana a,b , Shiv Kumar a,c , Ajay Rana c , Vivek Sharma d , Preeti Katoch b , Yogendra Padwad a,c , Shashi Bhushan a,b,∗ a
Academy of Scientific and Innovative Research, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh 176061, India Division of Biotechnology, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh 176061, India c Food and Nutraceuticals Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh 176061, India d Department of Plant Pathology, CSK-HPKV, Palampur, Himachal Pradesh 176061, India b
a r t i c l e
i n f o
Article history: Received 27 February 2016 Received in revised form 21 June 2016 Accepted 22 June 2016 Keywords: Phenolics Antioxidant Antimicrobial Cytotoxicity Apple leaves Antiinflammatory
a b s t r a c t Apple (Malus domestica Borkh.) leaves are good source of polyphenols. Considering the increasing demand of such phytochemicals, particularly in healthcare sector, the objective of this study was to evaluate the bioactivity of apple leaves phenolics. Different solvent mediated extracts obtained from the apple leaves were assessed for presence of phenolic compounds. Among different extracts, the highest phenolic content of 30.38 ± 0.50 mg/g were recorded in 70% aqueous ethanol (ALE-7) with subsequent high antioxidant value (IC50 49.16 g/mL) by ABTS assay. RP-HPLC-DAD phenolic profiling of leaves extract, irrespective of solvent used for extraction, revealed presence of five major compounds with maximum yield of phloridzin (24.43 ± 0.05 mg/g), followed by quercitrin (2.06 ± 0.05 mg/g), quercetin3-O-glucoside (1.55 ± 0.001 mg/g), epicatechin (0.37 ± 0.07 mg/g) and phloretin (0.15 ± 0.05 mg/g). ALE-7 extract was further fractionated with hexane (ALH) and ethyl acetate (ALEA), which were evaluated for their in vitro biological activities. ALEA extract exhibited higher nitric oxide (NO) scavenging activity (63.3%) at 200 g/mL. This fraction also showed maximum lymphocyte proliferation (34%) at 25 g/mL after 48 h. The antimicrobial testing of isolated fractions revealed that ALH fraction (MIC value ranging from 1.18–2.37 g/mL) could be a good candidate, especially for controlling food borne pathogen. Furthermore, the in vitro cytotoxicity assessment of different apple leaves fractions was also performed against human cancer cell lines (KB, SiHa and A-549), but none of the fraction was found cytotoxic against selected cell line. In conclusion, the presence of biologically active phenolics in apple leaves makes it a feasible renewable bioresource for extraction of such phytochemicals for the development of nutraceuticals particularly against inflammation and microbial infections. © 2016 Elsevier B.V. All rights reserved.
1. Introduction In addition to dietary constituents, fruits and vegetables also enriched human diet with specific non-nutritious biologically active constituents like carotenoids, flavonoids, isoflavonoids and phenolic acids. These phytochemicals hold a range of activities and provide array of health benefits through prevention of oxida-
∗ Corresponding author at: Division of Biotechnology, CSIR-Institute of Himalayan Bioresource Technology, Palampur 176 061, Himachal Pradesh, India. E-mail addresses: [email protected] (S. Rana), [email protected] (S. Kumar), [email protected] (A. Rana), [email protected] (V. Sharma), [email protected] (P. Katoch), [email protected] (Y. Padwad), [email protected], [email protected] (S. Bhushan). http://dx.doi.org/10.1016/j.indcrop.2016.06.027 0926-6690/© 2016 Elsevier B.V. All rights reserved.
tive burst, hypolipidemic and inflammatory damage, regulation of immune response and protection against various chronic diseases (Alissa and Ferns, 2012; George et al., 2009; Liu, 2003). The potential as well as demand of such phytochemicals and plant extracts can be gauzed from their overall global market worth, valued at around $2.5 billion in previous year (M&M, 2014). However, there is an unprecedented gap between the demand and supply of these molecules, particularly in healthcare sector. This deficit is mainly owned by dwindling natural resources, which drives the scientific predilection to look for alternative renewable resources. Among different phytochemicals, polyphenols hold prime position among various health promoting natural ingredients owing to their protective role during oxidative damage of cellular tissues (Pandey and Rizvi, 2009). These compounds play significant role in regulating metabolic disorders like coronary heart disease
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(CHD)/Cardiovascular disease (CVD), a topmost killer disease of previous decade (WHO, 2014). Recently, few reports on exploring compounds from the leaves of various plants are published using different extraction solvents and techniques (Dahmoune et al., 2014; Kazan et al., 2014; Melguizo-Melguizo et al., 2014). Similarly, apple (Malus domestica Borkh.) leaves can be a potential source for extraction of valuable bioactives. Surprisingly, except fruits and pomace, limited information is available on leaves and stem of apple plants. A study on scab (Venturia inaequalis) resistance in different apple cultivars revealed that apple leaves contain variety of polyphenolic compounds (Piccinelli et al., 1995). In a similar study, higher content of hydroxycinnamic acid, flavanols and phloridzin were reported from V. inaequalis infected leaves as compared to healthy leaves of Golden delicious cultivar (Mayr et al., 1997; Petkovsek et al., 2011). It was revealed that ethanolic extracts of apple leaves possess comparable antioxidant activity to that of the fruit, as evident from the degree of lipid peroxidation (BonarskaKujawa et al., 2011). A number of reports are available exhibiting numerous biological activities such as antioxidant, anti-inflammatory, antimicrobial, anticancer and cardioprotective effects of the phenolic compounds extracted from apple fruits (Barreca et al., 2014; Chang et al., 2012; Olson et al., 2007; Rezik et al., 2002; Wu et al., 2009). However, information pertaining to such diverse activities from extracts of apple leaves is lacking. Hence, the efforts were made in this work to evaluate mature apple leaves as a source of polyphenols along with their possible in vitro biological activities. 2. Material and methods 2.1. Plant material Malus domestica leaves (Red Chief) were collected from the orchard situated in the mid-hills of the Northwestern Himalayas at Bulsan, District Shimla, Himachal Pradesh, India. The mature leaves were collected from the orchard and dried at 55 ◦ C until constant weight was attained in an oven (Macro scientific). The dried leaves were pulverized in Retsch cutting mill (1 mm size) and stored in an air tight polybags till further evaluation. 2.2. Chemical and reagents Epicatechin, phloridzin, phloretin, quercitrin, quercetin-3-Oglucoside, quercetin, gallic acid and trolox were purchased from Sigma Aldrich. All other chemicals and solvents were of analytical grade (Merck, India). 2.3. Extraction of plant material The extraction of phenolics from dried leaves powder was done using methanol and ethanol solvents along with their aqueous concentration (70 and 50%). In brief, 100 mg leaf powder was taken in 15 mL centrifuge tube, with addition of 2 mL of respective solvents. Tubes were vortexed for 2 min followed by centrifugation at 5000 rpm for 10 min at room temperature (RT). The extraction process was repeated twice with 1.5 mL solvent and supernatant was collected and pooled to make final volume 5 mL with respective solvent. The extracts were filtered using 0.45 m filter and were labeled as ALE (ethanol), ALE-5 (50% Ethanol), ALE-7 (70% Ethanol), ALM (Methanol), ALM-5 (50% Methanol) and ALM-7 (70% Methanol). 2.4. Total phenolic content The total phenolic contents of apple leaves extracts obtained using various solvents (ALE-7, ALE-5, ALE, ALM-7, ALM-5 and ALM)
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were determined by spectrophotometric method (Swain and Hillis, 1959; Rana et al., 2013). The absorbance of reaction mixture was measured at 735 nm using spectrophotometer (T 90+ , PG instruments Ltd). The total phenolics content was expressed as mg gallic acid equivalent (GAE)/g dry plant material. All measurements were done in triplicate.
2.5. Total flavonoid content Total flavonoid content in different solvent extracts of apple leaves was measured spectrophotometrically (Kosalec et al., 2004). The absorbance of the reaction mixture was measured at 415 nm using spectrophotometer (T 90+ , PG instruments Ltd). The total flavonoid content of samples was expressed as mg quercetin equivalent (QE)/g dry plant material. All measurements were done in triplicate.
2.6. In vitro antioxidant activity 2.6.1. 2,2 -Azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay The antioxidant activity of apple leaves extracts was determined spectrophotometrically by previously reported method with some modifications (Re et al., 1999). Different concentration of apple leaves extracts were mixed with 2.0 mL of ABTS solution and absorbance was read at 734 nm using spectrophotometer (T 90+ , PG instruments Ltd.) exactly after 4 min. The radical scavenging activity (% inhibition) was calculated as: % inhibition = [(AB − AA)/AB] × 100, where AA – absorption of respective extract and AB – absorption of blank sample. The percent inhibitions were plotted against respective concentrations and IC50 values were calculated.
2.7. RP-HPLC-DAD profiling of phenolics Profiling of phenolic constituents in apple leaf extracts was performed using previously reported Reverse Phase- High Performance Liquid Chromatography (RP-HPLC-DAD) method (Rana et al., 2014). The separation of phenolic constituents was done using Synergi MAX RP80, C12 column (4.6 × 250 mm length, 4 m particle size). Acetonitrile (A) and 0.01% trifluoroacetic acid (B) was used as mobile phase at a flow rate of 1.0 mL/min and phenolics spectral data was recorded at 280 nm. For the quantification of phenolics, five different concentrations (5–25 g/mL) of each standard were prepared from respective stock solution (1 mg/mL in HPLC grade methanol).
