Synthesis, Characterization, and Antifungal Activity of Schiff Bases of Inulin Bearing Pyridine ring

As a renewable, biocompatible, and biodegradable polysaccharide, inulin has a good solubility in water and some physiological functions. Chemical modification is one of the important methods to improve the bioactivity of inulin. In this paper, based on 6-amino-6-deoxy-3,4-acetyl inulin (3), three kinds of Schiff bases of inulin bearing pyridine rings were successfully designed and synthesized. Detailed structural characterization was carried out using FTIR, 13C NMR, and 1H NMR spectroscopy, and elemental analysis. Moreover, the antifungal activity of Schiff bases of inulin against three plant pathogenic fungi, including Botrytis cinerea, Fusarium oxysporum f.sp.niveum, and Phomopsis asparagi, were evaluated using in vitro hypha measurements. Inulin, as a natural polysaccharide, did not possess any antifungal activity at the tested concentration against the targeted fungi. Compared with inulin and the intermediate product 6-amino-6-deoxy-3,4-acetyl inulin (3), all the synthesized Schiff bases of inulin derivatives with >54.0% inhibitory index at 2.0 mg/mL exhibited enhanced antifungal activity. 3NS, with an inhibitory index of 77.0% exhibited good antifungal activity against Botrytis cinerea at 2.0 mg/mL. The synthesized Schiff bases of inulin bearing pyridine rings can be prepared for novel antifungal agents to expand the application of inulin.


Introduction
In developing countries, there are a large number of economic losses caused by plant pathogenic fungi every year [1]. It has been reported that Botrytis cinerea (B. cinerea) can cause the grey mold disease which could cause fruits and vegetables to rot after harvesting [2]. Fusarium oxysporum f.sp.niveum (F. oxysporum f.sp.niveum) can cause Fusarium wilt of watermelon [3,4]. Phomopsis asparagi (P. asparagi) can blight the asparagus stems, which has been considered as a great threat to asparagus [5]. In general, chemical pesticides were widely used to control pathogenic diseases. However, abuse of chemical fungicides causes harm to the environment and human health [6]. The demand for developing efficacious, environmentally friendly, and natural alternatives has been growing in recent years.
Inulin is usually obtained from low-requirement crops, such as Helianthus tuberosus, chicory, yacon, and so on [7]. It primarily consists of β (2→1)-fructosyl fructose units (Fm) and a glucopyranose unit at the reducing end (GFn) [8]. As a kind of natural and biodegradable polysaccharide, inulin is a prebiotic ingredient and is being increasingly used in food as a fat alternative [9]. As a renewable source, inulin also has some advantages in unique physicochemical characteristics and biological effects, such as non-toxicity, biodegradability, biocompatibility, being liquid phase adsorption, anticancer properties, immunomodulatory, and so on, which suggests that inulin can be widely applied in

Antifungal Assay
The antifungal assay was evaluated against B. cinerea, F. oxysporum f.sp.niveum, and P. asparagi in vitro by measuring the growth rate of mycelium according to the method of Guo [35]. Briefly, the compounds (inulin and the inulin derivatives) were dissolved in distilled water at a concentration of 6.0 mg/mL at room temperature. Then, the test sample solution was added to the sterilized potato dextrose agar (PDA) medium to get a final concentration of 0.1, 0.5, 1.0, and 2.0 mg/mL, respectively, and then the solution was poured into the sterilized Petri dishes (6.0 cm). Identical volume distilled water substituting samples were poured into control plates. Finally, the fungi mycelia disk with a diameter of 5.0 mm was placed into the center of the PDA Petri dishes and incubated at 27 • C for 2-3 days. When the diameter of the fungi mycelium reached to the edges of the control plate (without the sample), the inhibitory index was calculated as follows: where Da is the diameter of the growth zone in the test plates, and Db is the diameter of the growth zone in the control plate. The experiments were performed three times, and all the data were averaged and expressed as means ± SD (n = 3).

