Extraction of cellulose nano-crystals from Polyalthia longifolia and Terminalia catappa leaf litter and the synthesis of low-cost CNC-based hydrogel for articular cartilage

Due to the rise in demand for biodegradable and renewable materials, the synthesis of CNCs from lignocellulosic biomass opens up a new avenue for the creation and application of novel materials in nanotechnology. The CNC-based hydrogels appear to be a favorable material in various applications due to their excellent mechanical strength, biodegradability, biocompatibility, and low toxicity. This work aimed to utilize the fallen leaves for the extraction of Cellulose Nano-crystals (CNC) from Polyalthia longifolia and Terminalia catappa leaf litter. Leaves mainly consist of cellulose hence used for the extraction of nanocellulose. Alkali treatment was performed with aqueous sodium hydroxide, followed by bleaching with aqueous sodium chlorite. Sulphuric acid hydrolysis was used for the extraction of CNC. The morphology, structure, functional groups, and crystallinity of the retrieved CNC were studied using a Transmission Electron Microscope (TEM), Fourier Transformed Infrared spectroscopy (FTIR), and X-Ray Diffraction (XRD). The shape was rod-like for both P. longifolia and T. catappa and the CNC’s crystallinity index was enhanced to 72.40% and 73.95%, respectively. The TEM micrographs revealed that the impurities present on the leaf fibres were successfully removed by alkali treatment and subsequent bleaching further purified the fibres, leaving behind mostly cellulose while the hemicellulose and lignin were removed, which was revealed in FTIR spectra. The obtained CNC was used in the preparation of hydrogel by cross-linking with natural polymers like sodium alginate and gelatin. A Freeze-thawing process was carried out for the fabrication of hydrogel. The resulting hydrogel can be used as a substitute for cartilage applications.


Introduction
Over recent decades, fossil fuels have been increasingly scarce, green renewable resources arose wide attention, especially cellulose; the most abundant natural polymer [1]. Both rural and urban areas are burdened by the wastes that are generated from plants. Though the degradation process of the fallen leaves happens naturally but, it is a time consuming phenomenon. In urban settings the degradation process is difficult because the majority of soil is capped with concrete and results in the clogging of the drainage system. The disposal of this precious resource for making compost is generally done through burning which leads to air pollution or by dumping the litters into landfills [2,3].
Cellulose is mainly present in the cell wall of the plants and is composed of a linear chain of β-1,4-linked anhydro-Dglucose units [4]. Cellulose is composed of the both amorphous and crystalline regions and the CNCs are obtained from the crystalline region [5]. The extraction of the CNCs is done by hydrolysis process which results in the needle-like crystallites commonly called as Cellulose Nano-Crystals or Cellulose Nano-whiskers [6]. The CNCs are several nanometres in length and 10 -30 nm in width having excellent biological properties such as biocompatibility, biodegradability, and non-toxicity [7]. Nano-crystals of cellulose also has important characteristics such as a specific surface, high aspect ratio (length/width), high crystallinity, and excellent mechanical properties which makes them useful in various applications like packaging, food additives, cosmetics, drug delivery systems, nanocomposite materials as reinforcing filler for the automotive industry, constructions, electronics and biomedical purposes [7,8].
The CNCs have been extracted by various methods such as acid hydrolysis, alkali hydrolysis, enzymatic hydrolysis, TEMPO oxidation, steam explosion, ultrasonication, and grinding, high-pressure homogenization. Depending on the method of extraction in different compounds the physicochemical characteristics of the CNCs may differ [9,10,11,12,13].
The current investigation focuses on the extraction of CNCs from Polyalthia longifolia and Terminalia catappa leaf litters and used to prepare hydrogel. Since cellulose is rich with hydroxyl groups, it is used to perform physical cross-linking of hydrogen bonding to obtain gel formation. These gels are created by cross-linking hydrophilic polymer chains, which can absorb and retain large amounts of water in an aqueous environment without disintegrating. The bio-based hydrogels are biocompatible, non-toxic, and have a highly hydrated 3D porous structure, making them suitable for wound dressing, cell therapy, and tissue engineering. The fabrication of hydrogel networks is done via various processes which include homogenization (straight forward mixing), freeze-thawing process, UV/ion mediated cross-linking, free radical polymerization and 3D printing of natural (gelatin, alginate, chitosan) and synthetic (PVA, PAM, PEG) polymers [14,15].
The CNC-based hydrogels can be used in medical implants due to its high mechanical strength. To date, CNCs have not been extracted from Polyalthia longifolia and Terminalia catappa leaf litters and therefore this study was undertaken. The samples were powdered using a grinder, then the powder was treated with sodium hydroxide for alkaline treatment to remove the impurities like pectin, wax and also helps in altering the hemicellulose and lignin structure. Sodium chlorite was used as a bleaching agent and sulphuric acid for hydrolysis. The characterization of the samples was performed via Transmission Electron Microscopy, Fourier Transform infrared spectroscopy, and X-Ray diffraction. The low-cost hydrogel is prepared using the extracted CNCs in combination with other natural polymers (sodium alginate and gelatin). Freeze-thawing technique was followed and calcium chloride was used as a cross-linking agent. The CNCbased hydrogels improves the mechanical properties of hydrogels, which makes them a promising material to use for cartilage applications, especially for the easy and rapid repair of injured cartilage.

