Bio-assisted synthesized Ag(0) nanoparticles stabilized on hybrid of sepiolite and chitin: efficient catalytic system for xanthene synthesis

In this work, with the use of two natural compounds, chitin and sepiolite clay, a novel covalent hybrid is fabricated and applied as a support for the stabilization of silver nanoparticles with the aid of Kombucha extract as a natural reducing agent. The resultant catalytic system, Ag@Sep-N–CH, was characterized via XRD, TEM, FTIR, ICP, SEM, TGA, UV–Vis and BET. It was found that fine Ag(0) nanoparticles with mean diameter of 6.1 ± 1.8 nm were formed on the support and the specific surface area of the catalyst was 130 m2 g−1. The study of the catalytic performance of Ag@Sep-N–CH for catalyzing synthesis of xanthenes in aqueous media under mild reaction condition confirmed that Ag@Sep-N–CH exhibited high catalytic activity and could promote the reaction of various substrates to furnish the corresponding products in high yields. Moreover, the contribution of both chitin and sepiolite to the catalysis has been affirmed. It was found that hybridization of these two components led to synergistic effects and consequently improved the observed catalytic activity. Notably, the catalyst was recyclable up to several reaction runs.

In the continuation of research on heterogeneous catalysts based on clays and clay-carbohydrate hybrids [25][26][27][28][29] , herein we wish to report a covalent hybrid of sepiolite clay and chitin (CH), Fig. 1, and its utility as a support for the stabilization of silver nanoparticles with assistance of Kombucha extract as a natural reducing agent. The catalytic activity of the resulting hybrid, Ag@Sep-N-CH, was appraised for one-pot synthesis of xanthenes in aqueous media and under mild reaction condition. Furthermore, the roles of sepiolite and chitin in catalysis were evaluated and the recyclability of the catalyst has been examined.

characterization
To assess the morphological change of sepiolite after incorporation of CH and Ag(0) nanoparticles, the TEM image of sepiolite ( Fig. 2A) and Ag@Sep-N-CH (Fig. 2B,C) were recorded. As shown in Fig. 2A, the TEM analysis of sepiolite revealed that this clay showed rod like morphology. In Ag@Sep-N-CH TEM images (Fig. 2B,C), the sepiolite rods can be discerned, indicating that upon covalent grafting with CH and immobilization of silver nanoparticle, sepiolite morphology did not alter. On the other hand, in some parts of sepiolite rods, fine sheet can be observed that can be ascribed to CH. Furthermore, the spherical black spots on the sepiolite rods are ascribed to the silver nanoparticles. Measurement of silver nanoparticle sizes, Fig. 2D, showed that the mean diameter of these particles was 6.1 ± 1.8 nm.
In Fig. 3, the SEM images of sepiolite (Fig. 3A), Sep-N-CH (Fig. 3B) and Ag@Sep-N-CH (Fig. 3C) are illustrated. As shown, the SEM image of sepiolite is distinguished from Sep-N-CH. This observation indicated that introduction of CH induced morphological change to some extent. The morphology of Ag@Sep-N-CH is almost similar to that of Sep-N-CH, implying that immobilization of silver nanoparticles has slight effect on the morphology. This issue can be due to the low content of silver nanoparticles in the structure of the catalyst.
To confirm grafting of CH to sepiolite and formation of Ag@Sep-N-CH, TG curve of Ag@Sep-N-CH was compared with that of CH and sepiolite, Fig. 4. As illustrated, among three TG curves shown in Fig. 4, CH and sepiolite showed the lowest and the highest thermal stability respectively. This is a rational result as sepiolite is an inorganic clay with instinctive high thermal stability, while CH is a carbohydrate with low thermal resistance. The thermal stability of Ag@Sep-N-CH is between the two aforementioned samples, implying the conjugation of low thermally stable CH on sepiolite. In more detail, in the TG curve of Ag@Sep-N-CH, the first weight loss, discerned below 200 °C is due to the loss of structural water, while the second one at 380 °C is the sign of degradation of CH. According to TGA results, the content of CH was evaluated as 28 wt%.
To verify the structure of the inorganic-organic hybrid of Ag@Sep-N-CH, FTIR spectrum of Ag@Sep-N-CH was achieved and compared with that of each components, Fig. 5. In the spectrum of sepiolite, the absorbance band at 1,014 cm −1 is observed due to the Si-O stretching. Additionally, the absorbance bands discerned at 3,560, 3,428, and 645 cm −1 are representative of stretching and bending vibrations of -OH functionality 30 . In the FTIR spectrum of CH, the absorbance bands of CH at 3,444 cm −1 , 1662 cm −1 can be observed. In the case of the FTIR spectrum of Ag@Sep-N-CH, all the discussed absorbance bands can be detected. However, as the distinctive bands of CH overlapped with that of sepiolite, FTIR spectroscopy cannot provide enough proofs for  www.nature.com/scientificreports/ confirming the formation of the hybrid. Notably, the shifts of the bands can be attributed to the interactions between functionalized CH and sepiolite. As the specific surface area is one of the most important features of catalysts, this value was estimated for Ag@Sep-N-CH via BET method. Moreover, the specific surface area of the catalyst was compared with that of sepiolite. The results approved that upon grafting CH, the specific surface area of sepiolite reduced from 161 to 130 m 2 g −1 . Moreover, upon introduction of silver nanoparticles, the pore volume and pore diameter reduced to 30.0 cm 3 g −1 and 2.2 nm respectively. Furthermore, the recorded nitrogen adsorption-desorption isotherm was of the type II with H3 hysteresis loops, Figure S1 31 .
The XRD patterns of sepiolite and Ag@Sep-N-CH are illustrated in Figure S2. As shown the two XRD patterns are similar and displayed similar bands that can be assigned to sepiolite structure 8 . The reason for not observing the bands of silver nanoparticles can be accredited to low content of silver nanoparticles 32 .
To verify the potential of the extract for reducing Ag(I) to Ag(0), UV-Vis spectra of Kombucha extract and the mixture of Kombucha extract and AgNO 3 were obtained, Fig. 6. It is well-established that Ag(0) nanoparticles displayed a specific band in the UV-Vis spectrum at λ max = 430 nm 33,34 that can approve the reduction of Ag(I) to Ag(0). As shown, in Fig. 6, in the UV-Vis spectrum of the mixture of AgNO 3 and Kombucha extract, the so-called characteristic band can be discerned, while this band is not appeared in the spectrum of Kombucha extract. This result indicated the successful reduction of silver salt via Kombucha extract.
The loading of silver nanoparticles on Sep-N-CH was measured by ICP analysis as 0.5 w/w%.

