3.1. Extraction yield
After maceration of Fig in twelve distinct combinations of solvents, each infusion/extract was dried and yield calculated. Extraction yields for 100%, 80%, and 50% of acetone, ethanol, methanol and DMF extracts were determined to be 35.76 ± 1.0b, 160.76 ± 13.3a, 165.66 ± 15.5a, 44.43 ± 1.8b, 153.66 ± 12.8a, 154.63 ± 12.7a, 163.10 ± 4.3a, 165.73 ± 6.1a, 152.05 ± 10.7a, 184.16 ± 10.7a, 166.36 ± 3.4a, 162.46 ± 6.3a mg/g respectively. The values (mean ± SEM) superscripted with different alphabets are significantly different (P < 0.05) from each other. These values demonstrated that solvents of the lowest polarity that is absolute acetone (35.76 ± 1.0 mg/g) and absolute ethanol (44.43 ± 1.8 mg/g) had significantly (P < 0.05) lower yield compared to the other ten solvents of high polarity. Though the differences were not statistically significant, yet observations indicates that addition of water in less polar solvents (acetone and ethanol) increased the yield, but additional water decreased the yield for high polarity solvents (methanol and DMF). It was thus infered that medium polar combination of solvents were optimum for extraction yield, which is in agreement with earlier reports. Polar compounds such as carbohydrate, organic acids, and protein increase the yield with increasing fraction of water in solvents (Turkmen et al., 2006; Do et al., 2014).
3.2. Total phenolic content (TPC)
TPC of various solvent extracts (Fig. 1) ranged from 67.03 ± 0.9 to 220.9 ± 12.59 mg GAE/100g. Nearly three to four-fold higher TPC was observed as compared to those reported earlier on fresh figs (Kamiloglu & Capanoglu, 2014; Slatnar et al., 2011). The TPC in fig is comparatively higher than many other fruits like pear, orange, lemon, peach, banana, pineapple, strawberry, red grape and vegetables such as cucumber, celery, lettuce, potato, cabbage, carrot, red pepper, onion, spinach and broccoli (Chu, Sun, Wu, & Liu, 2002; Sun, Chu, Wu, & Liu, 2002).
TPC was highest in 100% DMF extract followed by 50% acetone, 80% DMF, and 80% methanol extract sequentially. Solvents of intermediate TPC yield as followes: 100% methanol > 50% DMF > 50% ethanol > 80% acetone > 80% ethanol > 50% methanol. TPC was least in absolute acetone and slightly higher in absolute ethanol (Fig. 1). The trend demonstrates an equivalent increase in TPC with increasing polarity/fraction of water for less polar organic solvents i.e. ethanol (100% < 80% < 50%) and acetone (100% < 80% < 50%). In case of methanol, 50% methanol extract showed highest TPC followed by 80% methanol and least in absolute methanol. Addition of water in absolute DMF was reducing the TPC yield as it was decreasing in the order: 100% > 80% > 50% (Fig. 1). Thus, the nature of solvent and content of water in the solvent affected TPC yield. Medium polar combination of solvents was found appropriate for TPC recovery. These observations were in accordance with previous reports and suggested an optimum TPC recovery by solvent combinations of medium polarity (Bachir Bey et al., 2014; Turkmen et al., 2006).
Moreover, TPC result shows that binary hydro-organic solvent is better for TPC recovery as compared to mono-component solvent system. Increasing fraction of water alters density, viscosity and dielectric constant of solvents. Solvation of hydrophilic matrix by water loosens cell wall matrix, hence facilitating penetration of extraction solvent, and enabling better mass transfer of the compounds. Organic solvent makes hydrophobic interaction weaker and solubilises phenolics and other phytochemicals. Conversely, addition of water increases the polarity of solvent mixture, reducing the solubility of relatively hydrophobic phenolics (Boeing et al., 2014).
3.3. Antioxidant assays
Methods for antioxidant capacity measurement can be broadly categorised as hydrogen atom transfer (HAT) based assays and electron transfer (ET) based assays. ORAC is an example of HAT-based assay in which antioxidant molecule act as hydrogen donor. ET-based assays (for example DPPH, ABTS, FRAP and TPC by Folin-Ciocalteu method) are based on the degree of colour change of respective synthetic oxidants after receiving electron from antioxidants (Prior, Wu, & Schaich, 2005).
