2.1 Quantification of bioactive compounds
Phenolic compounds are considered to be the most important antioxidants and are widely distributed among various plant species. The presence of the phenolic compounds could contribute to the protective properties and therefore it is quite important to assess the total phenolic content in the extracts tested. The content of phenolic acids is reported in µg of gallic acid equivalents (GAE)/mg of extract. The total phenols content in the different varieties was between 325,19±7,4 and 490, 91±5,1 µg of GAE/mg extract. Statistical analyses showed that, at 95%confidence level, the extracts were statistically different (Table1). Among the studied varieties, Rojo B showed the highest amount of total phenols with a value of 490,91±5,1 µg of GAE/mg extract, whereas the Jiro variety had the lowest total phenols content (325,19 µg of GAE/mg extract). Lower values have been reported in the other scientific literature. For instance, Esteban-Muñoz et al., [11], found that the astringent persimmons (Rojo B) and the non-astringent persimmons (Triumph) have lower concentrations of total phenolic with values of 380.786and 84.568µg of GAE/g respectively.
The flavonoid content in the different varieties of persimmon leaves were calculated using the standard curve for catechin. It has been observed that the flavonoid content was126.1± 6.3, 223.1 ± 4.4 and 293.2 ± 4.8 µg CE/mg of extract for respectively Rojo B, Triumph and Jiro varieties.
The Rojo B variety has the highest flavonoids content at 293.25± 4.8 µg of CE/mg of extract followed by the Triumph variety. However, the Jiro variety had the lowest flavonoids content compared to the others with 126.06 ± 6.3 µg of CE/mg of extract (Table 1). Statistical analysis showed that, at 95% confidence level, the flavonoid content in the three varieties was statistically different. Comparatively, a study conducted by Sun et al. [36], demonstrated that Persimmon (D. kaki L. folium) leaves possesses considerable amounts of flavonoids (192 μg of Catechin equivalent / mg of extract). [36].
Table1. Total phenols and total flavonoids contents in persimmon leaves extracts of three different varieties collected in Tunisia.
Variety
|
Total phenols (µg GAE/mg extract).
|
Total flavonoids (µg CE/mg extract).
|
Triumph
|
359,77±6,42a
|
223,05±4,498a
|
Jiro
|
325,19±7,48b
|
126,063±6,377b
|
Rojo B
|
490,91±5,19c
|
293,252±4,894c
|
a, b, cDifferent letters mean significantly different at 0.05
2.2 LC-MS-Qtof analysis
Persimmon leaves are interesting for their bioactive compounds that may exert beneficial effects on human health [2]. The phenolic fraction of the three varieties of Persimmon leaves was analyzed by LC-MS/MS. Extracts were analyzed in positive and negative mode but the identification was carried out only in negative mode where a higher number of compounds was found, also with higher intensity. Chromatograms of the three persimmon cultivars are shown in Figure 1. In total, 28 polyphenols were identified, including 1 benzoic acid, 15 flavonols, 7 flavones, 3isoflavonoides, 1 flavanones and 1 methoxyphenols (Table 2). Qualitative polyphenol screening of persimmon extracts revealed variation in phenolic constituents with regards to type of cultivars whereby the differences was detected in the presence or absence of fragmentation compounds as summarized in Table 2. Our results show that persimmon leaves have a complex phenolic profile that may help to explain the beneficial effects of their traditional use as a medicinal herb.
Other literature mentions the presence of simple phenolic compounds (hydroxybenzoicacids and hydroxycinnamic acids), tannins, procyanidins, flavonoids, and tyrosols [37-39, 11].
Table2: Identification of compounds in persimmon cultivars based on their spectral LC-MS characteristics in negative ion mode.