catalytic activity
To appraise the catalytic performance of Ag@Sep-N-CH, synthesis of xanthene derivatives from reaction of aldehydes and dimedone was targeted. In the onset, by using reaction of benzaldehyde and dimedone as a model reaction, the reaction parameters such as solvent, catalyst loading and temperature have been optimized. In this regard, first, the model xanthene derivative was synthesized by using 0.02 g Ag@Sep-N-CH in water as solvent at room temperature. It was revealed that (Table S1) in this condition only moderate yield of the desired product has been achieved. To improve the yield of the product, the reaction was examined in different solvents. It was www.nature.com/scientificreports/ determined that the best result was obtained in H 2 O: EtOH (1:2). Next, to elucidate the influence of the reaction temperature on the yield of the product, the model reaction was repeated at 50 and 70 °C. As listed in Table S1, the optimum reaction temperature was found as 50 °C. Finally, the influence of the loading of Ag@Sep-N-CH was scrutinized by using 0.02-0.04 g catalyst. The results demonstrated that increase of the catalyst loading from 0.02 to 0.03 g gave rise to the increase of the reaction yield. However, increase of this value to 0.04 g had no effect on the yield of the product. After finding the optimum reaction condition, more precise study on the role of the hybrid components has been carried out. First, the necessity of silver nanoparticles on the structure of the catalyst was appraised. In this context, a control catalyst, Sep-N-CH was prepared similar to the reported procedure and its activity for catalyzing the synthesis of the model xanthene was examined. The result, Table 1, affirmed that Sep-N-CH exhibited moderate catalytic activity and led to the formation of the desired product in 50% yield. This observation confirmed the necessity of incorporation of silver nanoparticles for achieving high catalytic activity. Next, to elucidate the efficiency of CH-NH 2 and Sep-Cl as catalyst supports and investigate whether there is any synergism between functionalized chitin and sepiolite, two samples, Ag@Sep-Cl and Ag@CH-NH 2 , were fabricated through wet impregnation of silver nanoparticles on CH-NH 2 and Sep-Cl as supports and then the activities of these samples for the model reaction were measured. As shown in Table 1, these two samples displayed moderate catalytic activities that were inferior compared to Ag@Sep-N-CH. This observation confirms that hybrid of CH-NH 2 and Sep-Cl is a more effective support compared to sole CH-NH 2 and Sep-Cl. This can be due to the synergism between the hybrid components.
In the next section, the scope of the present procedure is evaluated. In this regard, various aldehydes with electron-withdrawing and electron-donating groups were applied as starting materials for the synthesis of the corresponding xanthenes. Moreover, heterocyclic aldehyde, furfural has also been utilized as a substrate. The results, Table 2, established that all the used substrates could undergo the reaction using Ag@Sep-N-CH catalyst and led to the high yields of the corresponding xanthene derivatives. This result confirmed that this protocol can be generalized to diverse range of aldehydes.

comparison of the catalytic activity of Ag@Sep-n-cH with other catalysts
In the next part, Ag@Sep-N-CH performance for catalyzing the synthesis of the model xanthene was compared with some other catalysts, Table 3. Considering the fact that xanthenes are biologically-active compounds, various researchers focused on evolving efficient catalysts for their synthesis. Among the reported procedures in Table 3, some of them were not efficient and gave rise to low yields of the desired xanthene. Other tabulated catalysts www.nature.com/scientificreports/ showed competitive efficiency compared to Ag@Sep-N-CH. Although due to the different reaction condition, a precise comparison cannot be achieved, it can be inferred that Ag@Sep-N-CH can be recognized as an effective catalyst with comparable performance with reported catalysts. In detail, in the present protocol, high yield of the product can be achieved in aqueous media with use of relatively low catalyst loading at low temperature.