Total antioxidant capacity (TAOC) was quantified by DPPH and ABTS assay. In both the assays antioxidant activity of various extracts followed a similar trend (Table 1). It was highest in 100% DMF, followed by 80% DMF, 50% acetone and 80% methanol extract. On the other hand, it was the least in 100% acetone and slightly high in 100% ethanol extract. Other extracts had intermediate antioxidant capacity in decreasing sequence; 100% methanol > 50% DMF > 80% acetone > 50% ethanol > 50% methanol > 80% ethanol. It is thus infered that as the polarity of the three solvents (acetone, ethanol, and methanol) enhanced with increasing proportion of water, their antioxidant capacity also increased, except 50% methanol extract, which has lower antioxidant capacity than 80% methanol extract. Any additional water in DMF result in decrease of antioxidant capacity of the extracts. Medium polarity of solvents favoured antioxidant’s recovery. Previous studies also reported that moderately polar solvents yield a high antioxidant capacity (Bachir Bey et al., 2014; Turkmen et al., 2006).
ABTS and DPPH antioxidant capicities of extracts show strong positive correlation (r = 0.926) (Table 2). ET based mechanism of action in both the assays might be the reason for the strong correlation. A strong correlation of TPC with DPPH (r = 0.808) and ABTS (r = 0.889) antioxidant capcities demonstrated that phenolics may be major antioxidants present in fig extract (Slatnar et al., 2011).
In FRAP assay, antioxidants donate an electron to TPTZ-Fe3+ complex and reduce it to TPTZ-Fe2+. High level of FRAP activity was found in fig ranging from 28 to 73 µM FeSO4 equivalents/g (µM FeSO4 E/g) (Table 1) in various solvent extracts. FRAP values were comparable to that of common dry fruits of Indian diet i.e. almond, apricot, brown raisins,
Table 1. Antioxidant capacities and IC50 values of dry fig extracted in different solvents
Solvent
|
ABTS assay α
|
DPPH assay α
|
FRAP assay β
|
ORAC α
|
% SRSAγ
|
IC50δ
|
Acetone
|
|
|
|
|
|
|
100%
|
13.9±0.8c
|
18.1±1.0d
|
28.68±6.6def
|
78.7±18d
|
32.3±1.1
|
80.4±6.8
|
80%
|
217.4±7.2ab
|
282.6±9.3ac
|
53.03±1.7abcf
|
182.7±16cd
|
51.6±3.1bd
|
41.2±3.4
|
50%
|
263.3±1.5ad
|
342.3±1.9a
|
63.70±5.1ab
|
267.9±19ab
|
62.7±0.3d
|
36.4±2.4
|
Ethanol
|
|
|
|
|
|
|
100%
|
44.1±1.4c
|
57.3±1.8d
|
28.31±6.2de
|
89.1±23d
|
42.0±0.8ca
|
43.3±2.2
|
80%
|
141.5±12.8b
|
183.9±16.6c
|
55.23±6.9abcd
|
192.2±18cd
|
48.5±0.3ab
|
38.1±2.3
|
50%
|
185.7±24.4bd
|
241.2±31.7c
|
62.26±9.0abc
|
227.0±23a
|
47.6±1.6d
|
30.3±1.3
|
Methanol
|
|
|
|
|
|
|
100%
|
251.1±8.3a
|
326.4±10.8a
|
57.05±1.4a
|
280.2±36a
|
45.7±1.6a
|
36. 8±3.2
|
80%
|
259.5±15.2a
|
337.4±19.8ab
|
62.52±5.9ab
|
250.3±28ab
|
68.4±0.4
|
17.9±1.2
|
50%
|
183.2±0.7b
|
238.1±1.0c
|
42.50±2.3ac
|
159.5±14bc
|
51.5±0.1b
|
38.3±3.2
|
DMF
|
|
|
|
|
|
|
100%
|
353.7±11.5
|
459.9±15.0a
|
73.74±4.5ab
|
328.6±43a
|
50.6±0.6ab
|
22.0±3.3
|
80%
|
264.1±11.2ab
|
343.4±14.6ab
|
66.52±3.4abf
|
276.7±25ade
|
45.6±0.5ac
|
30.1±2.2
|
50%
|
230.7±2.1a
|
299.9±3.0bc
|
41.85±6.1ec
|
156.4±17e
|
38.9±0.6c
|
62.3±3.4
|
Range
|
14 -354
|
18-460
|
28.7-66.5
|
79-328
|
32-68
|
17.9-80.4
|
Note: Values are given as mean ± SEM (n = 9); values with different letters are significantly different from each other (p < 0.05)
α values are presented as micromole of trolox equivalents per gram of fruit (µM TE/g);
β values are expressed as micromole of FeSO4 equivalents per gram of fruit (µM FeSO4 E/g).
