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GC/MS profiling, in vitro antioxidant, antimicrobial and haemolytic activities of Smilax macrophylla leaves
⁎Corresponding authors. Tel.: +92 3008923442 (M. Zubair), tel.: +60 389467393; fax: +60 389467006 (U. Rashid). zubairmkn@yahoo.com (Muhammad Zubair), umer.rashid@yahoo.com (Umer Rashid)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
The current study has been designed to appraise the antioxidant, antimicrobial and haemolytic potential of Smilax macrophylla leaves. The n-hexane fraction was analysed by Gas Chromatography/Mass Spectrometer which revealed the presence of 38 compounds. All examined extracts and fractions of plant leaves showed significant antimicrobial activity. The haemolytic effect of the plant was found to be in a range of 3.41–8.48%. S. macrophylla leaves contained substantial level of total phenolic contents (2.2–6.2 Gallic acid equivalent mg/g) and total flavonoid contents (1.2–4.5 Catechin, mg/g) of dry plant matter. Leaf extract and fractions also exhibited a good antioxidant potential when measured by DPPH radical scavenging assay (Inhibitory concentration 50% = 33.4–72.3 μg/mL). The antioxidant activity of plant extracts was also studied using sunflower oil as an oxidative substrate and found that it stabilized the oil. Significant (p < 0.05) variations were observed in the results. The correlation between the results of different antioxidant assays and oxidation parameters of oil indicated that leaf extracts and fractions, exhibit considerable total phenolic contents, total flavonoid contents and scavenging power, along with more potent for enhancing the oxidative stability of sunflower oil. Considering these results, S. macrophylla could be used as a source for the exploration of new antimicrobial, antioxidant agents, functional food and nutraceutical applications.
Keywords
Smilax macrophylla leaves
Phenolics
Haemolytic
Antioxidant activity
Antimicrobial activity
1 Introduction
Smilax is a genus which has more than 300 species, which is found on temperature zones, tropic and subtropics worldwide (Fnaec, 2002). The plants of genus Smilax are nutritionally important as they have nectar-rich flowers. Smilax rhizomes have different kinds of pharmacological behaviours such as antibacterial, antifungal, antioxidant and others activities (Ozsoy et al., 2008). The tubers of Smilax have been broadly used in usual medicine for treatment of different diseases, especially for pelvic inflammation and chronic pelvic (Venkidesh et al., 2010). The leaves and fruit of the Smilax are used for treating syphilis and rheumatism.
Smilax has been reported to have many pharmacological properties as used in curing the diseases like cancer, diabetes mellitus, skin ailments including wounds, inflammations, boils and ulcers (Damayanthi et al., 2011). It is also used for the treatment of fever, gout, diuretic, ophthalmia, infertility and as a source of antioxidants.
Smilax macrophylla belongs to the family Smilacaceae. The plant grows as shrubs, thorny, flowering and ornament and forming dense impenetrable thickets. The leaves are heart shaped. S. macrophylla is widely distributed in the southern area of China. It is a herb indigenous to Hunan, Hubei, Jiangxi, Zhejiang, Jiangsu and Guangxi Provinces of China (Shu et al., 2006). The root of S. macrophylla has been used in traditional Chinese medicine to dispell windevil, eliminate damp and detoxicate (Ageel et al., 1989). It is explored for its importance in ethno medicine in African countries and other parts of the world. It is used to cure fever, skin and veneral diseases. It is used as a remedy for gout in Latin American countries, ophthalmia, diuretic, to relieve labour, and for the cure of infertility in Tanzania (Cabra et al., 1993).
In our previous studies we explored the medicinal importance of various plants (Bari et al., 2012; Rizwan et al., 2012; Rasool et al., 2011a,b; Zubair et al., 2011). S. macrophylla has been subjected to investigate the chemical composition of n-hexane fraction of the plant leaves and the in vitro antimicrobial, antioxidant, haemolytic activities of the methanol extract and various organic fractions thereof. To the best of our knowledge, there is no such previous study on the exploration of antioxidant, antimicrobial, haemolytic activities and oil characterization of S. macrophylla plant. This study will provide base-line data for further detailed investigations of various biological activities of S. macrophylla plant and of its use as a functional food.
2 Material and methods
2.1 Plant material
The fresh leaves of plant S. macrophylla were collected on May 2011 from local areas of Qausoor, Pakistan. The plant specimens were further identified by Mansoor Hameed, Department of Botany University of Agriculture Faisalabad, (UAF) Pakistan where a voucher specimen has been deposited.