2.8. Extract preparation for antimicrobial, cytotoxicity and anti-inflammatory potential For determining the antimicrobial, in vitro cytotoxic activity and anti-inflammatory potential, aqueous ethanol (70%) was used as extraction solvent due to high polyphenolic yield obtained in previous experiments. Dried apple leaves powder (500 g) was extracted thrice with 70% aqueous ethanol (1 L × 24 h × 3) at room temperature. The obtained extract was pooled and concentrated in a rotary evaporator (Buchi 210). The aqueous portion was partitioned with hexane and ethyl acetate (3 × 200 mL). The hexane (ALH), ethyl acetate (ALEA) and aqueous (ALW) fractions were concentrated in a rotary evaporator and lyophilized until a constant weight was obtained. The lyophilized extract powder was stored at 4 ◦ C for further analysis.
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2.9. Antimicrobial activity 2.9.1. Screening Different fractions were screened on Nutrient Agar (NA, Himedia) (Bacillus subtilis MTCC No 721, Klebisella pneumoniae MTCC 109, Staphylococus aureus MTCC 3160, Micrococus luteus MTCC 2470, and Escherichia coli MTCC 43) and brain heart infusion agar media (BHFA,Himedia) (Listeria monocytogenes MTCC 657) using diffusion well plate method. The bacterial inoculum was uniformly spread using sterile cotton swab on a sterile petri dishes and 30 L (50 mg/mL of each extract dissolved in molecular biology grade DMSO) (Biobasic Inc.) were added to wells (7 mm diameter in the agar gel). The plates were incubated overnight at 37 ◦ C and observed for bacterial growth. Tests were repeated three times. 2.9.2. Determination of minimum inhibitory concentration (MIC) The MIC value of the different fractions was determined in 96 well microplate in a final volume of 300 L (Wiegand et al., 2008). The overnight grown culture of different bacterial strains (CFU count of approximate 104 ) was inoculated in 2X liquid medium. Then different concentrations (0.29–4.67 g/mL) prepared from original stock (50 mg/mL) of tested antimicrobial agent were added to it in triplicates and final volume was adjusted. The plates were incubated at 37 ◦ C for a period of 16–20 h and cell growth was recorded at 600 nm using MultiskanTM GO Microplate Spectrophotometer (Thermo Scientitifc). The MIC value is defined as lowest concentration that prevents bacterial growth (Wiegand et al., 2008). 2.10. In vitro cytotoxicity of apple leaves 2.10.1. Cell lines and culture Cytotoxicity was determined in human cancer cells using, SiHa (Cervical squamous cell carcinoma ATCC HTB-35), KB (Oral carcinoma; ATCC CCL-17) and A549 (Lung carcinoma, ATCC CCL-185) cell lines, which were procured from National Centre for Cell Sciences (NCCS), Pune, India. SiHa and KB were grown in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Invitrogen, USA), whereas A549 was grown in RPMI-1640 medium (Gibco, Invitrogen, USA). Media were supplemented with 10% fetal bovine serum (FBS; Gibco, Invitrogen) and 1% antibiotic antimycotic (Gibco, Invitrogen). Cells were maintained under optimum culturing conditions i.e. 37 ◦ C temperature with 5% CO2 and 95% relative humidity in CO2 incubator (Thermo Scientifics, USA). 2.10.2. Sulforodamine B (SRB) assay SRB assay unveil the cell cytotoxicity through the measurement of change in cellular protein content that are fixed by trichloroacetic acid (Skehan et al., 1990). Briefly, confluent monolayer of cells were trypsinized, counted and seeded into 96-well microtiter plate at 2 × 104 cells per well containing 100 L of culture medium. Plates were incubated in CO2 incubator at 37 ◦ C with 95% humidity and 5% CO2 , for 24 h. Apple leaves extracts were dissolved in PBS and mixed at specific concentrations (25, 50, 100 and 200 g/mL) to culture medium in triplicates. Vinblastine (10 M) was used as standard drug molecule. The treated plates were re-incubated for 24, 48 and 72 h. Thereafter, the cells were fixed by adding 50 L, 50% trichloroacetic acid (w/v) and re-incubated for 1 h at 4 ◦ C, which was followed by washing with distilled water and air dried at room temperature. The staining was done by adding 100 L of 0.4% (w/v) SRB solution in 1% (v/v) acetic acid into each well. The plates containing stained cells were kept at room temperature for 30 min and subsequently these plates were washed four times with 1% (v/v) acetic acid to remove unbound dye. The excess moisture was removed through air drying under dark condition. The proteinbound dye was dissolved by adding 100 L of Tris base (Sigma)
solution (10 mmol) and absorbance was read at 540 nm using a microplate reader (BioTeK Synergy H1 Hybrid Reader). The percentage of the cellular toxicity was determined as follows: %cytotoxicity = [(C − S)/C] ∗ 100 where C = OD540 of Control and S = OD540 of Sample 2.11. Anti-inflammatory assay 2.11.1. Animals C57BL/6 j mice used in the study were housed and maintained in the animal house of CSIR-Institute of Himalayan Bioresource Technology, Palampur (H.P), India. The animals were fed with pelleted fed and distilled water ad libitum. Prior to study, the animals were acclimatized and maintained in animal house at 12 h light/dark cycle, 40–60% relative humidity and 21–25 ◦ C temperature. All animal studies were approved by institutional ethical committee (IHBT-10/Feb 2013) and were conducted in accordance with the guidelines of CPCSEA, Government of India. 2.11.2. Isolation of murine peritoneal macrophages C57BL/6J mice were injected with 5 mL of 3% brewer thioglycollate medium (w/v) (Cohn, 1978) into the peritoneal cavity for inflammatory response for 5 days. The animals were euthanized by CO2 asphyxiation at the end of experiment. The mice were carefully injected with ice cold phosphate buffer saline (PBS) supplemented with 3% fetal bovine serum (FBS) in peritoneal cavity. The abdomen was massaged gently to dislodge the macrophages into the PBS. The peritoneal fluid containing macrophages was aspirated and collected in polypropylene tube kept on ice. The contents were centrifuged at 1500 rpm for 8 min and the pellet was resuspended in RPMI-1640 medium supplemented with 10% FBS and 1% antibiotic antimycotic solution (Life Technologies, India). Thereafter, 0.5 × 106 cells per well were seeded in 96-well flat bottom microtiter plate, incubated at 37 ◦ C with 5% CO2 and 95% of relative humidity in CO2 incubator (Thermo Scientifics, USA) until cells attained morphology and becomes differentiated (Ray and Dittel, 2010). 2.11.3. Preparation of mice spleen cell suspension Following the sacrifice of mice by CO2 asphyxiation, spleen was excised aseptically in RPMI-1640 medium. Spleen tissue was pulverized using a rubber syringe plunger and centrifuged twice at 2000 rpm for 10 min each time. Pellet was suspended in a mixture of medium and red blood cell lysis buffer (Sigma, India) in 1:1 ratio followed by 5 min incubation at 37 ◦ C. Before cell count, cells were washed twice by centrifugation at 1500 rpm for 5 min. The final cell pellet was suspended in medium, counted and seeded (2 × 105 cells per well) in 96-well microtiter plate containing 100 L of culture medium (Lin et al., 2005). 2.11.4. Stimulation of peritoneal macrophages and determination of NO production The residual medium was replaced with IFN-␥ (100U/mL) (Merck Millipore) and LPS (Lipopolysaccharide) (1 g/mL) (Sigma Aldrich, USA) containing RPMI-1640 supplemented with 2% FBS, and cells were incubated in CO2 incubator for 24 h. After stimulation, the total NO released in the culture medium was determined as NO2 − by Griess Reagent method (Padwad et al., 2005) using Griess Reagent System (Promega) (Ding et al., 1988). In Griess reaction, 50 L of Griess reagent 1% Sulphanalamide solution and 0.1% N-(1-napthyly ethylenediamine) was added to the 50 L of culture medium and the absorbance was measured at 535 nm using microplate reader (BioTeK Synergy H1 Hybrid Reader). For standard curve, 0.1 M sodium nitrite solution was used.
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Fig. 1. Major phenolics in 70% ethanolic extract (ALE-7).