Structure of Schiff Bases of Inulin
The structures of inulin and the inulin derivatives were determined using FTIR, 13 C NMR, and 1 H NMR spectroscopy. The FTIR of inulin and all the inulin derivatives was shown in Figure 1. As shown in Figure 1, the spectrum of inulin showed that the saccharide mainly contains the following characteristic band: 3385 cm −1 , 1030 cm −1 , and 868 cm −1 [36]. In the 6-bromo-6-deoxy-3,4-acetyl inulin (1), the new peaks at 1735 cm −1 and 653 cm −1 were assigned to C=O and C-Br, respectively. After the chemical reaction between 6-bromo-6-deoxy-3,4-acetyl inulin and NaN 3 , a new strong peak at 2107 cm −1 was attributed to the stretching vibration of the azido group [18]. After reduction of the azido group using Ph 3 P, the peak at 2107 cm −1 disappeared in 6-amino-6-deoxy-3,4-acetyl inulin (3). Meantime, the new peak at 1593 cm −1 was assigned to the absorption of NH 2 [18]. In the spectra of Schiff bases of inulin, except for the carbonyl peak at 1740 cm −1 , the new peaks at 1610, 1538, 828 cm −1 , 1612, 1538, 822 cm −1 , and 1600, 1547, 820 cm −1 were assigned to the pyridine rings in 2NS, 3NS, and 4NS, respectively [7]. The characteristic peak of C=N was observed at 1667 or 1666 cm −1 [7]. The above results preliminarily demonstrated that Schiff bases of inulin were obtained. where Da is the diameter of the growth zone in the test plates, and Db is the diameter of the growth zone in the control plate. The experiments were performed three times, and all the data were averaged and expressed as means ±SD (n = 3).

Structure of Schiff Bases of Inulin
The structures of inulin and the inulin derivatives were determined using FTIR, 13 C NMR, and 1 H NMR spectroscopy. The FTIR of inulin and all the inulin derivatives was shown in Figure 1. As shown in Figure 1, the spectrum of inulin showed that the saccharide mainly contains the following characteristic band: 3385 cm −1 , 1030 cm −1 , and 868 cm −1 [36]. In the 6-bromo-6-deoxy-3,4-acetyl inulin (1), the new peaks at 1735 cm −1 and 653 cm −1 were assigned to C=O and C-Br, respectively. After the chemical reaction between 6-bromo-6-deoxy-3,4-acetyl inulin and NaN3, a new strong peak at 2107 cm −1 was attributed to the stretching vibration of the azido group [18]. After reduction of the azido group using Ph3P, the peak at 2107 cm −1 disappeared in 6-amino-6-deoxy-3,4-acetyl inulin (3). Meantime, the new peak at 1593 cm −1 was assigned to the absorption of NH2 [18]. In the spectra of Schiff bases of inulin, except for the carbonyl peak at 1740 cm −1 , the new peaks at 1610, 1538, 828 cm −1 , 1612, 1538, 822 cm −1 , and 1600, 1547, 820 cm −1 were assigned to the pyridine rings in 2NS, 3NS, and 4NS, respectively [7]. The characteristic peak of C=N was observed at 1667 or 1666 cm −1 [7]. The above results preliminarily demonstrated that Schiff bases of inulin were obtained.  Figure 2 shows the 13 C NMR spectra of inulin and 6-bromo-6-deoxy-3,4-acetyl inulin (1). The signals above 60.0 ppm were assigned to the chemical shift of 13 C NMR of inulin [18,37]. After reacting with acetic anhydride, there were new peaks at 170 ppm and 22 ppm in 1, which were related to the carbon of C=O and COCH3. The degree of substitution of 6-bromo-6-deoxy-3,4-acetyl inulin (1) was evaluated on the basis of the integral values of the 13 C NMR spectrum in Figure 2 [36]. The formula to determine DS1 of the inulin derivative 1 is shown in the following