2.2 Extraction of plant material
500 g fresh leaves of the plant S. macrophylla was washed with distilled water to remove dust particles. The shade dried leaves were powdered (80 mesh). The grinded fine powder (395 g) of the plant was extracted with absolute methanol (1.5 L) at room temperature (30 °C) for 3 days. The extract was filtered through Whatman No. 1 filter paper and then concentrated at 45 °C, using a rotary vacuum evaporator (Eyela, Tokyo Rikakikai Co., Ltd., Japan). This process was repeated thrice to obtain a sufficient quantity of absolute methanol extract. The methanolic extract (60 g) was dissolved in distilled water and then fractionation was performed by using different polarity based solvents and obtained successively n-hexane (20 g), chloroform (16 g) ethylacetate (12 g), and n-butanol (9 g) fractions. The remaining plant residue was further extracted with 95% methanol (95:05, methanol:water, v/v) (15 g) and 90% methanol (90:10, methanol:water, v/v) (13 g). All these obtained extracts and fractions were stored at −4 °C till further analysis.
2.3 Gas chromatography/mass spectrometry (GC/MS) analysis
The GC/MS analysis of n-hexane fraction was performed using GC 6850 Network gas chromatographic system equipped with 7683 B series auto injector and 5973 inert mass selective detector (Agilent Technologies USA). Compounds were separated on DB-5 MS capillary column having 5% phenyl polysiloxane as stationary phase, column length 30.0 m, internal diameter 0.25 mm and film thickness 0.25 μm. The temperature of injector was 300 °C and 1.0 μL of sample was injected in the split mode with a split ratio 30:1. Helium (He) was used as carrier gas, and the flow rate of gas was 1.5 mL/min. The temperature program was: initial temperature 150 °C and held for 1 min, then ramping at a rate of 10 °C/min up to 290 °C and finally held at this temperature for 5 min. The temperature of MSD transfer line was 300 °C. For mass spectra determination MSD was operated in electron ionization (EI) mode, with the ionization energy of 70 eV, while the mass range scanned was 3–500 m/z. The temperature of ion source was 230 °C and that of MS quadropole was 150 °C. The identification of components was based on comparison of their mass spectra with those of NIST mass spectral library (Massada, 1996; Mass Spectral Library, 2002).
2.4 Antimicrobial assay
2.4.1 Test microorganisms
Alternaria alternata ATCC 20084 and Ganoderma lucidum locally isolated, were used as the fungal tested organisms and Pasturella multocida locally isolated, Escherichia coli ATCC 25922, Bacillus subtilis JS 2004, Staphylococcus aureus API Staph tac 6736153 were used as bacterial tested organisms. The pure fungal and bacterial strains were obtained from the Department of Veterinary Microbiology, University of Agriculture, Faisalabad, Pakistan. The fungal strains were cultured overnight at 28 °C using potato dextrose agar while bacterial strains were cultured overnight at 37 °C in nutrient agar (Oxoid, Hampshire, UK).
2.4.2 Disc diffusion method
Antimicrobial activity of the plant’s different extracts and fractions was determined by using the disc diffusion method (NCCLS, 1997). All samples (dry residue) were dissolved in 10% sterile dimethyl sulfoxide. The discs (6 mm diameter) were impregnated with 20 mg/mL extract/fractions (100 μL/disc) placed aseptically on the inoculated agar. Discs injected with 100 μL of respective solvents served as negative controls, rifampcin (100 μL/disc) (Oxoid) and fluconazole (100 μL/disc) (Oxoid) were used as a positive reference for bacteria and fungi, respectively. The petri dishes were incubated at 37 ± 0.1 °C for 24 h and 28 ± 0.3 °C for 48 h for bacteria and fungi, respectively. At the end of time period, the inhibition zones were measured which formed on the media. The positive antimicrobial activity was read based on growth inhibition zone.
2.4.3 Resazurin microtitre-plate assay
The minimum inhibitory concentration (MIC) of plant extract/fractions was evaluated by a modified resazurin microtitre-plate assay (Sarker et al., 2007) with some modifications. Briefly, 100 μL of each extract and fraction solution in 10% dimethyl sulfoxide (DMSO, v/v) was transferred into the first row of the 96 well plates. To all other wells, 50 μL of nutrient broth and Muller Hinton broth for bacteria and fungi respectively was added. Twofold serial dilutions were performed such that each well had 50 μL of the test material in serially descending concentrations. Then to each well, 10 μL of resazurin indicator solution (prepared by dissolving 270 mg resazurin tablet in 40 mL of sterile distilled water) was added. Finally, 10 μL of bacterial/fungal suspension was added to each well. Aluminium foil was used to cover each plate. Each plate had a set of controls: a column with broad spectrum antibiotics (as positive control), a column with all solutions with the exception of the test samples, a column with all solutions with the exception of the bacterial/fungal solution adding 10 μL of broths instead and a column with respective solvents (as negative control). The plates were prepared in triplicate, and incubated at 37 ± 0.1 °C for 20–24 h and 28 ± 0.3 °C for 40–48 h for bacteria and fungi, respectively. The growth was indicated by colour changes (from purple to pink or colourless). The minimum concentration at which colour change appeared was taken as the MIC value.