2.12. Determination of lymphocyte proliferation, using MTT assay Cell viability was determined, using trypan blue dye exclusion technique and cell proliferation was assessed by 3-[4,5dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay using previously reported method with slight modification (Mosmann, 1983). Briefly, cells were seeded into 96-well round-bottom microtiter plate at 0.2 × 106 cells per well in 100 L complete RPMI 1640 media containing Concanavalin A (Sigma Aldrich, USA) (1 g/mL) and Lipopolysaccharide (2 g/mL) (Sigma Aldrich, USA) for priming T cells and B cells respectively. Thereafter, samples were mixed at the appropriate concentrations (25, 50 100 and 200 g/mL) with culture medium and final volume was adjusted to 200 L. Plates were then incubated in CO2 incubator at 37 ◦ C with 95% humidity and 5% CO2. After 24, 48 and 72 h, 20 L MTT (5 mg/mL) was added to each well and incubated for overnight. After incubation, the cells were centrifuged at 2000 rpm for 10 min, and suspended in 100 L formazan crystals pre-dissolved in dimethyl sulfoxide (DMSO). The absorbance was measured at 570 nm using a microplate reader (BioTeK Synergy H1 Hybrid Reader). 2.13. Statistical analysis The results were expressed as the mean ± standard deviation (SD) calculated using Microsoft Excel whereas, p value was calculated with the help of GraphPad QuickCalcs: t test calculator. The experiments were performed for three times, with three replicates per treatment, if not otherwise stated. 3. Results and discussion 3.1. Total polyphenol and flavonoid content The total phenolic contents in apple leaves extracts obtained using different solvent systems ranged from 12.50 ± 0.33 to 30.38 ± 0.50 mg/g dry leaf powder (Table 1). Among solvent sys-
Table 1 Quantification of phenolics and antioxidant activity in various extracts of apple leaves. Extracts
Total phenolics Total flavonoids (mg/g DW leaf powder)
ABTS (IC50 g/mL)
ALM-5 ALM-7 ALM ALE-7 ALE-5 ALE
24.10 ± 0.11 24.48 ± 0.29 21.38 ± 0.11 30.38 ± 0.50 25.38 ± 0.38 12.50 ± 0.33
136.73 110.52 311.92 49.16 67.11 316.81
16.72 ± 0.50 15.66 ± 0.52 15.85 ± 0.05 20.92 ± 0.26 20.12 ± 0.34 10.95 ± 0.87
Note: ALM-5, 50% Methanol extract; ALM-7, 70% Methanol extract; ALM, Methanol extract; ALE-7, 70% Ethanol extract; ALE-5, 50% Ethanol extract; ALE, Ethanol extract.
tems, highest phenolic content (30.38 ± 0.50 mg/g as GAE) was obtained in 70% aqueous ethanol (ALE-7) followed by 50% aqueous ethanol (ALE-5) and 70% aqueous methanol (ALM-7). Similarly, for total flavonoid content, the maximum yield was obtained in ALE7 extract (20.9 mg/g as quercetin equivalent), followed by ALE-5. In a previous study, the ethanolic extract of apple leaves of two cultivars (Cvs Golden and Royal Delicious) have been reported to contain an average of 36 mg polyphenols (mg gallic acid equivalent (GAE)/g DW plant material) and 30.7 mg flavonoids (per g DW plant material) (Walia et al., 2015). A high amount of total phenolic (avg. 132 mg GAE/g DW) and flavonoid (avg. 33 mg Rutin Equivalent (RE)/g DW) contents were also reported in ethanol (70%, v/v) leaves extract obtained from different varieties of apple grown in Lithuania region (Liaudanskas et al., 2014). It was also reported that methanolic extracts of Venturia inequalis infected apple leaves (cvs Jonagold and Golden Delicious) accumulated higher phenolic compounds (86–159 mg GAE/g DW of leaf) as compared to healthy plants (60–88 mg GAE/g) (Petkovsek et al., 2008). In the present study, the high yield of extraction of phenolic compounds and flavonoids may be attributed to their affinity with the waterethanol solvent system. However, the variation of the yield and composition of the extractible may be attributed to the varietal,
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Table 2 Regression equation, correlation coefficient of phenolic standards. Compounds
Regression equation
R2
Retention Time
max
Epicatechin Phloridzin Phloretin Quercetin-3-O-glucoside Quercitrin
Y = 0.005x + 0.007 Y = 0.018x − 0.036 Y = 0.039x + 0.001 Y = 0.005x − 0.030 Y = 0.008x − 0.010
0.994 0.996 0.999 0.999 0.998
4.8 14.2 24.1 7.9 11.6
205.5, 280.5 224.2, 286.7 225.4, 286.7 205.5, 257.2, 355.6 205.5, 257.2, 349.6
Table 3 Quantification of phenolics in various extracts of apple leaves. Extracts ALM-5 ALM-7 ALM ALE-7 ALE-5 ALE
Epicatechin (mg/g) 0.46 ± 0.02 0.28 ± 0.005 0.26 ± 0.002 0.37 ± 0.07 0.31 ± 0.02 0.07 ± 0.001
Quercetin-3-O-glucoside (mg/g) 1.53 ± 0.003 1.55 ± 0.002 1.60 ± 0.001 1.55 ± 0.001 1.19 ± 0.002 0.50 ± 0.034
Phloridzin (mg/g) 21.90 ± 0.02 18.79 ± 0.08 22.68 ± 0.10 24.43 ± 0.05 21.07 ± 0.06 19.67 ± 0.07
Phloretin (mg/g)
Quercitrin (mg/g)
0.17 ± 1.13 0.12 ± 0.29 0.15 ± 0.77 0.15 ± 0.05 0.13 ± 0.07 0.16 ± 0.05
1.75 ± 0.02 1.56 ± 0.03 1.93 ± 0.02 2.06 ± 0.05 1.50 ± 0.001 0.81 ± 0.009
Note: ALM-5, 50% Methanol extract; ALM-7, 70% Methanol extract; ALM, Methanol extract; ALE-7, 70% Ethanol extract; ALE-5, 50% Ethanol extract; ALE, Ethanol extract.
geographic conditions and extraction factors, as has been reported in previous works. 3.2. In vitro antioxidant potential The quality of polyphenols is generally associated with their antioxidant potential. ABTS is a widely used method for phenolic antioxidants determination. This assay is based on decolorization through reduction of ABTS• + radicals generated by the oxidation of ABTS with potassium persulphate. The different extracts of apple leaves evaluated under in vitro conditions by ABTS assay showed lower IC50 value in ALE-7 extract (49.16 g/mL) followed by ALE-5 and ALM-7 (Table 1). The results are in accordance with phenolics and flavonoids contents recorded in respective fractions. The comparative IC50 values of 47.10 g/mL and 66.53 g/mL have been reported in case of methanol extract of golden and royal delicious leaves respectively (Walia et al., 2015). The antioxidant potential of ethanolic extracts obtained from variety specific apple leaves was reported to be in a range of 150-355 moL Trolox equivalent/g DW of lyophilized leaf powder (Liaudanskas et al., 2014). 3.3. Quantification of phenolics in apple leaves Liquid chromatography (LC) is the most accurate technique for the characterization and quantification of phenolic constituents because of its very high sensitivity and selectivity. There are numerous reports available on profiling of phenolic bioconstituents by liquid chromatography (Liaudanskas et al., 2014; Walia et al., 2015). In present study, five phenolic compounds were identified in apple leaves using RP-HPLC system by comparing retention times, max and peak spiking with internal standards. Quantification of individual phenolics was accomplished with an external standard calibration. The regression equation, correlation coefficient, retention time and absorbance maxima of each standard are shown in Table 2. The results revealed the presence of phloridzin, quercetin-3-O-glucoside, phloretin, quercitrin and epicatechin in various solvent extracts of apple leaves. RP-HPLC chromatogram of ALE-7 extract is shown in Fig. 1. The results showed that total amounts of detected phenolic compounds were higher in ALE7 (extract) and predominant phenolic present in ALE-7 extract was phloridzin (24.43 ± 0.05 mg/g). Amount of individual phenolics present in the samples is summarized in Table 3. In our study, highest content of phloridzin was observed among all the extracts by RP-HPLC are in general agreement with those accounted by others (Walia et al., 2015). The phloridzin has been reported as major phenolic (20.3 mg/g) in both royal and golden varieties of apple
Table 4 Minimum inhibitory concentration (MIC, g/mL) of various fractionated extracts of apple leaves. Pathogens
ALH
ALEA
ALW
Bacillus subtilis Klebisella pneumoniae Staphylococus aureus Micrococus luteus Escherichia coli Listeria monocytogenes
1.18 1.18 1.18 1.18 1.18 2.37
– – – – – –
– – – – – –
Note: ALH, Hexane fraction; ALEA, Ethyl acetate fraction; ALW, aqueous fraction.