equation:  Figure 2 shows the 13 C NMR spectra of inulin and 6-bromo-6-deoxy-3,4-acetyl inulin (1). The signals above 60.0 ppm were assigned to the chemical shift of 13 C NMR of inulin [18,37]. After reacting with acetic anhydride, there were new peaks at 170 ppm and 22 ppm in 1, which were related to the carbon of C=O and COCH 3 . The degree of substitution of 6-bromo-6-deoxy-3,4-acetyl inulin (1) was evaluated on the basis of the integral values of the 13 C NMR spectrum in Figure 2 [36]. The formula to determine DS1 of the inulin derivative 1 is shown in the following equation: where A represents the integration areas of carbons in CH 3 group of inulin derivative 1, and B represents the integration areas of carbons of at C 2 (δ = 98.7 to 101.1 ppm) of furanose rings. The 1 H NMR spectra of inulin and all Schiff bases of inulin are shown in Figure 3. It has been known that the peaks at δ = 3.0-5.2 ppm were assigned to the absorption peaks of protons in the fructose and glucose skeleton of inulin [38]. The peak at δ = 5.2 ppm was assigned to the α-anomeric forms of free glucose [37]. The signal of protons at 1.8 ppm was assigned to the primary amino groups in 3 [18]. In 2NS, 3NS, and 4NS, multiple peaks at δ = 7.7-8.6 ppm were assigned to the pyridine ring and CH=N [7]. Besides, the peak at δ = 2.1 ppm revealed the presence of the CH 3 -C=O group. The absorption peak at δ = 1.9 ppm indicated the residual amino group. The 1 H NMR spectra further confirmed the successful synthesis of Schiff bases of inulin.

Antifungal Activity
Inulin, 2NS, 3NS, and 4NS all had good solubility in water ( Figure 4) and were prepared as solutions at room temperature. The antifungal activities of inulin, 6-amino-6-deoxy-3,4-acetyl inulin   Figure 5 shows the inhibitory indices of inulin, 6-amino-6-deoxy-3,4-acetyl inulin (3), 2NS, 3NS, and 4NS against B. cinerea at all the tested concentrations. According to the graph, we concluded the results as follows. As a positive control, carbendazim with an inhibitory index of 100% could totally inhibit the growth of B. cinerea, even at 0.1 mg/mL. However, inulin didn't show any antifungal activity even at 2.0 mg/mL, which was in accordance with the earlier report [7]. Besides, the antifungal indices of the inulin derivatives was enhanced upon increasing the concentration. The inhibitory index of 6-amino-6-deoxy-3,4-acetyl inulin (3) was 69.5% at 2.0 mg/mL. It has been reported that the amino group could contribute to the antifungal activity since the amino group would interact with anionic components of the cell membranes, such as glucan, mannan, proteins, and lipids, to destroy the cell membranes or to form an impervious layer preventing the transport of essential nutrients from entering the cell [18]. Besides, the inhibitory indices of Schiff bases inulin 2NS, 3NS, and 4NS were 72.2%, 77.0%, and 69.9% at 2.0 mg/mL, respectively. It was obvious that all Schiff bases of inulin bearing pyridine rings exhibited excellent antifungal activity compared with inulin, especially at 2.0 mg/mL. The aromatic moieties as favorable factors could contribute to the antifungal activity [39]. Compared with 6-amino-6-deoxy-3,4-acetyl inulin (3), the Schiff bases of inulin, such as 3NS, showed relatively better antifungal activity. In addition, the antifungal activity of Schiff bases of inulin with the different position of the nitrogen atom on the pyridine ring didn't show significant difference.