2.5 Antioxidant activity of S. macrophylla leaves
2.5.1 Determination of total phenolic contents (TPC)
The amount of TPC was assessed using the Folin–Ciocalteu reagent (Chaovanalikit and Wrolstad, 2004). Briefly, 1 mg of dry mass of the crude extract/fractions was mixed with 0.5 mL of Folin–Ciocalteu reagent and 7.5 mL of deionized water. The mixture was kept at room temperature for 10 min, then 1.5 mL of 20% sodium carbonate (w/v) was added. In water bath at 40 °C the mixture was heated for 20 min, and then cooled in an ice bath, and finally the absorbance was measured at 755 nm (Hitachi U-2001 spectrophotometer). Amounts of TP were calculated using a calibration curve for gallic acid (10–100 ppm) (R2 = 0.9986). The results were expressed as gallic acid equivalents (GAE) of dry plant matter.
2.5.2 Determination of total flavonoid contents (TFC)
The total flavonoid contents (TFC) in plant extract and fractions were determined following the procedure as described by Dewanto et al. (2002). Plant extract/fractions of each material (1 mL containing 0.1 g/mL) were placed in a 10 mL volumetric flask, and then added distilled water (5 mL) and 0.3 mL of 5% NaNO2 to each volumetric flask initially, after 5 min., 0.6 mL of 10% AlCl3 was added. After another 5 min, 2 mL of 1 M NaOH was added and volume made up with distilled water. Then solution was mixed. At 510 nm absorbance of the reaction mixture was taken using a spectrophotometer. TFC were evaluated as catechin equivalents (g/100 g of dry plant matter).
2.5.3 Evaluation of antioxidant activity by DPPH radical scavenging assay
The 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) assay was carried out as described by Tepe et al. (2005). Aliquots (50 μl) of various concentrations (10–100 μg/mL) of the plant samples were added to a 5 mL of a 0.004% methanolic solution of DPPH. After incubation period (30 min) at room temperature, the absorbance was recorded against a blank at 517 nm: where Ablank is the absorbance of the control reaction and Asample is the absorbance of the test compound. Extract concentration providing 50% inhibition (IC50) was calculated from a graph plotting percentage inhibition against extract concentration.
2.6 Determination of antioxidant efficacy using sunflower oil as oxidation substrate
2.6.1 Stabilization of sunflower oil
The crude concentrated various extracts and fractions of the plant were separately added into the preheated (50 °C) refined, bleached and deodorizer sunflower oil (SO) at a concentration of 300 ppm (w/w). The oil samples were at 50 °C stirred for 30 min to obtain a uniform dispersion. All oil samples were separately stabilized and stored in an airtight bottle of 100 mL. Control sample was also prepared (excluding extract) under the same set of analytical conditions. At room temperature samples were stored. BHT (Synthetic antioxidant) was employed at its limit of 200 ppm to compare the efficacy of extracts. Stabilized and control oil samples (100 mL) were placed in dark coloured airtight glass bottles and subjected to storage in an electric hot air oven (IM-30, Irmeco, Germany) at 60 °C for 28 days. All oil samples were prepared in triplicate. Oil samples were taken after every 7 day intervals.
2.6.2 Measurement of oxidation parameters of sunflower oil
The oxidative deterioration level was assessed by the measurement of peroxide value (PV), free fatty acids (FFA) conjugate diene (CD), conjugate triene (CT) and p-anisidine values. Determination of the FFA and PV of stabilized and control sunflower oil samples was made following the AOCS official methods, AOCS Official methods (1997), Cd 8-53 and F 9a-44 respectively. The oxidation products such as conjugated dienes and conjugated trienes were analysed by following the IUPAC method (IUPAC, 1987), II D.23. The absorbance was noted at 232 and 268 nm respectively. The determination of the p-anisidine value was made following an IUPAC method II. D. 26.