leaves by UPLC–MS-MS. (Walia et al., 2015). In another study, Liaudanskas et al. reported the occurrence of phloretin, catechin, caffeic acid, chlorogenic acid, epicatechin, isoquercitrin, rutin, hyperoside, quercitrin and avicularin along with phloridzin in ethanolic extracts of the Aldas, Auksis, Ligol, and Lodel cultivars. A higher content of dihydrochalcones phloridzin was also reported in different varieties of apple leaves (Liaudanskas et al., 2014). Petkovsek et al. reported the presence of hydroxycinnamic acids, flavanols, dihydrochalcones, and quercetin in apple leaves and fruits grown in organic and integrated conditions (Petkovsek et al., 2010). The results attained during above studies will be helpful for the selection of extraction solvent and cultivars for the efficient extraction of phenolic rich fraction so as to measure the in vitro cytotoxicity, anti-inflammatory potential and antimicrobial activity. 3.4. Antimicrobial activity The antimicrobial activity of the different fractions (ALH, ALEA and ALW) was studied against six pathogenic bacterial strains (Bacillus subtilis, Klebisella pneumoniae, Staphylococus aureus, Micrococus luteus, Listeria monocytogenes and E. coli). Initial screening of different fractions was done using diffusion well plate method. Among all the fractions, ALH fraction showed significant zone of inhibition against all the selected pathogens. Furthermore, ALH was screened for the MIC using different concentrations 0.29–4.67 g/mL. Results showed that ALH fraction posses 1.8 g/mL MIC against Bacillus subtilis, Klebisella pneumoniae, Staphylococus aureus, Micrococus luteus, Escherichia coli and 2.37 g/mL against Listeria monocytogenes. The results of the antibacterial activity are presented in Table 4. Phenolics constituents present in apple and its plant parts are mainly responsible for their biological activity. Phloretin, the major dihydrochalcone present in apple fruit was also reported as chief phenolics in the leaves of Malus domestica responsible for antibacterial activity in
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present study. In earlier study, phloretin and its glycosides have been evaluated for antimicrobial activity against gram positive and gram negative bacterial strains. Dihydrochalcone phloretin has been reported for its antibacterial action against Staphylococcus aureus, Listeria monocytogenes and Salmonella typhimurium. Phloretin showed the inhibitory effects at concentration of 7.81, 62.5 and 125 g/mL against S. aureus, L. monocytogenes, S. typhimurium respectively (Barreca et al., 2014). Park et al. suggested that at low concentration (30–35 g/mL) phloretin inhibit biofilm formation of enterohemorrhagic E. coli O157, and thus acts as a biofilm inhibitor (Park et al., 2012). Apple peel phenolics also showed significant antimicrobial activity against Bacillus cereus and Escherichia coli serotype O157:H7 and suggested to have potential for treating specific microbial infections (Fratianni et al., 2011). 3.5. Assessment of cytotoxicity on human cancer cell lines The various fractions of apple leaves were assessed in vitro against SiHa, KB and HT-29 human cancer cells. It was revealed that maximum (24.47 ± 010%) cytotoxicity was observed in ALH extract after 48 h against KB cell line, but neither the concentration nor the time point of all the fractions prove more than 50% cytotoxic and the IC50 value remains >200 g/mL. The results of cytotoxicity are shown in Table 5. In previous studies, cytotoxic activity of essential oil obtained from apple leaves was reported against glioma cells, human lung carcinoma, chinese hamster ovary and human acute monocytic leukemia cell line. The highest % cytotoxicity of essential oil of apple leaves was observed in glioma cells lines (98.2%) at 2000 g/mL (Walia et al., 2012). 3.6. Cellular NO inhibitory effect of fractions on pre-treated peritoneal macrophages In the present study, different fractions of apple leaves were evaluated for NO inhibition production on pre-treated peritoneal macrophages. After LPS treatment, NO production was increased in peritoneal macrophages. Results of present investigation showed that all the fractions inhibited NO production at different concentrations (25–200 g/mL) (Fig. 2). ALEA (63.3%) and ALH (61.8%) found to inhibit significantly (p < 0.05) higher percentage of NO radicals as compared to ALW (28.7%) at 200 g/mL concentration. On the other hand, ALW showed 28.7% inhibition at same concentration. Among all the extracts minimum % inhibition was showed by
Fig. 2. Nitric oxide (NO) scavenging activity of hexane (ALH), ethyl acetate (ALEA) and aqueous (ALW) fraction of apple leaves.
ALH (7.01%) at 25 g/mL. The higher NO inhibitory effect of ALEA may be attributed to the presence of dihydrochalcones (phloridzin and phloretin). In the previous study, it was reported that pretreatment of RAW264.7 cells with 10 M phloretin significantly inhibited the levels of NO, COX-2, PGE2, TNF-␣, iNOS and IL-6 (Chang et al., 2012). 3.7. Effect of extracts on proliferation of murine splenocytes MTT-based splenocytes proliferation was assayed at different incubation periods. The apple leaves fractions were tested in an array of concentrations, towards lipopolysaccharide (LPS) and concanavalin A (Con A) stimulated splenocytes. Results revealed that ALH showed maximum proliferation 10.1% (p < 0.05) at 25 g/mL after 24 h, whereas it showed 8.87% (p < 0.05) proliferation at same concentration after 48 h and no proliferation after 72 h (Fig. 3). Similar to this ALW showed decrease in proliferation with increase in concentration after 24 h with maximum proliferation 17.27% (p < 0.05) at 25 g/mL concentration, whereas 10.7% (p < 0.05) proliferation was found after 48 h at the same concentration with no more proliferation up to 72 h. However, ALEA showed delayed response as no proliferation was observed after 24 h, but a significant increase i.e. 33.91% (p < 0.05) in proliferation after 48 h at same concentration was observed. After 72 h it showed 3.80% (p < 0.05) proliferation at 25 g/mL concentration, which further decreases with increase in concentration. Results of the present investigation
Fig. 3. Depicting lymphocyte proliferation of hexane (ALH), ethyl acetate (ALEA) and aqueous (ALW) fraction of apple leaves.
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Table 5 Cytotoxicity of apple leaves extracts on different human cancer cell lines. Extracts Conc (g/mL)
Cytotoxicity (%) SiHa 24 h
KB 48 h
72 h
24 h
A549 48 h
72 h
24 h
48 h
72 h
ALH
50 100 200 400
3.10 ± 0.04 0.00 ± 0.00 0.00 ± 0.00 17.89 ± 0.05
4.77 ± 0.05 4.44 ± 0.02 3.33 ± 0.008 17.14 ± 0.027
12.27 ± 0.11 13.18 ± 0.03 13.70 ± 0.04 21.97 ± 0.08
2.23 ± 0.05 4.78 ± 0.05 8.72 ± 0.02 23.69 ± 0.08
5.18 ± 0.09 2.47 ± 0.05 2.08 ± 0.03 24.47 ± 010
0.12 ± 0.09 2.23 ± 0.03 3.26 ± 0.02 18.25 ± 0.05
4.19 ± 0.09 4.81 ± 0.12 4.68 ± 0.11 14.75 ± 0.01
3.02 ± 0.13 5.40 ± 0.05 35.09 ± 0.01 10.17 ± 0.009
5.21 ± 0.14 9.62 ± 0.05 8.94 ± 0.04 11.74 ± 0.12
ALEA
50 100 200 400
1.24 ± 0.05 0.00 ± 0.00 2.24 ± 0.04 14.03 ± 0.03
0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 7.33 ± 0.030
10.88 ± 0.09 13.61 ± 0.02 14.94 ± 0.07 18.30 ± 0.07
7.26 ± 0.05 5.11 ± 0.03 9.57 ± 0.01 16.47 ± 0.03
4.07 ± 0.15 1.56 ± 0.02 3.13 ± 0.04 11.00 ± 0.17
2.58 ± 0.13 4.27 ± 0.01 10.64 ± 0.05 16.01 ± 0.002
4.54 ± 0.02 7.17 ± 0.06 6.55 ± 0.06 11.72 ± 0.05
0.25 ± 0.08 0.00 ± 0.00 0.00 ± 0.00 0.000 ± 0.00
0.42 ± 0.06 5.55 ± 0.04 7.76 ± 0.08 10.47 ± 0.13
ALW
50 100 200 400
0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 3.29 ± 0.03
3.21 ± 0.04 2.97 ± 0.01 9.45 ± 0.01 22.88 ± 0.8
4.54 ± 0.05 2.63 ± 0.04 5.96 ± 0.06 12.69 ± 0.01
0.00 ± 0.00 0.69 ± 0.14 3.72 ± 0.03 10.68 ± 0.02
0.00 ± 0.00 0.00 ± 0.00 5.27 ± 0.02 12.03 ± 0.05
4.54 ± 0.08 3.70 ± 0.05 4.94 ± 0.02 10.20 ± 0.01
0.55 ± 0.03 3.49 ± 0.10 0.00 ± 0.00 2.64 ± 0.01
2.33 ± 0.09 5.13 ± 0.04 9.16 ± 0.08 10.22 ± 0.03
Note: ALH, Hexane fraction; ALEA, Ethyl acetate fraction; ALW, aqueous fraction; SiHa, Cervical squamous cell carcinoma; KB, Oral carcinoma; A549, Lung carcinoma.
showed good pharmacological activity of apple leaves suggests that it can be used as a potential source for the extraction of valuable bioactive molecules. 4. Conclusions The present study concludes that leaves of Malus domestica are promising source of phenolic compounds (dihydrochalcones and flavonol glycosides) and also possess appreciable free radical scavenging activity. Ethyl acetate fraction showed highest potential in the inhibition of nitric oxide production in pretreated peritoneal macrophages. Results of antimicrobial activity revealed the potential of hexane fraction as strong antimicrobial agent against selected pathogenic strains and suggested to have capability for treating specific microbial infections. Hexane fraction was also found to play significant role in proliferation of murine splenocytes at low concentration. The findings of this study clearly provide evidence that underutilized apple leaves can be used for the treatment of inflammatory diseases and pathogenic infections in the form of various health promoting nutraceuticals and dietary supplements. 5. Conflict of interest Authors declare that they have no conflict of interest including any financial, personal or other relationships with other people or organizations. Acknowledgements The authors are thankful to the Director (CSIR-IHBT), Palampur for providing support during the course of work. Authors are also grateful to CSIR, New Delhi, India for funding through network project (BSC 105) under which this work was carried out. References Alissa, E.M., Ferns, G.A., 2012. Functional foods and nutraceuticals in the primary prevention of cardiovascular diseases. J. Nutr. Metab., 16, Article ID 569486. Barreca, D., Bellocco, E., Lagana, G., Ginestra, G., Bisignano, C., 2014. Biochemical and antimicrobial activity of phloretin and its glycosilated derivatives present in apple and kumquat. Food Chem. 160, 292–297. Bonarska-Kujawa, D., Cyboran, S., Oszmianski, J., Kleszczynska, H., 2011. Extracts from apple leaves and fruits as effective antioxidants. J. Med. Plant Res. 5, 2339–2347. Chang, W.-T., Huang, W.-T., Liou, C.-J., 2012. Evaluation of the anti-inflammatory effects of phloretin and phlorizin in lipopolysaccharide-stimulated mouse macrophages. Food Chem. 134, 972–979. Cohn, Z., 1978. The activation of mononuclear phagocytes: fact, fancy, and future. J. Immunol. 121, 813–816.
