Determination of total polyphenol content
The total polyphenol content of different solvent extracts of fig leaves was shown in figure 2a. It was obvious that the ethyl acetate extract showed the highest content of polyphenol compounds, which was (1.72±0.01) mg/g. In contrast, the content of polyphenol compounds measured in water and petroleum ether extracts was significantly lower, which might be related to the low solubility of polyphenol in petroleum ether and water.
Determination of total flavonoids content
The total flavonoids content of different solvent extracts of fig leaves was shown in figure 2b. The content of total flavonoids in the extracts was mainly related to different extraction solvents. The content of total flavonoids in the extracts of different solvents was determined. Total flavonoids content in the ethyl acetate part was the highest, which was (83.92±0.01) mg/g, and that in the petroleum ether part was the lowest, which was (18.71±0.11) mg/g.
Antioxidant capacity
DPPH, ABTS and FRAP were used to evaluate the antioxidant capacity of extracts from fig waste leaves. All the extracts of fig leaves with three different solvents all had the ability to scavenge DPPH free radicals, and with the increase of extract concentration, the DPPH scavenging rate gradually increased. The scavenging ability of ethyl acetate extract varied greatly with concentration. As is known, the lower the IC50 value, the higher the antioxidant activity of the antioxidants. The IC50 value of ethyl acetate extract was 0.54 mg/mL, which did not exceed the scavenging ability of Vc on DPPH free radical, nevertheless, the difference was not significant (P>0.05). It can be seen from figure 3a that the scavenging ability of three extracts on DPPH free radical decreased in the following order: ethyl acetate phase > water phase > petroleum ether phase. This may be related to the fact that the ethyl acetate extract contained more flavonoids and polyphenol compounds.
It can be seen from figure 3b that each fig leaves extract had the ability of scavenging ABTS free radicals, which was similar to that of DPPH free radical. The changing trend of sample scavenging ability was the same as that of sample concentration. The ethyl acetate extract had the strongest scavenging rate at different concentration of three extracts and the scavenging rate was 80.28% at the concentration of 2.5 mg/mL. The ABTS free radical scavenging activity decreased in the following order: ethyl acetate phase > water phase > petroleum ether phase, which also proved that the flavonoids of fig leaves had strong antioxidant activity. Flavonoids and polyphenols mostly have phenolic hydroxyl groups, which show moderate polarity and can bind with free radicals, showing strong antioxidant activity.
When the total antioxidant capacity was measured by FRAP method, the FRAP value was represented by the concentration of FeSO4 solution. The higher the concentration of extracts, the stronger the antioxidant activity of the substance. The total antioxidant capacity was positively correlated with the concentration of extracts. Among the three extracts, ethyl acetate phase showed the highest FRAP value, 3.46 mmol/g and the strongest reducing ability to ferric ion, which was significantly higher than that of water and petroleum ether phase. The ethyl acetate extract has a strong ferric-ion reducing ability, which is related to the polarity of the solvent. The greater the polarity, the stronger the reducing ability, however, the water extract had the weakest reducing ability, which might be due to the extraction of water-soluble chemical components with water as the extraction solvent, resulting in the reduced antioxidant activity of the extract.
Flavonoid characterization of fig leaves by HPLC-DAD-ESI-MS
Flavonoids from fig waste leaves were characterized by HPLC-DAD-ESI-MS, as is shown in figure 4, 11 negative peaks of ethyl acetate extract in fig leaves were observed at 517 nm. By scanning in negative ion mode, 11 compounds were analyzed by mass spectrometry. The larger the area of the negative peak, the stronger the antioxidant activity of the compound. The chemical structures of 11 compounds are elucidated in figure 5. Among them, 1, 6 and 7 are the main substances with high antioxidant activity. It is speculated that they are 3-O-(rhamnopyranosyl-glucopyranosyl)-7-O-(glucopyranosyl)-quercetin, isoschaftoside and rutin, respectively.
Under the condition of negative ions, the total ion flow diagram of flavonoids in fig leaves is shown in figure 6. Through the analysis of the information in the first-level mass spectrometry, it can be preliminarily inferred that the molecular weight of each compound is shown in Table 1. By comparing the fragmentation information of the target compounds in the secondary mass spectrometry and searching the computer standard mass spectrometry database, combining with the references, the experimental results were comprehensively analyzed [34].
Radical scavenging capacity of fig flavonoids by online HPLC-DPPH
The isolated compound reacted with DPPH in post-column, and the antioxidants in fig leaves were screened. 11 components of fig leaves extract have negative peak can be observed in DPPH free radical detection spectrum at wavelength 517 nm (figure 4). Three main peaks (1, 6 and 7) were clearly observed as the main contributors of antioxidants. The results showed that there were abundant antioxidant substances scavenging DPPH free radical in fig leaves. When extracted with ethyl acetate, the negative peak had a better front shape, a stable baseline and a large signal noise ratio (SNR).