2.7 In vitro haemolytic activity
Haemolytic activity of plant extracts and fractions was checked by the reported method of Powell et al. (2000). 3 ml fresh heparinized human blood was mixed, poured into a sterile 15 mL polystyrene screw-cap tube and centrifuged for 5 min, at 850g. The supernatant was decanted and the viscous pellet was washed 3 times with 5 mL of chilled (4 °C) sterile isotonic phosphate-buffered saline (PBS) solution, adjusted to pH 7.4. The washed cells were suspended in a final volume of 20 ml of a chilled, sterile PBS and to count cell hemocytometer was used. The blood cell suspension was maintained in ice, and diluted with sterile PBS to 7.068 × 108 cells mL−1 for each assay. Aliquots of 20 μL of plant extract/fractions were placed into 2.0 mL microfuge tubes. For each assay, 0.1% Triton X-100 was used as the positive control, (100% lysis) and PBS as negative control (0% lysis). Aliquots of 180 μL diluted blood cell suspension were placed into each 2 mL tube and mixed. For 35 min at 37 °C tubes were incubated with stirring (80 rpm). After incubation, the tubes were placed on ice for 5 min, and then centrifuged for 5 min to 1310g. Aliquots of 100 μL of supernatant were carefully collected and placed in a 1.5 mL microfuge tube, and diluted with 900 μL chilled, sterile PBS. All tubes were maintained on ice after dilution. Then 200 μL was placed into 96 well plates, and 3 replicates were taken in well plate which contains one positive and one negative. By using microquant absorbance at 576 nm was then measured. The experiment was performed in triplicate. Percent haemolysis was calculated by the following formula:
2.8 Statistical analysis
Samples were analysed individually in triplicate and data were reported as mean (n = 3 × 3 × 1) ± standard deviation (n = 3 × 3 × 1). Data were also analysed by analysis of variance (ANOVA) using Minitab 2000 Version 13.2 statistical software (Minitab Inc. Pennysalvania, USA).
3 Results and discussion
3.1 Chemical composition of n-hexane fraction
The GC/MS analysis of n-hexane fraction from methanol extract enabled the identification of 38 components (Table 1). This volatile fraction consisted of a mixture of different classes of compounds. The mass spectrum of each compound was compared with the NIST 05 library. The major constituents (>4%) were found to be 2,2,3-trimethylbutane (10.30%), not identified (6.30%), not identified (5.49%), 2,4,6-trimethylheptane (4.97%), 2-nitrobutane (4.72%), 3-methyl pentane (4.47%), 2,4-dimethylpentane (4.30%), 5-ethyl-4-methyl-3-heptanone (4.12%) etc. along with other major and minor constituents also being reported. Compounds in n-hexane fraction were identified by GC/MS analysis on the basis of retention time (RT), Kovats indices, fragmentation patterns and data comparison with NIST mass spectral library giving a complete structure identification. The n-hexane fraction may have some fatty acids/methyl esters, straight chain alkanes which can be implicated in some antioxidant and antimicrobial activities. It was found that the secondary metabolites and bioactive phyto-constituents identified by GC/MS in different plants have been previously reported which have antimicrobial, anti-inflammatory, antioxidant and antiproliferative activities (Kumar et al., 2010; Hussain et al., 2010; Abad et al., 2012; Marzoug et al., 2011). Therefore the chemical constituents found in S. macrophylla leaves may play major roles in the biological activities and pharmacological properties.
Kovats
Chemical compounds
% composition
545
3-Methyl-1-pentene
3.16
–
Not identified
2.12
579
3-Methyl pentane
4.47
597
2-Nitrobutane
4.72
614
3,4-Dimethylpentane
3.24
618
2,2-Dimethylpentane
2.45
621
2,4-Dimethylpentane
4.30
630
2,2,3-Trimethylbutane
10.30
676
2,3-Dimethylpentane
2.60
729
2,4-Dimethylhexane
3.57
–
Not identified
6.30
739
2,3,3-Trimethylbutane
2.56
773
3,4-Dimethylhexane
2.74
777
1,2,4-Trimethylcyclopentane
3.14
820
2,2,3,4-Tetramethylpentane
1.70
875
2,4,6-Trimethylheptane
4.97
915
3-Ethyl-2,5-dimethylhexane
4.06
933
2,6-Dimethyloctane
0.34
999
n-Decane
3.95
1010
5-Ethyl-4-methyl-3-heptanone
4.12
–
Not identified
t⁎
–
Not identified
0.33
1115
5-Ethyl-4-methyl-3-heptanone
1.71
1372
3-Methyltridecane
1.12
–
Not identified
3.35
2061
11, 14, 17-Eicosatrienoic acid, methyl ester
0.46
–
Not identified
5.49
–
Not identified
1.92
–
Not identified
0.59
2100
Heneicosane
0.40
2111
Trans-Phytol
2.50
–
Not identified
0.42
2173
9,12-Octadecadienoic acid (Z,Z)
0.39
2175
(6Z)-6-Octadecadienoic acid
1.53
2784
Alpha-Tocopherol-beta-d-mannoside
0.47
3408
Gamma-Sitosterol
0.35
3600
n-Hexatriacontane
0.62
3654
1-(+)-Ascorbic acid 2,6-dihexadecanoate
0.70
3.2 Antimicrobial activity
The antimicrobial activity of various organic extracts and fractions of S. macrophylla leaves was evaluated against some pathogenic microorganisms. The plant exhibited considerable antimicrobial activity against bacterial and fungal strains. The result from the disc diffusion method measured in inhibition zone (IZ in mm) and minimum inhibitory concentration (MIC in mg/mL), of plant extracts and fractions against bacterial and fungal strains ranged from 12 to 27.5 mm and 30.4 to 54.2 mg/mL, respectively (Table 2). Data are expressed as the mean ± standard deviation of three separate experiments (p < 0.05). Different letters in superscript indicate significant differences among different extracts and fractions. Rifampcin and Fluconazole used as control for bacterial and fungal strains respectively.