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Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop
Phenolic constituents from apple tree leaves and their in vitro biological activity Shalika Rana a,b , Shiv Kumar a,c , Ajay Rana c , Vivek Sharma d , Preeti Katoch b , Yogendra Padwad a,c , Shashi Bhushan a,b,∗ a
Academy of Scientific and Innovative Research, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh 176061, India Division of Biotechnology, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh 176061, India c Food and Nutraceuticals Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh 176061, India d Department of Plant Pathology, CSK-HPKV, Palampur, Himachal Pradesh 176061, India b
a r t i c l e
i n f o
Article history: Received 27 February 2016 Received in revised form 21 June 2016 Accepted 22 June 2016 Keywords: Phenolics Antioxidant Antimicrobial Cytotoxicity Apple leaves Antiinflammatory
a b s t r a c t Apple (Malus domestica Borkh.) leaves are good source of polyphenols. Considering the increasing demand of such phytochemicals, particularly in healthcare sector, the objective of this study was to evaluate the bioactivity of apple leaves phenolics. Different solvent mediated extracts obtained from the apple leaves were assessed for presence of phenolic compounds. Among different extracts, the highest phenolic content of 30.38 ± 0.50 mg/g were recorded in 70% aqueous ethanol (ALE-7) with subsequent high antioxidant value (IC50 49.16 g/mL) by ABTS assay. RP-HPLC-DAD phenolic profiling of leaves extract, irrespective of solvent used for extraction, revealed presence of five major compounds with maximum yield of phloridzin (24.43 ± 0.05 mg/g), followed by quercitrin (2.06 ± 0.05 mg/g), quercetin3-O-glucoside (1.55 ± 0.001 mg/g), epicatechin (0.37 ± 0.07 mg/g) and phloretin (0.15 ± 0.05 mg/g). ALE-7 extract was further fractionated with hexane (ALH) and ethyl acetate (ALEA), which were evaluated for their in vitro biological activities. ALEA extract exhibited higher nitric oxide (NO) scavenging activity (63.3%) at 200 g/mL. This fraction also showed maximum lymphocyte proliferation (34%) at 25 g/mL after 48 h. The antimicrobial testing of isolated fractions revealed that ALH fraction (MIC value ranging from 1.18–2.37 g/mL) could be a good candidate, especially for controlling food borne pathogen. Furthermore, the in vitro cytotoxicity assessment of different apple leaves fractions was also performed against human cancer cell lines (KB, SiHa and A-549), but none of the fraction was found cytotoxic against selected cell line. In conclusion, the presence of biologically active phenolics in apple leaves makes it a feasible renewable bioresource for extraction of such phytochemicals for the development of nutraceuticals particularly against inflammation and microbial infections. © 2016 Elsevier B.V. All rights reserved.
1. Introduction In addition to dietary constituents, fruits and vegetables also enriched human diet with specific non-nutritious biologically active constituents like carotenoids, flavonoids, isoflavonoids and phenolic acids. These phytochemicals hold a range of activities and provide array of health benefits through prevention of oxida-
∗ Corresponding author at: Division of Biotechnology, CSIR-Institute of Himalayan Bioresource Technology, Palampur 176 061, Himachal Pradesh, India. E-mail addresses: [email protected] (S. Rana), [email protected] (S. Kumar), [email protected] (A. Rana), [email protected] (V. Sharma), [email protected] (P. Katoch), [email protected] (Y. Padwad), [email protected], [email protected] (S. Bhushan). http://dx.doi.org/10.1016/j.indcrop.2016.06.027 0926-6690/© 2016 Elsevier B.V. All rights reserved.
tive burst, hypolipidemic and inflammatory damage, regulation of immune response and protection against various chronic diseases (Alissa and Ferns, 2012; George et al., 2009; Liu, 2003). The potential as well as demand of such phytochemicals and plant extracts can be gauzed from their overall global market worth, valued at around $2.5 billion in previous year (M&M, 2014). However, there is an unprecedented gap between the demand and supply of these molecules, particularly in healthcare sector. This deficit is mainly owned by dwindling natural resources, which drives the scientific predilection to look for alternative renewable resources. Among different phytochemicals, polyphenols hold prime position among various health promoting natural ingredients owing to their protective role during oxidative damage of cellular tissues (Pandey and Rizvi, 2009). These compounds play significant role in regulating metabolic disorders like coronary heart disease
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(CHD)/Cardiovascular disease (CVD), a topmost killer disease of previous decade (WHO, 2014). Recently, few reports on exploring compounds from the leaves of various plants are published using different extraction solvents and techniques (Dahmoune et al., 2014; Kazan et al., 2014; Melguizo-Melguizo et al., 2014). Similarly, apple (Malus domestica Borkh.) leaves can be a potential source for extraction of valuable bioactives. Surprisingly, except fruits and pomace, limited information is available on leaves and stem of apple plants. A study on scab (Venturia inaequalis) resistance in different apple cultivars revealed that apple leaves contain variety of polyphenolic compounds (Piccinelli et al., 1995). In a similar study, higher content of hydroxycinnamic acid, flavanols and phloridzin were reported from V. inaequalis infected leaves as compared to healthy leaves of Golden delicious cultivar (Mayr et al., 1997; Petkovsek et al., 2011). It was revealed that ethanolic extracts of apple leaves possess comparable antioxidant activity to that of the fruit, as evident from the degree of lipid peroxidation (BonarskaKujawa et al., 2011). A number of reports are available exhibiting numerous biological activities such as antioxidant, anti-inflammatory, antimicrobial, anticancer and cardioprotective effects of the phenolic compounds extracted from apple fruits (Barreca et al., 2014; Chang et al., 2012; Olson et al., 2007; Rezik et al., 2002; Wu et al., 2009). However, information pertaining to such diverse activities from extracts of apple leaves is lacking. Hence, the efforts were made in this work to evaluate mature apple leaves as a source of polyphenols along with their possible in vitro biological activities. 2. Material and methods 2.1. Plant material Malus domestica leaves (Red Chief) were collected from the orchard situated in the mid-hills of the Northwestern Himalayas at Bulsan, District Shimla, Himachal Pradesh, India. The mature leaves were collected from the orchard and dried at 55 ◦ C until constant weight was attained in an oven (Macro scientific). The dried leaves were pulverized in Retsch cutting mill (1 mm size) and stored in an air tight polybags till further evaluation. 2.2. Chemical and reagents Epicatechin, phloridzin, phloretin, quercitrin, quercetin-3-Oglucoside, quercetin, gallic acid and trolox were purchased from Sigma Aldrich. All other chemicals and solvents were of analytical grade (Merck, India). 2.3. Extraction of plant material The extraction of phenolics from dried leaves powder was done using methanol and ethanol solvents along with their aqueous concentration (70 and 50%). In brief, 100 mg leaf powder was taken in 15 mL centrifuge tube, with addition of 2 mL of respective solvents. Tubes were vortexed for 2 min followed by centrifugation at 5000 rpm for 10 min at room temperature (RT). The extraction process was repeated twice with 1.5 mL solvent and supernatant was collected and pooled to make final volume 5 mL with respective solvent. The extracts were filtered using 0.45 m filter and were labeled as ALE (ethanol), ALE-5 (50% Ethanol), ALE-7 (70% Ethanol), ALM (Methanol), ALM-5 (50% Methanol) and ALM-7 (70% Methanol). 2.4. Total phenolic content The total phenolic contents of apple leaves extracts obtained using various solvents (ALE-7, ALE-5, ALE, ALM-7, ALM-5 and ALM)
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were determined by spectrophotometric method (Swain and Hillis, 1959; Rana et al., 2013). The absorbance of reaction mixture was measured at 735 nm using spectrophotometer (T 90+ , PG instruments Ltd). The total phenolics content was expressed as mg gallic acid equivalent (GAE)/g dry plant material. All measurements were done in triplicate.
2.5. Total flavonoid content Total flavonoid content in different solvent extracts of apple leaves was measured spectrophotometrically (Kosalec et al., 2004). The absorbance of the reaction mixture was measured at 415 nm using spectrophotometer (T 90+ , PG instruments Ltd). The total flavonoid content of samples was expressed as mg quercetin equivalent (QE)/g dry plant material. All measurements were done in triplicate.
2.6. In vitro antioxidant activity 2.6.1. 2,2 -Azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay The antioxidant activity of apple leaves extracts was determined spectrophotometrically by previously reported method with some modifications (Re et al., 1999). Different concentration of apple leaves extracts were mixed with 2.0 mL of ABTS solution and absorbance was read at 734 nm using spectrophotometer (T 90+ , PG instruments Ltd.) exactly after 4 min. The radical scavenging activity (% inhibition) was calculated as: % inhibition = [(AB − AA)/AB] × 100, where AA – absorption of respective extract and AB – absorption of blank sample. The percent inhibitions were plotted against respective concentrations and IC50 values were calculated.
2.7. RP-HPLC-DAD profiling of phenolics Profiling of phenolic constituents in apple leaf extracts was performed using previously reported Reverse Phase- High Performance Liquid Chromatography (RP-HPLC-DAD) method (Rana et al., 2014). The separation of phenolic constituents was done using Synergi MAX RP80, C12 column (4.6 × 250 mm length, 4 m particle size). Acetonitrile (A) and 0.01% trifluoroacetic acid (B) was used as mobile phase at a flow rate of 1.0 mL/min and phenolics spectral data was recorded at 280 nm. For the quantification of phenolics, five different concentrations (5–25 g/mL) of each standard were prepared from respective stock solution (1 mg/mL in HPLC grade methanol).