Peak
|
Rt (min)
|
Compound (subclass)
|
Molecular formula
|
Accurate [M-H]-m/z
(Δ ppm)
|
Fragments m/z (% intensities)
|
Availability
|
Rojo B
|
Jiro
|
Triumph
|
1
|
1.1
|
8-Hydroxydaidzein (Isoflavones).
|
C15H10O5
|
269.0434 (-3.7)
|
88.9824 (100)
|
|
|
×
|
1
|
1.1
|
6,8-Di-C-methylkaempferol 3,7-dimethyl ether (Flavonols)
|
C15H18O9
|
341.1005
(-7.62)
|
165.0355 (100)
|
|
×
|
×
|
1
|
1.1
|
7-Hydroxy-3',4', 5, 6,8-pentamethoxyflavone (Flavones)
|
C 20H20O8
|
387.1038
(-6.4)
|
89.0192 (100); 119.0536 (64)
149.0397 (24); 179.0503 (42)
|
×
|
×
|
×
|
1
|
1.1
|
Benzoyl benzoate (Benzoic acid)
|
C14H10O3
|
225.0547
0.44
|
59.0091 (100); 89.0192 (30)
|
×
|
×
|
×
|
2
|
1.2
|
5-Hydroxy-3,7,8,2',4'-pentamethoxyflavone(Flavones)
|
C20H20O8
|
387.1047 (0.51)
|
59.0085 (100); 89.0192 (84)
341.1023 (32)
|
|
×
|
|
2
|
1.2
|
5,7,8-Trihydroxyflavone 7-galactoside (Flavones)
|
C21H20O10
|
431.0921 (-11.8)
|
89.9844 (100)
|
|
×
|
×
|
2
|
1.2
|
7,2'-Dihydroxy-3',4'-dimethoxyisoflavone 7-O-glucoside (Flavones)
|
C23H24O11
|
475.1173 (-12.8)
|
133.0090 (100); 114.9983(40)135.0244 (38)
|
|
|
×
|
3
|
1.3
|
5,2',5'-Trihydroxy-3, 6,7, 4’-tetramethoxyflavone 5'-glucoside (Flavones)
|
C 25H28O14
|
551.1310 (-15.4)
|
95.9487 (100); 78.9517 (60)
|
|
×
|
|
3
|
1.3
|
Kaempferol 3-rhamnoside (Flavonols)
|
C21H20O10
|
431.0924
(-13.91)
|
88.9827 (100)
|
|
×
|
×
|
4
|
1.5
|
Quercetagetin 3, 5, 6, 7,3’-pentamethyl ether(Flavonols)
|
C 20H20O 8
|
387.1085 (2.0)
|
89.0190 (100); 341.0999 (58)
179.0502(41); 113.0196 (20)
|
|
×
|
×
|
5
|
5.1
|
(+)-Catechin(Flavonols)
|
C15H14O6
|
289.0635 (-24.5)
|
109.0242 (100); 123.0400 (80)
203.0649 (57); 151.0353 (40)
|
×
|
×
|
×
|
5
|
5.1
|
Kaempferol 3-O-xylosyl-glucoside (Flavonols)
|
C26H28O15
|
579.1339 (0.86)
|
289.0640 (100); 245.0750 (11)
|
×
|
×
|
×
|
6
|
5.2
|
3,5,8-Trimethoxy-6,7:3',4'-bis(methylenedioxy)flavones (Flavones)
|
C20H16O9
|
399.0827 (29.3)
|
191.0287 (100); 152.0162 (13)
|
×
|
×
|
×
|
7
|
5.4
|
5,3',5'-Trihydroxy-3, 6, 7, 8,4’-pentamethoxyflavone (Flavones )
|
C20H20O10
|
419.1085 (26.9)
|
89.0190 (100); 141.0141 (82) 71.0084(74); 119.0301 (57)
|
×
|
|
|
7
|
5.4
|
Daidzein 6’’-Oacetate (Isoflavonoids) |
C23H22O10
|
457.1240
(24.3)
|
119.0450 (100) 163.043 (83)
|
×
|
×
|
×
|
8
|
6.6
|
Quercetagetin 3'-methylether 6-glucoside (Flavonols)
|
C22H22O13
|
493.0869
(-21.7)
|
315.0062 (100); 287.0124 (84)
330.0273 (50); 301.0257 (43)
|
×
|
×
|
×
|
8
|
6.6
|
Quercetin 7-(6''-galloylglucoside) (Flavonols)
|
C 28H24O16
|
615.0812 (-27.3)
|
301.0282 (100); 150.9985 (17)
|
×
|
×
|
|
8
|
6.6
|
6-Hydroxykaempferol 3-glucoside (Flavonols)
|
C 21H20O12
|
463.0758
(-24.4)
|
300.0212 (100);271.0176 (20)
463.0765 (19) ;243.0236 (13)
|
×
|
×
|
×
|
8
|
6.6
|
Oolonghomobisflavan B (Flavonols)
|
C 45H36O22
|
927.1555
(-6.4)
|