Materials
The biomasses used in this study are Polyalthia longifolia and Terminalia catappa leaf litters collected from the soil surface during the summer season in R. V. College of Engineering campus.

Sample processing
First, the leaf litters of P. longifolia and T. catappa were collected separately and washed with tap water to remove the dirt. The leaves are then rinsed with distilled water and air dried for 24hrs. Milling or grinding are common methods for reducing particle size to facilitate material handling and increase the surface-to-volume ratio. So, the dried samples were crushed by hand and dried in the oven at 90ᵒC. After drying, the samples were ground using a mixer grinder and sieved through a #60 mesh sieve. The samples were stored in an airtight container separately and stored at room temperature.

Extraction of Cellulose fibers
The cellulose fibres were extracted from the samples using an alkali treatment, which consisted of dissolving 50g of P. longifolia leaves powder in 1 litre of 1M (4% w/v) NaOH. The mixture was kept under stirring conditions at 80ᵒC for 2 hours. The alkali treatment was repeated 3 times and washed several times with distilled water to remove the alkali contents. Also, the pH was neutralized. The residues were then oven-dried at 50ᵒC for 24 hours. The same procedure was carried out for the T. catappa sample as well. Then, bleaching treatment was carried using 2% w/v NaClO2 at 100ᵒC for 1 hours under stirring conditions. The bleaching treatment was repeated several times till the colour changed to white. In the end, the fibres were washed with distilled water to neutralize the pH and oven-dried at 50ᵒC for 24 hours. The same procedure was carried out for T. catappa sample as well.

Acid hydrolysis
The extracted cellulose fibres were hydrolysed to obtain Cellulose nano-crystal through the acid hydrolysis method. 10g of the extracted cellulose fibres were added into 200 ml of 64% w/w sulphuric acid and was hydrolysed at 45ᵒC for 1 hour at stirring condition. The reaction was stopped by adding 5 fold distilled water and then being centrifuged at 10,000 rpm for 10 mins and the gel suspension was collected. Filtering the collected gel suspension via a dialysis membrane soaked in distilled water neutralised it. The dialysis membrane used was of 12kDa molecular weight cut off. Finally, the neutralized CNC suspensions were then sonicated by using an ultrasonic sonicator for 20 mins and then stored in a refrigerator at 4ᵒC.

CNC-based Hydrogel
In a beaker containing distilled water, 1% sodium alginate was dissolved at 60ᵒC for 2 hours kept on a magnetic hotplate stirrer. In another beaker, 1% of homogeneous solution of gelatin was prepared at 35ᵒC under constant stirring. The solution was then heated to 60ᵒC stirring continuously. To this, 30% glycerol and 0.2% NaCl were added. This mixture was then poured into the above beaker containing the Sodium alginate solution.
Finally, 2% of CNC was heated at 40ᵒC by stirring continuously and added to the prepared mixture under stirring for 2 hours until a homogenous dispersion was obtained. After that, the mixture was cast and dried in a 40ᵒC oven for two days. The dried mixture was then immersed for 1 hour in a 1% CaCl2 aqueous solution to allow cross-linking. To eliminate the unbound cross-linking agents, the samples were rinsed with distilled water. Finally, froze at -20ᵒC for 24 hours and then thawed at room temperature. The Freeze-thawing process was repeated 4 times to obtain cross-linked sponges.