Leaf extracts and fractions
Tested microorganisms
(Diameter of inhibition zone (IZ), mm)
B. subtilis
P. multocida
S. aureus
E. coli
A. alternate
G. lucidum
n-Hexane
23.0 ± 0.23b
13.5 ± 0.18d
13.0 ± 0.20d
28.4 ± 0.25a
16.0 ± 0.41c
22.0 ± 0.41b
Chloroform
19.5 ± 0.19c
24.5 ± 0.25b
0
24.0 ± 0.44bc
22.0 ± 0.44b
12.5 ± 0.77b
Ethylacetate
22.0 ± 0.41b
0
27.5 ± 0.22a
25.5 ± 0.11b
18.0 ± 0.41c
23.0 ± 2.236b
n-Butanol
0
13.5 ± 0.38d
19.5 ± 0.25c
22.5 ± 0.35cd
23.5 ± 0.48b
0
Absolute methanol
18.0 ± 0.22c
21.0 ± 0.23c
0
0
0
22.5 ± 0.65b
95% Methanol
23 ± 0.26b
0
17.5 ± 0.38c
19.5 ± 0.65de
12.0 ± 0.45d
19.0 ± 0.23c
90% Methanol
0
22.5 ± 0.65bc
22.0 ± 0.41b
20.0 ± 0.41cd
22.0 ± 1.41b
18.0 ± 0.41c
Control
29.0 ± 0.33a
34.0 ± 0.44a
28.5 ± 0.25a
28.5 ± 0.61a
32.2 ± 0.34a
27.2 ± 0.38a
Minimum inhibitory concentration (MIC) mg/mL
n-Hexane
37.2 ± 0.33
53.1 ± 0.87
52.4 ± 0.53
30.4 ± 0.66
44.2 ± 0.20
39.1 ± 0.33
Chloroform
41.2 ± 0.53
35.2 ± 0.89
0
35.7 ± 0.12
38.1 ± 0.38
54.2 ± 0.37
Ethylacetate
38.1 ± 0.83
0
31.0 ± 0.71
33.2 ± 0.12
42.1 ± 0.47
33.2 ± 0.41
n-Butanol
0
47.2 ± 0.56
40.2 ± 0.98
38.2 ± 0.22
36.1 ± 0.55
0
Absolute methanol
42.1 ± 0.37
39.2 ± 0.45
0
0
0
33.2 ± 0.44
95% Methanol
33.2 ± 0.34
0
41.2 ± 0.74
41.0 ± 0.22
54.2 ± 0.54
40.2 ± 0.64
90% Methanol
0
38.1 ± 0.10
38.1 ± 0.43
40.2 ± 0.18
38.1 ± 0.22
40.2 ± 0.23
Control
30.1 ± 0.252
9.1 ± 0.16
30.1 ± 0.70
30.2 ± 0.23
10.2 ± 0.13
31.1 ± 0.40
The n-hexane fraction showed potent inhibitory activity against E. coli (IZ = 28.4 mm, MIC = 30.4 mg/mL), B. subtilis (IZ = 23.0 mm, MIC = 37.2 mg/mL) and G. lucidum (IZ = 22.0 mm, MIC = 39.1 mg/mL), respectively. The chloroform fraction was inactive against S. aureus and it showed strong inhibitory effects against P. multocida (IZ = 24.5 mm, MIC = 35.2 mg/mL) and E. coli (IZ = 24.0 mm, MIC = 35.7 mg/mL). Ethylacetate fraction and 95% methanol extract were inactive against P. multocida, while both these organic extracts strongly inhibited the growth of S. aureus, E. coli and B. subtilis. Furthermore, n-butanol fraction of the plant did not show any inhibitory activity against B. subtilis and G. lucidum while strong inhibitory action was observed against A. alternata (IZ = 23.5 mm, MIC = 36.1 mg/mL) and E. coli (IZ = 22.5 mm, MIC = 38.2 mg/mL). Absolute methanol extract was inactive against S. aureus, E. coli and A. alternata while it showed significant activity against G. lucidum (IZ = 22.5 mm, MIC = 33.2 mg/mL) and P. multocida (IZ = 21.0 mm, MIC = 39.2 mg/mL). Fluconazole and Rifampcin were used as standard antifungal and antibacterial agents to compare the potential of plant extracts and fractions (Table 2). All experiments against various microbes were conducted in triplicate. The standard antibiotics were refined industrial products so their activity was more as compared to crude extract and fractions.