463.0744 (100); 300.0196 (87)
|
×
|
|
×
|
8
|
6.6
|
Quercetin 3galacturonide(Flavonols)
|
C 21H18O13
|
477.0540
(-25.7)
|
301.0266 (100); 150.9981 (15)
|
|
|
×
|
9
|
6.8
|
Kaempferol 7-O-glucoside (Flavonols)
|
C21H20O11
|
447.0815 (-22.2)
|
284.0261 (100); 227.0288 (71)
|
×
|
×
|
×
|
10
|
6.9
|
Kaempferol 7-(6''-galloylglucoside) ( Flavonols)
|
C28H24O15
|
599.0872
(-26.5)
|
285.0329 (100); 313.0478 (33)
|
×
|
×
|
×
|
10
|
6.9
|
Quercetin 3-O-xyloside(Flavonols)
|
C20H18O11
|
433.0664 (-23.3)
|
300.0197 (100); 271.0166 (19)
|
|
×
|
×
|
11
|
7
|
Kaempferol 5-glucuronide (Flavonols)
|
C 21H18O12
|
461.0596
(25.5)
|
285.0335 (100)
|
|
|
×
|
12
|
7.5
|
5,6,7,2',3',4',5'-Heptamethoxyflavanone (Flavanones )
|
C 22H26O9
|
433.1505 (2.7)
|
91.0348 (100); 249.0602 (98)
|
×
|
×
|
×
|
12
|
7.5
|
Daidzein 7-O-glucoside-4'-O-apioside (Isoflavonoids)
|
C26H28O13
|
547.1393 (-12.4)
|
249.0605 (100); 363.0864 (84)
160.0095 (51)
|
|
×
|
×
|
13
|
11.6
|
Nordihydrocapsiate (Methoxyphenols)
|
C17H26O4
|
293.1677 (-23.8)
|
221.1469 (100); 236.0979(36) 148.0476 (11)
|
×
|
×
|
×
|
14
|
13.3
|
5,6-Dimethoxy-[2'',3'':7,8] furanoflavanone
(Flavanones )
|
C19H 16O5
|
323.0987 (22.6)
|
265.1403 (100)
|
|
×
|
|
2.3 Antioxidant activity
Oxidative stress inside the cell may be the cause of several diseases [31, 32]. The use of antioxidants to prevent the formation of oxidative stress inside cells has been recommended [40]. The antioxidant activity of the extract of the different varieties of persimmon leaveswas evaluated by the DPPH radical scavenging assaywhich is a good in vitro model to investigate the antioxidant activity [41].
The results from the radical scavenging assays for all extracts are presented in Table 3. The highest antioxidant activity was obtained with Rojo B extract (EC50 =3.79±0.19 µg/mL). These obtained results suggest that the three varieties possess very high antioxidant activity and better than the activity of (BHT) that has an EC50of 25.42±1.46 µg/mL [21]. The statistical analysis showed a that the three varieties are significantly different at 95% confidence level (α=0.05).
The observed differences in the antioxidant activity can be attributed to the structure and content of phenolic compounds [42]. In fact, the highest antioxidant activity was attributed to the extract that has higher polyphenols and flavonoids. In addition, previous descriptive structure-radical scavenging activity relationships of flavonoids demonstrated that the positions of phenolic OH groups could be more important for the radical scavenging activity than the number of phenolic OH groups [43, 44] and it has been shown that glycosylated polyphenols have a reduced ability to donate hydrogen and are less effective in the antioxidant capacity compared to their free forms as aglycones [45].