2.8. Extract preparation for antimicrobial, cytotoxicity and anti-inflammatory potential For determining the antimicrobial, in vitro cytotoxic activity and anti-inflammatory potential, aqueous ethanol (70%) was used as extraction solvent due to high polyphenolic yield obtained in previous experiments. Dried apple leaves powder (500 g) was extracted thrice with 70% aqueous ethanol (1 L × 24 h × 3) at room temperature. The obtained extract was pooled and concentrated in a rotary evaporator (Buchi 210). The aqueous portion was partitioned with hexane and ethyl acetate (3 × 200 mL). The hexane (ALH), ethyl acetate (ALEA) and aqueous (ALW) fractions were concentrated in a rotary evaporator and lyophilized until a constant weight was obtained. The lyophilized extract powder was stored at 4 ◦ C for further analysis.
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2.9. Antimicrobial activity 2.9.1. Screening Different fractions were screened on Nutrient Agar (NA, Himedia) (Bacillus subtilis MTCC No 721, Klebisella pneumoniae MTCC 109, Staphylococus aureus MTCC 3160, Micrococus luteus MTCC 2470, and Escherichia coli MTCC 43) and brain heart infusion agar media (BHFA,Himedia) (Listeria monocytogenes MTCC 657) using diffusion well plate method. The bacterial inoculum was uniformly spread using sterile cotton swab on a sterile petri dishes and 30 L (50 mg/mL of each extract dissolved in molecular biology grade DMSO) (Biobasic Inc.) were added to wells (7 mm diameter in the agar gel). The plates were incubated overnight at 37 ◦ C and observed for bacterial growth. Tests were repeated three times. 2.9.2. Determination of minimum inhibitory concentration (MIC) The MIC value of the different fractions was determined in 96 well microplate in a final volume of 300 L (Wiegand et al., 2008). The overnight grown culture of different bacterial strains (CFU count of approximate 104 ) was inoculated in 2X liquid medium. Then different concentrations (0.29–4.67 g/mL) prepared from original stock (50 mg/mL) of tested antimicrobial agent were added to it in triplicates and final volume was adjusted. The plates were incubated at 37 ◦ C for a period of 16–20 h and cell growth was recorded at 600 nm using MultiskanTM GO Microplate Spectrophotometer (Thermo Scientitifc). The MIC value is defined as lowest concentration that prevents bacterial growth (Wiegand et al., 2008). 2.10. In vitro cytotoxicity of apple leaves 2.10.1. Cell lines and culture Cytotoxicity was determined in human cancer cells using, SiHa (Cervical squamous cell carcinoma ATCC HTB-35), KB (Oral carcinoma; ATCC CCL-17) and A549 (Lung carcinoma, ATCC CCL-185) cell lines, which were procured from National Centre for Cell Sciences (NCCS), Pune, India. SiHa and KB were grown in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Invitrogen, USA), whereas A549 was grown in RPMI-1640 medium (Gibco, Invitrogen, USA). Media were supplemented with 10% fetal bovine serum (FBS; Gibco, Invitrogen) and 1% antibiotic antimycotic (Gibco, Invitrogen). Cells were maintained under optimum culturing conditions i.e. 37 ◦ C temperature with 5% CO2 and 95% relative humidity in CO2 incubator (Thermo Scientifics, USA). 2.10.2. Sulforodamine B (SRB) assay SRB assay unveil the cell cytotoxicity through the measurement of change in cellular protein content that are fixed by trichloroacetic acid (Skehan et al., 1990). Briefly, confluent monolayer of cells were trypsinized, counted and seeded into 96-well microtiter plate at 2 × 104 cells per well containing 100 L of culture medium. Plates were incubated in CO2 incubator at 37 ◦ C with 95% humidity and 5% CO2 , for 24 h. Apple leaves extracts were dissolved in PBS and mixed at specific concentrations (25, 50, 100 and 200 g/mL) to culture medium in triplicates. Vinblastine (10 M) was used as standard drug molecule. The treated plates were re-incubated for 24, 48 and 72 h. Thereafter, the cells were fixed by adding 50 L, 50% trichloroacetic acid (w/v) and re-incubated for 1 h at 4 ◦ C, which was followed by washing with distilled water and air dried at room temperature. The staining was done by adding 100 L of 0.4% (w/v) SRB solution in 1% (v/v) acetic acid into each well. The plates containing stained cells were kept at room temperature for 30 min and subsequently these plates were washed four times with 1% (v/v) acetic acid to remove unbound dye. The excess moisture was removed through air drying under dark condition. The proteinbound dye was dissolved by adding 100 L of Tris base (Sigma)
solution (10 mmol) and absorbance was read at 540 nm using a microplate reader (BioTeK Synergy H1 Hybrid Reader). The percentage of the cellular toxicity was determined as follows: %cytotoxicity = [(C − S)/C] ∗ 100 where C = OD540 of Control and S = OD540 of Sample 2.11. Anti-inflammatory assay 2.11.1. Animals C57BL/6 j mice used in the study were housed and maintained in the animal house of CSIR-Institute of Himalayan Bioresource Technology, Palampur (H.P), India. The animals were fed with pelleted fed and distilled water ad libitum. Prior to study, the animals were acclimatized and maintained in animal house at 12 h light/dark cycle, 40–60% relative humidity and 21–25 ◦ C temperature. All animal studies were approved by institutional ethical committee (IHBT-10/Feb 2013) and were conducted in accordance with the guidelines of CPCSEA, Government of India. 2.11.2. Isolation of murine peritoneal macrophages C57BL/6J mice were injected with 5 mL of 3% brewer thioglycollate medium (w/v) (Cohn, 1978) into the peritoneal cavity for inflammatory response for 5 days. The animals were euthanized by CO2 asphyxiation at the end of experiment. The mice were carefully injected with ice cold phosphate buffer saline (PBS) supplemented with 3% fetal bovine serum (FBS) in peritoneal cavity. The abdomen was massaged gently to dislodge the macrophages into the PBS. The peritoneal fluid containing macrophages was aspirated and collected in polypropylene tube kept on ice. The contents were centrifuged at 1500 rpm for 8 min and the pellet was resuspended in RPMI-1640 medium supplemented with 10% FBS and 1% antibiotic antimycotic solution (Life Technologies, India). Thereafter, 0.5 × 106 cells per well were seeded in 96-well flat bottom microtiter plate, incubated at 37 ◦ C with 5% CO2 and 95% of relative humidity in CO2 incubator (Thermo Scientifics, USA) until cells attained morphology and becomes differentiated (Ray and Dittel, 2010). 2.11.3. Preparation of mice spleen cell suspension Following the sacrifice of mice by CO2 asphyxiation, spleen was excised aseptically in RPMI-1640 medium. Spleen tissue was pulverized using a rubber syringe plunger and centrifuged twice at 2000 rpm for 10 min each time. Pellet was suspended in a mixture of medium and red blood cell lysis buffer (Sigma, India) in 1:1 ratio followed by 5 min incubation at 37 ◦ C. Before cell count, cells were washed twice by centrifugation at 1500 rpm for 5 min. The final cell pellet was suspended in medium, counted and seeded (2 × 105 cells per well) in 96-well microtiter plate containing 100 L of culture medium (Lin et al., 2005). 2.11.4. Stimulation of peritoneal macrophages and determination of NO production The residual medium was replaced with IFN-␥ (100U/mL) (Merck Millipore) and LPS (Lipopolysaccharide) (1 g/mL) (Sigma Aldrich, USA) containing RPMI-1640 supplemented with 2% FBS, and cells were incubated in CO2 incubator for 24 h. After stimulation, the total NO released in the culture medium was determined as NO2 − by Griess Reagent method (Padwad et al., 2005) using Griess Reagent System (Promega) (Ding et al., 1988). In Griess reaction, 50 L of Griess reagent 1% Sulphanalamide solution and 0.1% N-(1-napthyly ethylenediamine) was added to the 50 L of culture medium and the absorbance was measured at 535 nm using microplate reader (BioTeK Synergy H1 Hybrid Reader). For standard curve, 0.1 M sodium nitrite solution was used.
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Fig. 1. Major phenolics in 70% ethanolic extract (ALE-7).