γ percent superoxide radical scavenging activity is given for 10 mg IDF/ml of the extracts
δIC50 values are expressed as mg IDF/ml
|
cashew nut, dry dates, ground nut, piyal seeds, walnuts and fig, as reported earlier (Reddy et al., 2010). High level of FRAP activity was found in extracts of less polar combination (100% and 80%) of methanol and DMF and more polar combination (80% and 50%) of ethanol and acetone (Table 1). It was least in 100% ethanol, followed by 50% DMF, and 50% methanol extract. FRAP results demonstrated that medium polar solvents were suitable for antioxidant recovery. The positive correlation (r = 0.808) of FRAP assay with TPC (Table 2) signifies electron donating capacity of phenolic compounds (Reddy et al., 2010).
Oxygen radical antioxidant capacity (ORAC) assay measures the antioxidant capacity at biological pH (7.4) in phosphate buffer saline against biologically relevant synthetic oxidant, AAPH. High ORAC level, ranging from 7.9 to 32.8 µM TE/g (Table 1) was observed. The observation is in accordance with earlier report (Ammar, del Mar Contreras, Belguith-Hadrich, Bouaziz, & Segura-Carretero, 2015) in which 7.0 to 40.0 µM TE/g ORAC for different varieties of figs was found. An equivalent ORAC value was reported in dry fig and other dry fruits of Indian diet (Reddy et al., 2010). Acetone, ethanol and methanol extracts showed increasing ORAC with increasing proportion of water, but reverse trend was observed for DMF extracts. All the solvent extracts could be arranged in the decreasing order of respective ORAC as: 100% DMF > 100% methanol > 80% DMF > 50% acetone > 80% methanol > 50% ethanol > 80% ethanol > 80% acetone > 50% methanol > 50% DMF > 100% ethanol > 100% acetone. ORAC had a good correlation (r = 0.701) with TPC (Table 2), but was comperatively weaker than the correlation of DPPH or ABTS antioxidant capacities with TPC. The reason might be, different HAT based mechanism for ORAC as compared to ABTS or DPPH assay which rely on ET based mechanism.
SRSA ranged from 32 to 68% for various solvent extracts (10 mg IDF/ml) of fig (Table 1). A similar activity was shown in a previous study on fresh plums (Chun, Kim, & Lee, 2003). SRSA (Table 1) was the highest in 80% methanol extract and gradually reduced in the order: 50% acetone > 80% acetone > 50% methanol > 100% DMF > 80% ethanol > 50% ethanol > 100% methanol > 80% DMF > 100% ethanol > 50% DMF > 100% acetone extracts. The SRSA showed positive correlation with TPC and other antioxidant capacities (Table 2). SRSA result is in agreement with previous report which showed positive correlation between SRSA and TPC determined in Sonchus asper extracts (Khan, Khan, Sahreen, & Ahmed, 2012). Superoxide anion (O2.-) is a biologically relevant radical which can directly damage cellular macromolecules (Siddhuraju & Becker, 2003). Therefore, superoxide radical scavenging activity of the fig supports its role in various diseases (Lim, 2012) and antiproliferative activity shown in present study.
3.4. Correlation studies
Statistical analysis shows a good correlation (Table 2) between TPC and antioxidant activities as determined by ABTS (r = 0.701), DPPH (r = 0.885), FRAP (r = 0.808), ORAC (r = 0.793), and SRSA (r = 0.600) assay. Further, antioxidants capacities are well correlated among each other (Table 2). Similar correlations were reported earlier (Ammar et al., 2015; Solomon et al., 2006), which suggest that phenolic compounds might be major antioxidants present in fig extracts. Phenolic compounds have the ability to scavenge free radicals, chelate pro-oxidant metal ions and inhibit enzymes involved in oxidant production. The hydroxyl groups of phenolic compounds can easily transfer hydrogen ion to free radicals producing phenoxide radicals, comparatively stable products (Slatnar et al., 2011).