2.4 Acetylcholinesterase inhibition
In the present study, persimmon leaves extracts were tested to evaluate their anti-acetylcholinesterase activity. AChE inhibitor activity of extracts was found to increase dose-dependently, the results expressed as IC50(Table 3). The best inhibitory activity was exhibited by the leaf extract of variety Rojo B followed by the leaves of Triumph and Jiro varieties. For instance, the variety Rojo B had the highest inhibition with an IC50value of 58.35±0.69µg/mL, while the extract from persimmon leaves of Jiro variety exhibited the lowest activity with an IC50value of 210.21±28.9 µg/mL. The statistical analysis showed that the anti-acetylcholinesterase activity of leaf extract from Truimph and Rojo B was not significantly different at 95% confidence level whereas Jiro leaves extract was significantly different from Truimph and Rojo B leaves extracts.
The good inhibitory activity obtained with persimmon leaf extracts could be attributed to their chemical composition which mainly contain phenolic, as well as the possible synergistic interaction between these components. In fact, several studies showed that other phenolic compounds possess the ability to fit into the active site and act as acetylcholinesterase inhibitors [19, 21, 29].
Previous studies on other diospyros species tested for their inhibitory activities found that aqueous extract of Diospyros lotus fruit exhibited a low anti-AChE activity with an IC50 value of 16.75±0.11µg / mL whereas the ethanol extract of Diospyros lotus leaves had an inhibition of 23.53±2.06 % for 100µg/mL [46].
Table 3: Antioxidant activity and AChE inhibitory activity of three variety persimmon leaves
Variety
|
DPPH (EC50µg/mL)
|
AChE (IC50µg/mL)
|
Triumph
|
8.01±0.35a
|
95.98±16.3a
|
Jiro
|
12.05±0.11b
|
210.21±28.9b
|
Rojo B
|
3.79±0.19c
|
58.35±0.69c
|
a, b, cDifferent letters mean significantly different at 0.05
2.5 HMG-CoA reductase assay
A reduction in cholesterol synthesis is probably the most efficient way to lower plasma cholesterol levels. Persimmon leaves aqueous extracts, for a concentration of 100 µg/mL, showed a good inhibitory activity for HMGR(Figure2). The extracts had a percentage of inhibition of 61.44%, 57.35%, and 46.28% for Rojo B, Jiro and Triumph varieties, respectively on HMG-CoA. The obtained results showed the ability of persimmon leaves extract to have cholesterol lowering effect by inhibiting the rate-limiting enzyme of the cholesterol biosynthesis HMG-CoA reductase. Previous study using ethanol extract of persimmon fruits on HepG2 cells showed an inhibition of HMG-CoA reductase and a decrease in cholesterol level in HepG2 cells [47]. In addition, it has been shown that the effect of phenolic acids on HepG2 cell line at a molecular level is similar to pravastatin, a drug often prescribed to inhibit HMG-CoA reductase enzyme [48].
2.6 Cytotoxicity
The toxicity of persimmon leaves aqueous extract was tested in the human cell lines HepG2 and MCF7, using 5 serial concentrations ranging from 0.05 to 1 mg/mL of extract (Figure 3). The obtained results showed that an increase in the concentration of the extract decreased the viability of the treated cell. The concentration of extracts that killed 50% of the cells (IC50) was determined from the dose-response curves (Table 1). IC50 values for HepG2 cells were lower than those of MCF-7 cell lines for Rojo B and Triumph varieties, in contrast IC50 values for HepG2 cells were higher than those of MCF-7 cell lines for Jiro variety. The obtained results showed that none of the tested extracts have any cytotoxic effect on HepG2 and MCF-7 cells since the IC50% values were higher than the established one for this type of test which is 0.1 mg/mL except Hep G2 cells treated with Rojo B which had an IC50 in the limit of toxicity [49]. Other study of ethanolic extract of astringent persimmons on HepG2 cells showed no cytotoxic effect for the concentrations 50 and 100 μg/mL [50].
Table 4: Cytotoxicity towards HepG2 and MCF-7 cell lines of Disopyros kaki leaves aqueous extractIC50(mg/mL).
Cell line
|
RojoB
|
Triumph
|
Jiro
|
HepG2
|
0.96±0.05a
|
0.98±0.01a
|
1.18±0.02b
|
MCF7
|
0.85±0.008a
|
0.94±0.04b
|
0.84±0.001a
|
a, b, Different letters mean significantly different at 0.05