Characterization
The shape and size of the CNCs are examined using Transmission electron microscopy (TEM). The functional groups in the samples were investigated using Fourier Transform Infrared spectroscopy and the XRD patterns of the samples were recorded to calculate the crystallinity index (CrI) by using the equation: (%) = ( + )/ * 100 where, + is parts of crystalline and amorphous at 2Ɵ = 23ᵒ and is the intensity peak of the amorphous region at 2Ɵ = 16ᵒ [16,17,18].

Transmission Electron Microscope
The characteristic size and shape of cellulose nano-crystals hydrolysed by acid hydrolysis is shown in Figure 7 for (a) Polyalthia longifolia and (b) Terminalia catappa. The CNCs were discovered to be rod-like in shape, with an average ( ℎ × ℎ) of (296.02 ± 36.45 × 5.79 ± 2.08 ) and (258.85 ± 32.64 × 4.81 ± 4.47 ) for P. longifolia and T. catappa, respectively. It was observed that there were no impurities present in both samples. The Aspect ratios are mentioned in the below Table 1.   Figure 8 shows the FTIR spectra images. It was found that cellulose existed in all of the samples. The pure cellulose with characteristic peaks of -OH, C-H, and C-O stretching vibrations were observed at 3325cm -1 , 2854cm -1 , 1643cm -1 and 3390cm -1 , 2890cm -1 , 1630cm -1 for (a) P. longifolia and (b) T. catappa respectively. The glycosidic deformation was seen at 900cm -1 and 882cm -1 for P. longifolia and T. catappa respectively, as shown in Table 2.

X-ray Diffraction
The diffraction patterns were seen in the XRD spectrum of P. longifolia and T. catappa bleached and hydrolysed CNC. Figure 9 showing X-ray diffraction patterns after being bleached and hydrolyzed for (a) Polyalthia longifolia samples shows three peaks at 2θ = 16.2ᵒ, 23.0ᵒ, and 34.8ᵒ. The crystallinity index of the hydrolyzed CNC was increased to 72.40%. In contrast, (b) Terminalia catappa samples show three peaks at 2θ = 16.0ᵒ, 23.2ᵒ, and 34.9ᵒ and the crystallinity index of the hydrolyzed CNC was increased to 73.95%.

Conclusion
In this study, Cellulose nano-crystals were successfully extracted from Polyalthia longifolia and Terminalia catappa leaf litter via sulphuric acid hydrolysis. The Non-Cellulosic components were extensively removed by alkali treatment and bleaching treatment. The morphological study confirms that the chemical treatment used is appropriate to obtain CNC with a high aspect ratio of 51.12 and 53.81 also the CNCs exhibited rod-like shapes with crystallinity indexes of 72.40% and 73.95% for P. longifolia and T. catappa respectively. After the chemical treatment, the FTIR spectra showcased the elimination of hemicellulose and lignin. As a result, the Polyalthia longifolia and Terminalia catappa leaf litter can be used as a cost-effective source for the production of CNC which can be used as a reinforcing agent in various applications.
The extracted CNC from Polyalthia longifolia and Terminalia catappa leaf litter was successfully used in the preparation of hydrogel. CNC, in addition to sodium alginate, gelatin and glycerol were mixed together and for cross-linking, CaCl2 was used. The 3D hydrogel was fabricated by a freeze-thawing process. The prepared hydrogel is biocompatible, elastic, viscoelastic and flexible in nature. For people suffering from osteoarthritis, it can be used as articular cartilage.

Disclosure of conflict of interest
Both the authors, Drathi U K and Pushpa Agrawal declares that no conflict of interest in this article.

Statement of ethical approval
The present research work does not contain any studies performed on animals/humans subjects by any of the authors.