Plants are the significant source of fungi and bacteria toxic compounds and they may be a renewable source of valuable fungicides and bactericides. The effect of extracts and fractions against tested microorganisms was significant implying that S. macrophylla can be utilized as drugs against infections caused by pathogenic microorganisms.
There is alarming news of fungal and bacterial infections (Bauer et al., 1966) which illustrate that increased resistance of micro-organisms is due to the random use of commercial drugs which are commonly used for cure of infectious diseases. Several studies have been done to confirm that bacterial and fungal strains used in the current study are disease causing pathogens (Hughey and Johnson, 1987; Andrews et al., 1997). This condition forced the researchers to seek out for new antimicrobial agents from different natural sources (Bauer et al., 1966). The present study results showed that medicinal plant S. macrophylla can play an important part in fighting fungal and bacterial resistance.
3.3 Percent yield, total phenolic contents (TPC) and total flavonoid content (TFC)
From different extracts and organic fractions of S. macrophylla leaves highest amount (15.1%) of extractable matter with absolute methanol was obtained (Table 3). The extraction yield of antioxidant components varied 2.27–15.1% for leaves showing a significant variation among different extracts and organic fractions. Different solvents such as methanol, ethanol, and ethyl acetate were commonly used for extracting the antioxidant components from plants, however, extraction with absolute methanol, often results in a higher recovery of secondary metabolites (Manzoor et al., 2012). The extracts and fraction yield from S. macrophylla leaves as determined in the present study was found to be in close agreement with Bari et al., 2012. Values are mean ± SD of three separate experiments (p < 0.05).
Parameters→
% yield of extracts and fractions
TPC (GAE mg/g) of dry plant matter
TFC (CE mg/g) of dry plant matter
DPPH, IC50 (μg/mL)
leaf extracts, fractions and reference compound↓
n-Hexane
5.06 ± 0.04
2.2 ± 0.04
1.2 ± 0.03
72.3 ± 0.15
Chloroform
4.05 ± 0.03
4.1 ± 0.06
4.9 ± 0.04
55.2 ± 0.09
Ethylacetate
3.03 ± 0.01
5.2 ± 0.05
4.0 ± 0.03
42.4 ± 0.10
n-Butanol
2.27 ± 0.02
3.4 ± 0.05
4.1 ± 0.04
48.3 ± 0.07
Absolute methanol
15.1 ± 0.10
6.2 ± 0.10
5.4 ± 0.10
33.4 ± 0.06
95% Methanol
3.79 ± 0.02
5.5 ± 0.05
2.5 ± 0.05
38.1 ± 0.06
90% Methanol
4.30 ± 0.03
2.4 ± 0.06
3.4 ± 0.04
45.8 ± 0.05
BHT
–
–
–
19.1 ± 0.04
Total phenolic contents (TPC) and total flavonoid contents (TFC) of S. macrophylla leaves varied significantly (p < 0.05) among different extracts and fractions (Table 3). In the present investigation, the amount of TPC and TFC ranged 2.2–6.2 gallic acid equivalent (GAE) mg/g and 1.2–5.4 catechin equivalents (CE) mg/g of dry plant matter, respectively. TPC extracted in different solvents were found to decrease in the following order: Absolute methanol > 95% methanol > Ethylacetate > Chloroform > n-butanol > 90% methanol > n-hexane. The efficiency of different solvents for extraction of flavonoids was found to be in the order: Absolute methanol > Chloroform > n-butanol > Ethylacetate > 90% methanol > 95% methanol > n-hexane.