Table 2: Correlation among total phenolic content and antioxidant capacity determined by DPPH, ABTS, ORAC, FRAP assay, SRSA and IC50
|
TPC
|
ABTS
|
DPPH
|
FRAP
|
ORAC
|
% SRSA
|
IC50
|
TPC
|
Pearson correlation
|
1
|
|
|
|
|
|
|
Sig. (2-tailed)
|
|
|
|
|
|
|
|
ABTS
|
Pearson correlation
|
0.885
|
1
|
|
|
|
|
|
Sig. (2-tailed)
|
0.000
|
|
|
|
|
|
|
DPPH
|
Pearson correlation
|
0.889
|
0.926
|
1
|
|
|
|
|
Sig. (2-tailed)
|
0.000
|
0.000
|
|
|
|
|
|
FRAP
|
Pearson correlation
|
0.808
|
0.865
|
0.927
|
1
|
|
|
|
Sig. (2-tailed)
|
0.000
|
0.000
|
0.000
|
|
|
|
|
ORAC
|
Pearson correlation
|
0.701
|
0.888
|
0.817
|
0.697
|
1
|
|
|
Sig. (2-tailed)
|
0.011
|
0.000
|
0.001
|
0.011
|
|
|
|
% SRSA
|
Pearson correlation
|
0.177
|
0.038
|
0.132
|
0.249
|
-0.156
|
1
|
|
Sig. (2-tailed)
|
0.581
|
0.905
|
0.680
|
0.434
|
0.627
|
|
|
IC50
|
Pearson correlation
|
-0.244
|
-0.448
|
-0.350
|
-0.456
|
-0.432
|
-0.459
|
1
|
Sig. (2-tailed)
|
0.443
|
0.143
|
0.264
|
0.135
|
0.210
|
0.133
|
|
3.5. UPLC analysis of major phenolic compounds
UPLC-PDA method was applied for analysis of phenolics in fig extracts. Six Phenolic compounds- cyanidin 3-O-glucoside, quercetin 3-β-glucoside, trans-cinnamic acid, ellagic acid, rutin and ferulic acid were studied (Fig. 2). Retention time and quantities of the
analysed phenolic compounds are shown in Table 3. Positive correlation of individual compounds (Table 3) with TPC was observed, implying the influence of solvent combination on TPC recoveries. Quantities of rutin and cyanidin 3-O-glucoside of fig used in the study was slightly higher than previously reported fig varieties from Italy, Greece and Turkey on extraction in 80% aqueous-methanol (Russo, Caporaso, Paduano, & Sacchi, 2014).
Table 3. Quantities of cyanidin 3-O-glucoside, quercetin 3-β-D-glucoside, trans-cinnamic acid, ellagic acid, rutin, and ferulic acid determined by UPLC-PDA analysis in various solvent extract of dried fig and their correlation with TPC and IC50 values.