The inhibitory indices of inulin and the inulin derivatives against P. asparagi and F. oxysporum f.sp.niveum are shown in Figure 6 and Figure 7, respectively. Carbendazim also exhibited prominent antifungal activity against P. asparagi and F. oxysporum f.sp.niveum even at 0.1 mg/mL. Figure 6 shows the antifungal activity of inulin, 6-amino-6-deoxy-3,4-acetyl inulin (3), 2NS, 3NS, and 4NS against P. asparagi. The results were almost similar to the antifungal activity against B. cinerea. Firstly, inulin as a nutrition polysaccharide had no marked inhibition of fungi growth even at 2.0 mg/mL. Secondly, the inhibitory indices of all the inulin derivatives were in a concentration-dependent manner. Thirdly, the compound 3 and all Schiff bases of inulin exhibited enhanced antifungal activity than inulin especially at 2.0 mg/mL, which was attributed to the introduction of the active group such as the amino group and the pyridine ring. Besides, the Schiff bases of inulin especially 2NS and 3NS with inhibitory indices >67% showed relatively better antifungal activity than 6-amino-6-deoxy-3,4-acetyl inulin (3). The antifungal activities of inulin and the inulin derivatives against F. oxysporum f.sp.niveum are shown in Figure 7. The results were similar to the above results against B. cinerea and P. asparagi. Inulin didn't show any antifungal  Figure 5 shows the inhibitory indices of inulin, 6-amino-6-deoxy-3,4-acetyl inulin (3), 2NS, 3NS, and 4NS against B. cinerea at all the tested concentrations. According to the graph, we concluded the results as follows. As a positive control, carbendazim with an inhibitory index of 100% could totally inhibit the growth of B. cinerea, even at 0.1 mg/mL. However, inulin didn't show any antifungal activity even at 2.0 mg/mL, which was in accordance with the earlier report [7]. Besides, the antifungal indices of the inulin derivatives was enhanced upon increasing the concentration. The inhibitory index of 6-amino-6-deoxy-3,4-acetyl inulin (3) was 69.5% at 2.0 mg/mL. It has been reported that the amino group could contribute to the antifungal activity since the amino group would interact with anionic components of the cell membranes, such as glucan, mannan, proteins, and lipids, to destroy the cell membranes or to form an impervious layer preventing the transport of essential nutrients from entering the cell [18]. Besides, the inhibitory indices of Schiff bases inulin 2NS, 3NS, and 4NS were 72.2%, 77.0%, and 69.9% at 2.0 mg/mL, respectively. It was obvious that all Schiff bases of inulin bearing pyridine rings exhibited excellent antifungal activity compared with inulin, especially at 2.0 mg/mL. The aromatic moieties as favorable factors could contribute to the antifungal activity [39]. Compared with 6-amino-6-deoxy-3,4-acetyl inulin (3), the Schiff bases of inulin, such as 3NS, showed relatively better antifungal activity. In addition, the antifungal activity of Schiff bases of inulin with the different position of the nitrogen atom on the pyridine ring didn't show significant difference.
The inhibitory indices of inulin and the inulin derivatives against P. asparagi and F. oxysporum f.sp.niveum are shown in Figures 6 and 7, respectively. Carbendazim also exhibited prominent antifungal activity against P. asparagi and F. oxysporum f.sp.niveum even at 0.1 mg/mL. Figure 6 shows the antifungal activity of inulin, 6-amino-6-deoxy-3,4-acetyl inulin (3), 2NS, 3NS, and 4NS against P. asparagi. The results were almost similar to the antifungal activity against B. cinerea. Firstly, inulin as a nutrition polysaccharide had no marked inhibition of fungi growth even at 2.0 mg/mL. Secondly, the inhibitory indices of all the inulin derivatives were in a concentration-dependent manner. Thirdly, the compound 3 and all Schiff bases of inulin exhibited enhanced antifungal activity than inulin especially at 2.0 mg/mL, which was attributed to the introduction of the active group such as the amino group and the pyridine ring. Besides, the Schiff bases of inulin especially 2NS and 3NS with inhibitory indices >67% showed relatively better antifungal activity than 6-amino-6-deoxy-3,4-acetyl inulin (3). The antifungal activities of inulin and the inulin derivatives against F. oxysporum f.sp.niveum are shown in Figure 7. The results were similar to the above results against B. cinerea and P. asparagi. Inulin didn't show any antifungal activity against F. oxysporum f.sp.niveum under the tested condition. Besides, the inhibitory indices of the inulin derivatives mounted up with the increasing concentration. The inhibitory indices of 6-amino-6-deoxy-3,4-acetyl inulin (3), 2NS, 3NS, and 4NS could reach 52.4%, 57.4%, 70.9%, and 66.5% at 2.0 mg/mL, respectively. All the inulin derivatives showed more enhanced antifungal activity than inulin, especially at 2.0 mg/mL. Besides, as illustrated by the data, the introduction of Schiff bases was more significant for enhancing the antifungal activity of inulin.