Identification of flavonoid compounds
The active components in ethyl acetate from fig leaves were identified according to other characteristics of fragments in the mass spectrometry. 11 flavonoids in fig leaves were identified and their chemical structures were determined. Table 1 presented the retention time, MS fragmentation and molecular formula of compounds detected by HPLC-QTOF-MS/MS analysis. The first-level and secondary mass spectrometry of 11 flavonoids were shown in figure 7.
Compound 1: from the first-level mass spectrometry of compound 1, it can be found that there is an excimer ion peak at m/z 771.2027 in the negative ion mode. The main fragment ions were obtained by secondary scanning of the parent ion m/z 771.2027, which were 609.1530, 462.0838 and 301.0357, respectively. According to the mass spectrometry data in Table 1 and related literature [35], it was inferred that the compound is 3-O-(rhamnopyranosyl-glucopyranosyl)-7-O-(glucopyranosyl)-quercetin.
Compound 2: the m/z 365.0881 of the negative ion mode scan was a [M-H]- peak. The main fragments were 203.0352, 159.0454, 130.0422 by secondary mass spectrometry scanning of m/z 365.0881 parent ion. According to the fragmentation information combined with the literature [36], the compound was identified as 2-carboxyl-1, 4-naphthohydroquinone-4-O-glucopyranoside according to the mass spectrometry data in Table 1.
Compound 3: the m/z 579.1366 scanning in negative ion mode was [M-H]- peak. The main fragments were 519.1194, 489.1083, 429.0856, 399.0748, 369.0635 and 339.0521 by the secondary mass spectrometry scanning of the parent ion m/z 579.1419. It was found to be luteolin 6-C-glucopyranoside, 8-C-arabinopyranoside according to the mass spectrometry data in Table 1 and related literature [37].
Compound 4: in the first-level mass spectrometry of the compound, the m/z 563.1412 scanning in the negative ion mode of the compound was [M-H]- peak. The main secondary mass fragments were m/z 443.1001, 473.1115, 353.0670 and 383.0795 by the secondary mass spectrometry scanning of the parent ion m/z 563.1414. According to the mass spectrometric data in Table 1 and the information combined with the previous literature [38], the compound was presumed to be schaftoside.
Compound 5: the m/z 447.0934 scanning in negative ion mode was [M-H]- peak. Through the secondary mass spectrometry scanning of the parent ion m/z 447.0937, the main secondary mass spectrometry fragments can be obtained as follows: 369.0615, 357.0622, 327.0511, 297.0397, 285.0396 and 133.0280. According to the mass spectrometric data in Table 1 and related literature [39], it was speculated that compound 5 was isoorientin.
Compound 6: the m/z 563.1412 scanning in negative ion mode of the compound was [M-H]- peak. The main fragments were m/z 443.1001, 473.1115, 353.0670 and 383.0795 by the secondary mass spectrometry scanning of the parent ion m/z 563.1414. According to the mass spectrometric data in Table 1 and related literature [40], it was speculated that this compound was isoschaftoside.
Compound 7: the m/z 609.1470 of negative ion mode scanning is [M-H]- peak. The main secondary mass spectrometry fragments were 301.0362, 151.0031, 257.0450 and 273.0477 by the secondary mass spectrometry scan of the parent ion m/z 609.1470. According to the mass spectrum data in Table 1 and related literature [41], the compound was presumed to be rutin.
Compound 8: the negative ion mode scan m/z 577.1577 shows the [M-H]- peak. By the secondary mass spectrometry scanning of the parent ion m/z 577.1577, the main secondary mass spectrometry fragments were obtained as m/z 457.1164 and 293.0454. The compound was presumed to be 2''-O-rhamnosylvitexin according to the mass spectrum data in Table 1 and related literature [42].
Compound 9: scanning m/z 431.0989 in the negative ion mode of the compound showed [M-H]- peak. The main fragments of m/z 431.0981 were 283.0621, 311.0564 and 341.0673 by secondary mass spectrometry. According to the mass spectrum data in Table 1 and related literature [43], it could be speculated that the compound was isovitexin.
Compound 10: scanning in negative ion mode at m/z 463.0890 showed [M-H]- peak. m/z 463.0890 was scanned by secondary mass spectrometry, and the main fragment ions were m/z 301.0386 and 151.0033. According to the mass spectrum data in Table 1 and earlier studies [44], the compound was presumed to be isoquercetin.
Compound 11: scanning m/z 593.1524 in the negative ion mode of the compound showed [M-H]- peak. m/z 593.1576 was scanned by secondary mass spectrometry, and the main fragment ion was m/z 285.0403. The compound was presumed to be kaempferol-3-O-rutinoside according to the mass spectrum data in Table 1 and unified with the literature [45].