Solvent
|
εCyanidin 3-O-glucoside
|
ε Quercetin 3-β-D-glucoside
|
εTrans- cinnamic acid
|
ε Ellagic acid
|
εRutin
|
ε Ferulic acid
|
⅀επ
|
χRT
|
3.99
|
4.55
|
5.28
|
3.6
|
3.44
|
3.94
|
|
Acetone
|
|
|
|
|
|
|
|
100%
|
11.8±0.3
|
3.9±0.4ce
|
8±0.05a
|
17.3±0.4a
|
41.4±0.5d
|
7.1±0.1c
|
82.5
|
80%
|
36.8±0.7
|
9.4±0.4a
|
3.8±0.2a
|
31±0.5bd
|
39.1±0.5d
|
16.7±0.6a
|
136.9
|
50%
|
53±0.8c
|
23.8±0.3ce
|
4.9±0.1a
|
34.6±0.1b
|
94.3±1.5c
|
36.6±0.4d
|
247.4
|
Ethanol
|
|
|
|
|
|
|
|
100%
|
15.9±0.3c
|
5.3±0.2ace
|
0.8±0.1a
|
20.5±0.8ad
|
48.7±0.3
|
8.1±0.2c
|
99.6
|
80%
|
69.1±0.9ed
|
14.4±0.2af
|
5.4±0.1a
|
49.3±0.7c
|
59±0.4a
|
32.3±0.7d
|
229.8
|
50%
|
73±0.2ad
|
17.1±0.6cdf
|
6.5±0.2a
|
53.3±0.4bc
|
64.4±0.5a
|
42.3±0.5d
|
256.8
|
Methanol
|
|
|
|
|
|
|
|
100%
|
66.2±2.9a
|
10.7±0.5a
|
3.3±0.2a
|
21.3±1a
|
61.3±9a
|
21.5±1.2a
|
184.6
|
80%
|
78.3±1.5b
|
37.8±0.8b
|
13.9±0.1
|
29±0.8b
|
84±0.5b
|
24.5±0.8a
|
.8
|
50%
|
54.4±0.8c
|
10.6±0.4ac
|
3.2±0.3a
|
21.4±0.3a
|
61.4±0.8a
|
19.1±1.2a
|
170.4
|
DMF
|
|
|
|
|
|
|
|
100%
|
71.8±0.7ad
|
42.±0.6b
|
5.7±0.7a
|
51.8±0.8c
|
97.3±1c
|
57.6±0.4
|
326.5
|
80%
|
64.7±1.2ae
|
10.8±0.3ad
|
4.4±0.1a
|
22.5±0.9ad
|
79.9±0.2b
|
20.2±0.3a
|
202.7
|
50%
|
54.2±1.7bc
|
10.1±1.2a
|
4.1±0.4a
|
22±0.7af
|
63.6±1a
|
18.8±1.2a
|
173
|
ψr TPC
|
0.73
|
0.66
|
0.64
|
0.36
|
0.78
|
0.61
|
0.76
|
P
|
0.0062
|
0.0192
|
0.0236
|
0.2544
|
0.0025
|
0.0349
|
0.0036
|
λr IC50
|
-0.74
|
-0.60
|
-0.60
|
-0.47
|
-0.59
|
-0.57
|
-0.72
|
P
|
0.0056
|
0.0384
|
0.0391
|
0.1191
|
0.0399
|
0.0522
|
0.0082
|
Note: χRT = Retention time in minutes; εvalues are presented as mean ± SEM (n = 3) mg/100g dry fruit;
πsum of all quantified phenolics;
ψPearson correlation coefficient between quantity of given phenolic compound and TPC (Figure 1) at respective significance level; P is less than given value
λPearson correlation coefficient between given phenolic compound and corresponding IC50 at respective significance level - P is less than given value
|
Recoveries of ellagic acid, quercetin 3-β-glucoside, rutin and cyanidin 3-O-glucoside were more when compared to (Table 3) previous report on fig fruit extracted in 75% aqueous-methanol (Kamiloglu & Capanoglu, 2014) or hot water (Oliveira et al., 2009).
3.6. Antiproliferative activity
Antiproliferative activity of different fig solvent extracts were tested against triple negative breast cancer cells; MDA MB-468. An observable decrease in cell viabilities after 24 hr of treatment with each extract in a dose-dependent manner occured (Fig. 3). IC50 were 80.4, 41.2, 36.4, 43.31, 38.19, 30.37, 36.79, 17.97, 38.35, 22.0, 30.1, 62.3 mg IDF/ml for 100%, 80%, 50% of acetone, ethanol, methanol, and DMF extracts of the fig respectively (Table 1). IC50 values of dry residue was 3.93, 6.47, 5.87, 1.93, 5.72, 4.61, 6.04, 3.03, 5.76, 4.01, 5.05, and 9.99 µg/ml for aforementioned extracts respectively. Minimum effective dose (minimum dose causing significant decrease in cell viability as compared to control) were 30 (P < 0.01), 10 (P < 0.05), 5 (P < 0.05), 30 (P < 0.01), 10 (P < 0.001), 10 (P < 0.05), 10 (P < 0.001), 10 (P < 0.01), 50 mg IDF/ml (P < 0.01) for 100%, 80%, 50% of ethanol, methanol and DMF extracts sequentially. It was 30 (P < 0.05) and 30 mg IDF/ml (P < 0.01) for 80% and 50% for acetone extract respectively. However, there was no significant decrease in cell viability in case of 100% acetone extract (Fig. 3).