2.12. Determination of lymphocyte proliferation, using MTT assay Cell viability was determined, using trypan blue dye exclusion technique and cell proliferation was assessed by 3-[4,5dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay using previously reported method with slight modification (Mosmann, 1983). Briefly, cells were seeded into 96-well round-bottom microtiter plate at 0.2 × 106 cells per well in 100 L complete RPMI 1640 media containing Concanavalin A (Sigma Aldrich, USA) (1 g/mL) and Lipopolysaccharide (2 g/mL) (Sigma Aldrich, USA) for priming T cells and B cells respectively. Thereafter, samples were mixed at the appropriate concentrations (25, 50 100 and 200 g/mL) with culture medium and final volume was adjusted to 200 L. Plates were then incubated in CO2 incubator at 37 ◦ C with 95% humidity and 5% CO2. After 24, 48 and 72 h, 20 L MTT (5 mg/mL) was added to each well and incubated for overnight. After incubation, the cells were centrifuged at 2000 rpm for 10 min, and suspended in 100 L formazan crystals pre-dissolved in dimethyl sulfoxide (DMSO). The absorbance was measured at 570 nm using a microplate reader (BioTeK Synergy H1 Hybrid Reader). 2.13. Statistical analysis The results were expressed as the mean ± standard deviation (SD) calculated using Microsoft Excel whereas, p value was calculated with the help of GraphPad QuickCalcs: t test calculator. The experiments were performed for three times, with three replicates per treatment, if not otherwise stated. 3. Results and discussion 3.1. Total polyphenol and flavonoid content The total phenolic contents in apple leaves extracts obtained using different solvent systems ranged from 12.50 ± 0.33 to 30.38 ± 0.50 mg/g dry leaf powder (Table 1). Among solvent sys-
Table 1 Quantification of phenolics and antioxidant activity in various extracts of apple leaves. Extracts
Total phenolics Total flavonoids (mg/g DW leaf powder)
ABTS (IC50 g/mL)
ALM-5 ALM-7 ALM ALE-7 ALE-5 ALE
24.10 ± 0.11 24.48 ± 0.29 21.38 ± 0.11 30.38 ± 0.50 25.38 ± 0.38 12.50 ± 0.33
136.73 110.52 311.92 49.16 67.11 316.81
16.72 ± 0.50 15.66 ± 0.52 15.85 ± 0.05 20.92 ± 0.26 20.12 ± 0.34 10.95 ± 0.87
Note: ALM-5, 50% Methanol extract; ALM-7, 70% Methanol extract; ALM, Methanol extract; ALE-7, 70% Ethanol extract; ALE-5, 50% Ethanol extract; ALE, Ethanol extract.
tems, highest phenolic content (30.38 ± 0.50 mg/g as GAE) was obtained in 70% aqueous ethanol (ALE-7) followed by 50% aqueous ethanol (ALE-5) and 70% aqueous methanol (ALM-7). Similarly, for total flavonoid content, the maximum yield was obtained in ALE7 extract (20.9 mg/g as quercetin equivalent), followed by ALE-5. In a previous study, the ethanolic extract of apple leaves of two cultivars (Cvs Golden and Royal Delicious) have been reported to contain an average of 36 mg polyphenols (mg gallic acid equivalent (GAE)/g DW plant material) and 30.7 mg flavonoids (per g DW plant material) (Walia et al., 2015). A high amount of total phenolic (avg. 132 mg GAE/g DW) and flavonoid (avg. 33 mg Rutin Equivalent (RE)/g DW) contents were also reported in ethanol (70%, v/v) leaves extract obtained from different varieties of apple grown in Lithuania region (Liaudanskas et al., 2014). It was also reported that methanolic extracts of Venturia inequalis infected apple leaves (cvs Jonagold and Golden Delicious) accumulated higher phenolic compounds (86–159 mg GAE/g DW of leaf) as compared to healthy plants (60–88 mg GAE/g) (Petkovsek et al., 2008). In the present study, the high yield of extraction of phenolic compounds and flavonoids may be attributed to their affinity with the waterethanol solvent system. However, the variation of the yield and composition of the extractible may be attributed to the varietal,
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Table 2 Regression equation, correlation coefficient of phenolic standards. Compounds
Regression equation
R2
Retention Time
max
Epicatechin Phloridzin Phloretin Quercetin-3-O-glucoside Quercitrin
Y = 0.005x + 0.007 Y = 0.018x − 0.036 Y = 0.039x + 0.001 Y = 0.005x − 0.030 Y = 0.008x − 0.010
0.994 0.996 0.999 0.999 0.998
4.8 14.2 24.1 7.9 11.6
205.5, 280.5 224.2, 286.7 225.4, 286.7 205.5, 257.2, 355.6 205.5, 257.2, 349.6
Table 3 Quantification of phenolics in various extracts of apple leaves. Extracts ALM-5 ALM-7 ALM ALE-7 ALE-5 ALE
Epicatechin (mg/g) 0.46 ± 0.02 0.28 ± 0.005 0.26 ± 0.002 0.37 ± 0.07 0.31 ± 0.02 0.07 ± 0.001
Quercetin-3-O-glucoside (mg/g) 1.53 ± 0.003 1.55 ± 0.002 1.60 ± 0.001 1.55 ± 0.001 1.19 ± 0.002 0.50 ± 0.034
Phloridzin (mg/g) 21.90 ± 0.02 18.79 ± 0.08 22.68 ± 0.10 24.43 ± 0.05 21.07 ± 0.06 19.67 ± 0.07
Phloretin (mg/g)
Quercitrin (mg/g)
0.17 ± 1.13 0.12 ± 0.29 0.15 ± 0.77 0.15 ± 0.05 0.13 ± 0.07 0.16 ± 0.05
1.75 ± 0.02 1.56 ± 0.03 1.93 ± 0.02 2.06 ± 0.05 1.50 ± 0.001 0.81 ± 0.009
Note: ALM-5, 50% Methanol extract; ALM-7, 70% Methanol extract; ALM, Methanol extract; ALE-7, 70% Ethanol extract; ALE-5, 50% Ethanol extract; ALE, Ethanol extract.
geographic conditions and extraction factors, as has been reported in previous works. 3.2. In vitro antioxidant potential The quality of polyphenols is generally associated with their antioxidant potential. ABTS is a widely used method for phenolic antioxidants determination. This assay is based on decolorization through reduction of ABTS• + radicals generated by the oxidation of ABTS with potassium persulphate. The different extracts of apple leaves evaluated under in vitro conditions by ABTS assay showed lower IC50 value in ALE-7 extract (49.16 g/mL) followed by ALE-5 and ALM-7 (Table 1). The results are in accordance with phenolics and flavonoids contents recorded in respective fractions. The comparative IC50 values of 47.10 g/mL and 66.53 g/mL have been reported in case of methanol extract of golden and royal delicious leaves respectively (Walia et al., 2015). The antioxidant potential of ethanolic extracts obtained from variety specific apple leaves was reported to be in a range of 150-355 moL Trolox equivalent/g DW of lyophilized leaf powder (Liaudanskas et al., 2014). 3.3. Quantification of phenolics in apple leaves Liquid chromatography (LC) is the most accurate technique for the characterization and quantification of phenolic constituents because of its very high sensitivity and selectivity. There are numerous reports available on profiling of phenolic bioconstituents by liquid chromatography (Liaudanskas et al., 2014; Walia et al., 2015). In present study, five phenolic compounds were identified in apple leaves using RP-HPLC system by comparing retention times, max and peak spiking with internal standards. Quantification of individual phenolics was accomplished with an external standard calibration. The regression equation, correlation coefficient, retention time and absorbance maxima of each standard are shown in Table 2. The results revealed the presence of phloridzin, quercetin-3-O-glucoside, phloretin, quercitrin and epicatechin in various solvent extracts of apple leaves. RP-HPLC chromatogram of ALE-7 extract is shown in Fig. 1. The results showed that total amounts of detected phenolic compounds were higher in ALE7 (extract) and predominant phenolic present in ALE-7 extract was phloridzin (24.43 ± 0.05 mg/g). Amount of individual phenolics present in the samples is summarized in Table 3. In our study, highest content of phloridzin was observed among all the extracts by RP-HPLC are in general agreement with those accounted by others (Walia et al., 2015). The phloridzin has been reported as major phenolic (20.3 mg/g) in both royal and golden varieties of apple
Table 4 Minimum inhibitory concentration (MIC, g/mL) of various fractionated extracts of apple leaves. Pathogens
ALH
ALEA
ALW
Bacillus subtilis Klebisella pneumoniae Staphylococus aureus Micrococus luteus Escherichia coli Listeria monocytogenes
1.18 1.18 1.18 1.18 1.18 2.37
– – – – – –
– – – – – –
Note: ALH, Hexane fraction; ALEA, Ethyl acetate fraction; ALW, aqueous fraction.