In many studies, yield of phenolics and flavonoid extraction has shown a strong correlation with the polarity of the solvent used, high polarity solvents being the best for extraction (Lopez et al., 2011). It was observed that with the use of high polarity solvents, recovery of TPC and TFC was also improved and the highest concentration of phenolics and flavonoids was found in methanol extracts, confirming the ability of absolute methanol to solubilize a larger fraction of the phenolic and flavonoid components present in S. macrophylla leaves. All these observations suggest that most of the phenolic and flavonoid compounds are highly polar and are extractable in polar solvents. These results were in agreement with Spigno et al., 2007. Stankovic et al. (2011) determined that polar solvents are the best extracting media for flavonoids, which may be due to an increase in polarity of flavonoids upon conjugation through glycosides with hydroxyl groups that enhances their solubility in polar solvents (Mohsen and Ammar, 2009). In another study, Rizwan et al., 2012 reported the highest concentration of phenolics and flavonoids in polar solvents. All these previous reports are in agreement with our findings. From the results it was concluded that the plant leaves contained significant level of TPC and TFC, so the plant leaves can be used as a good source of phenolic and flavonoid compounds.
3.4 Antioxidant activity by DPPH radical scavenging assay
This method has been generally used for the antioxidant activity evaluation of biological samples including various plants. The leaf extracts and fractions exhibited different radical scavenging activity having IC50 value 33.4–72.3 μg/mL (Table 3). Absolute methanolic extract exhibited lowest IC50 (33.4 μg/mL) followed by 95% methanol extract (38.1 μg/mL), ethylacetate fraction (42.4 μg/mL), 90% methanol extract (45.8 μg/mL), n-butanol (48.3 μg/mL), chloroform (55.2 μg/mL) and n-hexane (56.2 μg/mL) fractions, respectively. The free radical scavenging activity of absolute methanol and 95% methanol extracts was superior to that of other solvent extracts. However, all extracts offered slightly less scavenging activity as compared to the synthetic antioxidant BHT (IC50 = 19.1 μg/mL). The experiments were conducted in triplicate. The nature and amount of secondary metabolites of the plant cause the variation in free radical scavenging ability (Sudjaroen et al., 2005). The free radical scavenging activity depends upon the chemical composition of extracts. The addition of S. macrophylla extracts and fractions to DPPH solution caused a quick decrease in the density at 517 nm. The scavenging capacity of the extracts and fractions indicated the degree of discolouration. Auto-oxidation of unsaturated lipids in food is caused by free radicals. The effect of antioxidant on DPPH radical scavenging was thought to be due to their hydrogen donating ability or radical scavenging activity. Therefore, formed stable end product does not permit further oxidation of lipid (Nilgun et al., 2007). Consequently, it is claimed that potent antioxidant activity was present in plant leaves due to which it can be used as a potential source of antioxidant agents.
3.5 Antioxidant potential of S. macrophylla leaves’ different extracts and fractions for the stabilization of sunflower oil
Rancidity of foods may occur due to formation of free fatty acids (FFA). Due to hydrolysis of triglycerides, FFAs are formed and this is promoted when reaction of oil with the moisture occurs (Frega et al., 1991). As storage time increases, FFA content went on increasing for all the samples but no regular pattern could be observed. Control exhibited the highest FFA, while sunflower oil stabilized with BHT exhibited the least (Fig. 1). All the oil samples stabilized with plant extracts and fractions were found to show a slow, followed by a gradual increase in free fatty acid contents. The lower values of free fatty acid contents of stabilized oil samples than control indicated the effectiveness of leaf extracts and fractions as natural antioxidant in retarding the free fatty acid contents.
Free fatty acid contents (%) of sunflower oil stabilized with S. macrophylla leaf extracts and organic fractions.
Peroxide value (PV) is usually used to evaluate the extent of primary oxidation products in oils. The highest PV was observed for control sample followed by n-hexane > Chloroform > Ethylacetate > n-butanol > 95% methanol > 90% methanol > Absolute methanol > BHT respectively (Fig. 2). All used extracts and fractions of the plant controlled peroxide value noticeably revealing the good antioxidant effectiveness of plant in stabilization of oil. Same trend in PV values is in accordance with reported results of Neff et al. (1994), Liu and White (1992) with some variations.
Peroxide values of sunflower oil stabilized with S. macrophylla leaf extracts and organic fractions.
The results for para-anisidine values (PAV) which usually determine the amount of aldehyde in oils are presented in Fig. 3. The control sample showed the maximum increase in para-anisidine values indicating a higher rate of secondary product formation. A slow increase in PAV of stabilized sunflower oil as compared with the control indicates the antioxidant potential of the plant leaves. A decreasing order of stability of oil treated with different extracts and fractions of plant regarding PAV was found to be: BHT > Absolute methanol > 90% methanol > n-butanol > 95% methanol > Ethylacetate > Chloroform > n-hexane > Control.