Reaction mechanism
According to the literature 37 , Ag@Sep-N-CH catalyzed synthesis of xanthene derivatives can be described as follow: Initially, Ag@Sep-N-CH activated the aldehyde. The activated aldehyde then reacted with the enol form of dimedone (Fig. 7) to generate an intermediate I. The latter underwent dehydration to form intermediate II.
In the next step, intermediated II reacted with the second molecules of dimedone to furnish compound III. The latter then tolerated dehydration followed by cyclization to produce xanthene.

catalyst reusability
To examine the reusability of Ag@Sep-N-CH, the synthesis of the model xanthene derivative was carried out by using the reused Ag@Sep-N-CH under the optimum condition. It was found out that by using the reused catalyst, the yield of the model reaction did not alter and remained as 95%. Encouraged by this result, the reused catalyst was recovered and applied for the third run of the same reaction. The result, Fig. 8A, demonstrated that upon third use, the reaction yield decreased from 95 to 80%. Reuse for the fourth time also confirmed further decrement of the yield of the model xanthene (75%). Finally, use of Ag@Sep-N-CH for the fifth run of the reaction resulted in significant drop of the efficiency of the catalyst to 50%. It was postulated that the reduction of the activity of Ag@Sep-N-CH can originate from leaching of silver nanoparticles that played the main role in the catalysis. To validate this assumption, Ag(0) loading of the recycled Ag@Sep-N-CH was measured via ICP method. It was determined that no leaching was occurred upon second  The morphological change of Ag@Sep-N-CH after fifth run of the model reaction was also appraised. As shown in Fig. 8B, the TEM image of the reused Ag@Sep-N-CH affirmed that reuse of the catalyst caused agglomeration and increase of the average size of Ag(0) nanoparticles to some extent (the estimated mean particle size was 6.8 ± 1.5 nm). This issue can also contribute to the observed lower activity of the reused Ag@Sep-N-CH. instrumentation and analysis. The applied instruments for the characterization of Ag@Sep-N-CH were follow: BRUKER, EQUINOX 55 apparatus for powder Fourier transform infrared (FT-IR) spectroscopy, MET-TLER TOLEDO instrument with heating rate of 10 °C min −1 for ), thermogravimetric analysis (TGA) under nitrogen atmosphere. Philips CM30300Kv instrument for transmission electron microscopy (TEM), BELSORP Mini II apparatus for Brunauer-Emmett-Teller (BET) analysis (degassing at 150 °C for 2 h). Siemens, D5000 apparatus using graphite monochromatic Cu-Kα for X-ray diffraction (XRD) analysis, UV-visible Spectrophotometer (PerkinElmer, Lambda 365) and Vista-pro for Inductively Coupled Plasma (ICP) analysis. Tescan instrument was used for recording SEM images.

catalyst fabrication
Synthesis of the catalyst. Synthesis of Cl-functionalized sepiolite (Sep-Cl). Sepiolite (2 g) was suspended in 80 mL toluene and mixed vigorously for 15 min. Subsequently, CPTES (2 mL) was added gradually and the mixture was sonicated for 30 min and then refluxed for 48 h at 110 °C under N 2 atmosphere. At the end, the solid was filtered, washed several times with toluene and dried in a vacuum oven at 90 °C overnight.
Synthesis of amine-functionalized chitin (CH-NH 2 ). A solution of APTES (2 mL) was added to a suspension of chitin (2 g) in dry toluene (40 mL). The mixture was then sonicated (power of 100 W) for 30 min and refluxed for 24 h at 110 °C. Afterwards, the resultant suspension was filtered and washed repeatedly with dry toluene and dried at 100 °C overnight. -N-CH). Sepiolite-Cl (1.5 g) in dry toluene (30 mL) was suspended. Meanwhile, CH-NH 2 (1.5 g) was crushed and well dispersed in toluene (30 mL) using ultrasonic irradiation of 100 W. Then, the two aforementioned suspension were mixed and refluxed for 24 h at 110 °C under N 2 atmosphere. Upon completion of the reaction, the solid was filtered and placed in water. Then, the aqueous suspension was centrifuged. The obtained Sep-N-CH was gathered, washed with toluene repeatedly and dried in a vacuum oven at 100 °C.