leaves by UPLC–MS-MS. (Walia et al., 2015). In another study, Liaudanskas et al. reported the occurrence of phloretin, catechin, caffeic acid, chlorogenic acid, epicatechin, isoquercitrin, rutin, hyperoside, quercitrin and avicularin along with phloridzin in ethanolic extracts of the Aldas, Auksis, Ligol, and Lodel cultivars. A higher content of dihydrochalcones phloridzin was also reported in different varieties of apple leaves (Liaudanskas et al., 2014). Petkovsek et al. reported the presence of hydroxycinnamic acids, flavanols, dihydrochalcones, and quercetin in apple leaves and fruits grown in organic and integrated conditions (Petkovsek et al., 2010). The results attained during above studies will be helpful for the selection of extraction solvent and cultivars for the efficient extraction of phenolic rich fraction so as to measure the in vitro cytotoxicity, anti-inflammatory potential and antimicrobial activity. 3.4. Antimicrobial activity The antimicrobial activity of the different fractions (ALH, ALEA and ALW) was studied against six pathogenic bacterial strains (Bacillus subtilis, Klebisella pneumoniae, Staphylococus aureus, Micrococus luteus, Listeria monocytogenes and E. coli). Initial screening of different fractions was done using diffusion well plate method. Among all the fractions, ALH fraction showed significant zone of inhibition against all the selected pathogens. Furthermore, ALH was screened for the MIC using different concentrations 0.29–4.67 g/mL. Results showed that ALH fraction posses 1.8 g/mL MIC against Bacillus subtilis, Klebisella pneumoniae, Staphylococus aureus, Micrococus luteus, Escherichia coli and 2.37 g/mL against Listeria monocytogenes. The results of the antibacterial activity are presented in Table 4. Phenolics constituents present in apple and its plant parts are mainly responsible for their biological activity. Phloretin, the major dihydrochalcone present in apple fruit was also reported as chief phenolics in the leaves of Malus domestica responsible for antibacterial activity in
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present study. In earlier study, phloretin and its glycosides have been evaluated for antimicrobial activity against gram positive and gram negative bacterial strains. Dihydrochalcone phloretin has been reported for its antibacterial action against Staphylococcus aureus, Listeria monocytogenes and Salmonella typhimurium. Phloretin showed the inhibitory effects at concentration of 7.81, 62.5 and 125 g/mL against S. aureus, L. monocytogenes, S. typhimurium respectively (Barreca et al., 2014). Park et al. suggested that at low concentration (30–35 g/mL) phloretin inhibit biofilm formation of enterohemorrhagic E. coli O157, and thus acts as a biofilm inhibitor (Park et al., 2012). Apple peel phenolics also showed significant antimicrobial activity against Bacillus cereus and Escherichia coli serotype O157:H7 and suggested to have potential for treating specific microbial infections (Fratianni et al., 2011). 3.5. Assessment of cytotoxicity on human cancer cell lines The various fractions of apple leaves were assessed in vitro against SiHa, KB and HT-29 human cancer cells. It was revealed that maximum (24.47 ± 010%) cytotoxicity was observed in ALH extract after 48 h against KB cell line, but neither the concentration nor the time point of all the fractions prove more than 50% cytotoxic and the IC50 value remains >200 g/mL. The results of cytotoxicity are shown in Table 5. In previous studies, cytotoxic activity of essential oil obtained from apple leaves was reported against glioma cells, human lung carcinoma, chinese hamster ovary and human acute monocytic leukemia cell line. The highest % cytotoxicity of essential oil of apple leaves was observed in glioma cells lines (98.2%) at 2000 g/mL (Walia et al., 2012). 3.6. Cellular NO inhibitory effect of fractions on pre-treated peritoneal macrophages In the present study, different fractions of apple leaves were evaluated for NO inhibition production on pre-treated peritoneal macrophages. After LPS treatment, NO production was increased in peritoneal macrophages. Results of present investigation showed that all the fractions inhibited NO production at different concentrations (25–200 g/mL) (Fig. 2). ALEA (63.3%) and ALH (61.8%) found to inhibit significantly (p < 0.05) higher percentage of NO radicals as compared to ALW (28.7%) at 200 g/mL concentration. On the other hand, ALW showed 28.7% inhibition at same concentration. Among all the extracts minimum % inhibition was showed by
Fig. 2. Nitric oxide (NO) scavenging activity of hexane (ALH), ethyl acetate (ALEA) and aqueous (ALW) fraction of apple leaves.
ALH (7.01%) at 25 g/mL. The higher NO inhibitory effect of ALEA may be attributed to the presence of dihydrochalcones (phloridzin and phloretin). In the previous study, it was reported that pretreatment of RAW264.7 cells with 10 M phloretin significantly inhibited the levels of NO, COX-2, PGE2, TNF-␣, iNOS and IL-6 (Chang et al., 2012). 3.7. Effect of extracts on proliferation of murine splenocytes MTT-based splenocytes proliferation was assayed at different incubation periods. The apple leaves fractions were tested in an array of concentrations, towards lipopolysaccharide (LPS) and concanavalin A (Con A) stimulated splenocytes. Results revealed that ALH showed maximum proliferation 10.1% (p < 0.05) at 25 g/mL after 24 h, whereas it showed 8.87% (p < 0.05) proliferation at same concentration after 48 h and no proliferation after 72 h (Fig. 3). Similar to this ALW showed decrease in proliferation with increase in concentration after 24 h with maximum proliferation 17.27% (p < 0.05) at 25 g/mL concentration, whereas 10.7% (p < 0.05) proliferation was found after 48 h at the same concentration with no more proliferation up to 72 h. However, ALEA showed delayed response as no proliferation was observed after 24 h, but a significant increase i.e. 33.91% (p < 0.05) in proliferation after 48 h at same concentration was observed. After 72 h it showed 3.80% (p < 0.05) proliferation at 25 g/mL concentration, which further decreases with increase in concentration. Results of the present investigation
Fig. 3. Depicting lymphocyte proliferation of hexane (ALH), ethyl acetate (ALEA) and aqueous (ALW) fraction of apple leaves.
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Table 5 Cytotoxicity of apple leaves extracts on different human cancer cell lines. Extracts Conc (g/mL)
Cytotoxicity (%) SiHa 24 h
KB 48 h
72 h
24 h
A549 48 h
72 h
24 h
48 h
72 h
ALH
50 100 200 400
3.10 ± 0.04 0.00 ± 0.00 0.00 ± 0.00 17.89 ± 0.05
4.77 ± 0.05 4.44 ± 0.02 3.33 ± 0.008 17.14 ± 0.027
12.27 ± 0.11 13.18 ± 0.03 13.70 ± 0.04 21.97 ± 0.08
2.23 ± 0.05 4.78 ± 0.05 8.72 ± 0.02 23.69 ± 0.08
5.18 ± 0.09 2.47 ± 0.05 2.08 ± 0.03 24.47 ± 010
0.12 ± 0.09 2.23 ± 0.03 3.26 ± 0.02 18.25 ± 0.05
4.19 ± 0.09 4.81 ± 0.12 4.68 ± 0.11 14.75 ± 0.01
3.02 ± 0.13 5.40 ± 0.05 35.09 ± 0.01 10.17 ± 0.009
5.21 ± 0.14 9.62 ± 0.05 8.94 ± 0.04 11.74 ± 0.12
ALEA
50 100 200 400
1.24 ± 0.05 0.00 ± 0.00 2.24 ± 0.04 14.03 ± 0.03
0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 7.33 ± 0.030
10.88 ± 0.09 13.61 ± 0.02 14.94 ± 0.07 18.30 ± 0.07
7.26 ± 0.05 5.11 ± 0.03 9.57 ± 0.01 16.47 ± 0.03
4.07 ± 0.15 1.56 ± 0.02 3.13 ± 0.04 11.00 ± 0.17
2.58 ± 0.13 4.27 ± 0.01 10.64 ± 0.05 16.01 ± 0.002
4.54 ± 0.02 7.17 ± 0.06 6.55 ± 0.06 11.72 ± 0.05
0.25 ± 0.08 0.00 ± 0.00 0.00 ± 0.00 0.000 ± 0.00
0.42 ± 0.06 5.55 ± 0.04 7.76 ± 0.08 10.47 ± 0.13
ALW
50 100 200 400
0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 3.29 ± 0.03
3.21 ± 0.04 2.97 ± 0.01 9.45 ± 0.01 22.88 ± 0.8
4.54 ± 0.05 2.63 ± 0.04 5.96 ± 0.06 12.69 ± 0.01
0.00 ± 0.00 0.69 ± 0.14 3.72 ± 0.03 10.68 ± 0.02
0.00 ± 0.00 0.00 ± 0.00 5.27 ± 0.02 12.03 ± 0.05
4.54 ± 0.08 3.70 ± 0.05 4.94 ± 0.02 10.20 ± 0.01
0.55 ± 0.03 3.49 ± 0.10 0.00 ± 0.00 2.64 ± 0.01
2.33 ± 0.09 5.13 ± 0.04 9.16 ± 0.08 10.22 ± 0.03
Note: ALH, Hexane fraction; ALEA, Ethyl acetate fraction; ALW, aqueous fraction; SiHa, Cervical squamous cell carcinoma; KB, Oral carcinoma; A549, Lung carcinoma.
showed good pharmacological activity of apple leaves suggests that it can be used as a potential source for the extraction of valuable bioactive molecules. 4. Conclusions The present study concludes that leaves of Malus domestica are promising source of phenolic compounds (dihydrochalcones and flavonol glycosides) and also possess appreciable free radical scavenging activity. Ethyl acetate fraction showed highest potential in the inhibition of nitric oxide production in pretreated peritoneal macrophages. Results of antimicrobial activity revealed the potential of hexane fraction as strong antimicrobial agent against selected pathogenic strains and suggested to have capability for treating specific microbial infections. Hexane fraction was also found to play significant role in proliferation of murine splenocytes at low concentration. The findings of this study clearly provide evidence that underutilized apple leaves can be used for the treatment of inflammatory diseases and pathogenic infections in the form of various health promoting nutraceuticals and dietary supplements. 5. Conflict of interest Authors declare that they have no conflict of interest including any financial, personal or other relationships with other people or organizations. Acknowledgements The authors are thankful to the Director (CSIR-IHBT), Palampur for providing support during the course of work. Authors are also grateful to CSIR, New Delhi, India for funding through network project (BSC 105) under which this work was carried out. References Alissa, E.M., Ferns, G.A., 2012. Functional foods and nutraceuticals in the primary prevention of cardiovascular diseases. J. Nutr. Metab., 16, Article ID 569486. Barreca, D., Bellocco, E., Lagana, G., Ginestra, G., Bisignano, C., 2014. Biochemical and antimicrobial activity of phloretin and its glycosilated derivatives present in apple and kumquat. Food Chem. 160, 292–297. Bonarska-Kujawa, D., Cyboran, S., Oszmianski, J., Kleszczynska, H., 2011. Extracts from apple leaves and fruits as effective antioxidants. J. Med. Plant Res. 5, 2339–2347. Chang, W.-T., Huang, W.-T., Liou, C.-J., 2012. Evaluation of the anti-inflammatory effects of phloretin and phlorizin in lipopolysaccharide-stimulated mouse macrophages. Food Chem. 134, 972–979. Cohn, Z., 1978. The activation of mononuclear phagocytes: fact, fancy, and future. J. Immunol. 121, 813–816.
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