Relative increase in p-anisidine values of sunflower oil stabilized with S. macrophylla leaf extracts and organic fractions.
The formation of conjugated diene (Fig. 4) and triene (Fig. 5) was analysed for the control and stabilized sunflower oil, respectively. Highest contents were observed for control showing greater intensity of oxidation. The determination of CD and CT is an excellent measure of the oxidative state of oils (Yoon et al., 1991) and thus a good indicator antioxidant efficacy. CD and CT contents increase with the increase in storage time. A slow increase in CD and CT of the stabilized sunflower oil as compared with those of the control indicated the antioxidant potential of the S. macrophylla leaves. Absolute methanolic extract showed the lowest values for CD and CT and n-hexane exhibited highest values after completion of storage period. The increasing trend of CT and CD in stabilized oil samples with passage of time is in accordance with the reported results of Yildiz et al. (2001). After standard compound BHT all extracts and fractions of plant leaves played a prominent role for stabilization of sunflower oil. In the present study by coupling the results of antioxidant activity with the oxidation parameters of stabilized sunflower oil, it is observed that all the extracts and fractions of S. macrophylla leaves showed good antioxidant activity. However, the antioxidant activity of the absolute methanolic extract was found to be significantly higher than others, which might be attributed to the high polarity effect of the absolute methanol. The results of the present study also revealed that leaves are an effective source of natural antioxidants, suggesting their use in medicines and functional food applications.
Relative increase in conjugated diene content (CD) of sunflower oil stabilized with S. macrophylla leaf extracts and organic fractions.
Relative increase in conjugated triene content (CT) of sunflower oil stabilized with S. macrophylla leaf extracts and organic fractions.
3.6 Evaluation of haemolytic activity for cytotoxicity studies
Heamolytic activity for cytotoxic study was analysed against human red blood cells (RBCs) using Triton X-100 as positive control. Haemolytic activity for cytotoxic study was evaluated because, even if the plant possesses potent antioxidant and antimicrobial activities, its use in medicine will be impossible in the presence of these haemolytic effects which confirm the presence of saponins (Mohamad et al., 2001). The % lysis of RBCs caused by the plant extracts and fractions was also observed as depicted in Table 4. Absolute methanol extract showed highest haemolytic effect (8.48%) followed by 90% methanol extract (5.23%), ethylacetate (4.58%), n-hexane (4.51%), n-butanol (4.01%), 95% methanol (3.90%), chloroform (3.41%) fractions respectively. As absolute methanol extract showed highest haemolytic effect this might be due to high amount of saponins extracted with this solvent (Rizwana et al., 2010; Kerem et al., 2005). The presence of saponins in genus Smilax has already been reported (Kim et al., 1989; Bernardo et al., 1996; Ivanova et al., 2009). The erythrocytic membrane stability is a good indicator of the effect of various in vitro studies by various compounds for the screening of cytotoxicity. All these results were in range. However it can be expected that the plant extracts have a minor cytotoxicity (Powell et al., 2000; Sharma and Sharma, 2001), therefore, pharmacologically this plant may be safe to use for human beings as a source of potential drug. Values are mean ± SD of three separate experiments (p < 0.05).
Leaf extracts and fractions
% of haemolysis
n-Hexane
4.51 ± 0.23
Chloroform
3.41 ± 0.11
Ethylacetate
4.58 ± 0.31
n-Butanol
4.01 ± 0.17
Absolute methanol
8.48 ± 0.21
95% Methanol
3.90 ± 0.08
90% Methanol
5.23 ± 0.17
Phosphate Buffer Saline (PBS)
0
Triton X-100
100 ± 0.99
4 Conclusions
The results of the present study revealed that S. macrophylla plant leaves contained considerable potential of antioxidant and antimicrobial activities so it may also be used to stabilize the edible sunflower oil and also to treat various diseases caused by pathogens. Haemolytic activity of the plant leaves against human erythrocytes was determined and it was found in safe range which indicated that S. macrophylla plant may be harmless for the use of pharmaceutical and natural therapies. S. macrophylla leaves could be used as a potential source for folk medicine, to preserve foods, for the exploration of new compounds as antimicrobial and antioxidant agents.
Acknowledgment
Authors would like to dedicate this article to world’s renowned scientist of Natural Product Research, Prof. Dr. Viqar Uddin Ahmad on his 73rd birthday.
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