Based on the results mentioned above, all Schiff bases of inulin enhanced the antifungal activity compared with inulin and 6-amino-6-deoxy-3,4-acetyl inulin (3), especially at 2.0 mg/mL. The introduction of the pyridine ring was an important factor to improve the antifungal activity. The lipophilic characteristic of the heterocyclic aromatic ring will be more likely to pass through the oil film outside the cell membranes based on the "like dissolves like" point, which will damage the cell membranes. Besides, there was no significant difference in antifungal activity between 2NS, 3NS, and 4NS. The inhibitory index showed extremely significant differences among the samples and the concentrations (p = 2e −16 < 0.01, p = 2e −16 < 0.01, two-way Analysis of Variance (ANOVA) test). Further analysis showed that the differences between samples were significant (p < 0.05, Tukey test). The differences between concentrations were extremely significant (p < 0.01, Tukey test). .5% at 2.0 mg/mL, respectively. All the inulin derivatives showed more enhanced antifungal activity than inulin, especially at 2.0 mg/mL. Besides, as illustrated by the data, the introduction of Schiff bases was more significant for enhancing the antifungal activity of inulin. Based on the results mentioned above, all Schiff bases of inulin enhanced the antifungal activity compared with inulin and 6-amino-6-deoxy-3,4-acetyl inulin (3), especially at 2.0 mg/mL. The introduction of the pyridine ring was an important factor to improve the antifungal activity. The lipophilic characteristic of the heterocyclic aromatic ring will be more likely to pass through the oil film outside the cell membranes based on the "like dissolves like" point, which will damage the cell membranes. Besides, there was no significant difference in antifungal activity between 2NS, 3NS, and 4NS. The inhibitory index showed extremely significant differences among the samples and the concentrations (p = 2e −16 < 0.01, p = 2e −16 < 0.01, two-way Analysis of Variance (ANOVA) test). Further analysis showed that the differences between samples were significant (p < 0.05, Tukey test). The differences between concentrations were extremely significant (p < 0.01, Tukey test).

Conclusions
In this study, a series of new Schiff bases of inulin were successfully designed, synthesized, and characterized using FTIR, 13 C NMR, and 1 H NMR spectroscopy, and elemental analysis. The antifungal activity against three kinds of phytopathogen, including B. cinerea, F. oxysporum f.sp.niveum, and P. asparagi were estimated using in vitro hyphal measurements. All Schiff bases of inulin had good solubility in water and showed enhanced antifungal activity when compared with inulin, especially at 2.0 mg/mL. The results indicated that the pyridine group should be the antifungal function group. The different position of N atom on the pyridine ring didn't show significant influence on the antifungal activity. The relationship between the structure and the activity needs further study in the future.

Conclusions
In this study, a series of new Schiff bases of inulin were successfully designed, synthesized, and characterized using FTIR, 13 C NMR, and 1 H NMR spectroscopy, and elemental analysis. The antifungal activity against three kinds of phytopathogen, including B. cinerea, F. oxysporum f.sp.niveum, and P. asparagi were estimated using in vitro hyphal measurements. All Schiff bases of inulin had good solubility in water and showed enhanced antifungal activity when compared with inulin, especially at 2.0 mg/mL. The results indicated that the pyridine group should be the antifungal function group. The different position of N atom on the pyridine ring didn't show significant influence on the antifungal activity. The relationship between the structure and the activity needs further study in the future.