Increasing percentage of water in methanol, ethanol, and acetone enhanced cytotoxic property, but for DMF the trend was reversed. Methanol 80% extract was the most effective with IC50 value of 17.97 mg IDF/ml. Results suggest that extracts of modest polar solvents had better antiproliferative activity. TPC and antioxidant capacity of the extracts might be contributing towards antiproliferative property. A fair negative correlation between IC50, respective TPC and antioxidant capacities was found (Table 2). Previous report on Theobroma cacao also exhibited similar correlation (Baharum, Akim, Taufiq-Yap, Hamid, & Kasran, 2014).
IC50 ranged from 1.93 to 9.99 µg/ml. Results of the present stduy showed a better antiproliferative activity than previous report on MDA MB-468 cells (Ayob, Mohd Bohari, Abd Samad, & Jamil, 2014). A similar study by Baharum et al. (2014) on MDA MB-231 reported that methanolic exctract of Theobroma cacao has comparatively low antiproliferatve activity. In addition, fig also has lower IC50 than several vegetables, namely cucumber, lettuce, celery, onion, cabbage, spinach, carrot, broccoli, red pepper (Chu et al., 2002).
3.7. Cell death analysis by flow cytometry
Fragmentation of nuclear DNA is an integral part of cell death process leading to hypodiploid condition. Propeidium iodied staining followed by flow-cytometry-scoring of treated cells was done to enable quantification of hypodiploid dead, and diploid live cell population (Fig. 4.a). Various solvent extracts induced cell death in dose dependent manner in MDA MB-468 cells; higher dose (40 mg IDF/ml) was more effective than lower dose (20 mg IDF/ml). All extracts induced cell death significantly (P < 0.05) at 40 mgIDF/ml treatment concentration as compared to control (Fig. 4.b). However, at same dose very very significant (P < 0.001) cell death took place as compared to control when absolute methanol, 80% methanol or 50% acetone extracts were applied (Fig. 4.b). At lower dose (20 mg IDF/ml) 100%, 80%, and 50% methanol, 100% DMF and 50% acetone extract triggered cell death significantly (P < 0.05) as compared to control. Among all, 80% methanol extract was most effective which induced death in 43 ± 4.5 and 64 ± 6.4% cells at 20 and 40 mg IDF/ml doses respectively (Fig. 4.b). The observation was in line with previous work reporting cell death in HeLa cells treated with Oroxylum indicum bark extracts (Moirangthem, Talukdar, Bora, Kasoju, & Das, 2013). Score of percent dead cells was positively correlated with TPC (r = 0.32 and 0.67 for 20 and 40 mg IDF/ml doses respectively) and negatively correlated with IC50 (r = -0.57 and -0.61 for 20 and 40 mg IDF/ml doses respectively). Present finding is in accordance with reports on other plants which showed efficacy of polyphenol rich extract against cancer cell proliferation (Cai, Luo, Sun, & Corke, 2004).
IC50 was negatively correlated and cell death was positively correlated with each of the analysed phenolic compounds (Table 3). It suggests that phenolic compounds i.e. trans-cinnamic acid, ferulic acid, ellagic acid, cyanidin 3-O-glucoside, quercetin 3-β-glucoside might be accounting for antiproliferative property (Table 3).
Antiproliferative activity of dry fig is supported by its anti-inflammatory property as it alleviates an elevated plasma cytokine (inteleukins: IL-2, IL-3, IL-4, IL-5, IL-9, IL-10; and Eotaxin) level by 22.27 to 30.49% in transgenic mice (Essa, Subash, Akbar, Al-Adawi, & Guillemin, 2015). Fig is reported to significantly increase blood plasma antioxidant capacity, and inhibit low-density lipoproteins oxidation in human (Vinson, Zubik, Bose, Samman, & Proch, 2005). Therefore, results of present study suggest fig as anticancer diet; moderately polar solvents (50% acetone, 50% ethanol, 80% methanol and absolute DMF) showed better antiproliferative activity. The antiproliferative activity is supported by respective antioxidant capacity and phenolics composition of the extracts.