Physicochemical Characterization of Cellulose and Microcrystalline Cellulose from Cordia africana Lam. Seeds

ABSTRACT This study aims to explore Cordia africana seeds as an alternative source of cellulose and MCC due to depletion of the major commercial sources such as wood pulp, and cotton. Cellulose was extracted from Cordia africana seeds employing a chlorine-free treatment approach, followed by partial depolymerization using acid hydrolysis to obtain microcrystalline cellulose (MCC). The untreated seeds, as-extracted cellulose, and MCC were investigated for yield, chemical composition, functionality, crystallinity, morphology, diameter, and thermal stability. The cellulose content increased from 30% (w/w) in the untreated seeds to 80.2% (w/w) in cellulose and 88.1% (w/w) in the MCC. The removal of non-cellulosic constituents was confirmed by the results obtained from Fourier-transform infrared spectroscopy, X-ray diffraction, Scanning electron microscopy, and Thermogravimetric analysis (TGA)/Derivative thermogravimetry (DTG). The crystallinity index of as-obtained cellulose and MCC increased from 32.38% (untreated seeds) to 68.28 (cellulose) and 73.19% (MCC) with chemical treatments. The extracted samples exhibited characteristic peaks of Cellulose I at around 15°, 16°, 22°, and 34°. Th e TGA/DTG results confirmed the cellulose and MCC had higher thermal stability than the untreated seeds. This study shows that cellulose and MCC can be obtained from unexploited source, Cordia africana seeds, for promising applications in various industries.


Introduction
Lignocellulosic materials have received wide attention for different industrial applications due to their availability, relative low-cost, environmental friendliness, and favorable sustainability (Naceur Abouloula et al. 2018;Gabriel et al. 2020;Veeramachineni et al. 2016).Cellulose is one of the most abundant, natural, renewable, and biodegradable lignocellulosic polymers produced in nature.It has been many centuries since mankind used cellulose in daily activities (Trache et al. 2016).It consists of both crystalline and amorphous regions which are bonded together by intra-and intermolecular bonds (Abdul Khalil et al. 2018).
Microcrystalline cellulose (MCC) is a white, fine, non-reactive, odorless, and crystalline powder, and a flowable excipient, obtained by purification and partial depolymerization by reacting alphacellulose with mineral acids to remove impurities (Terinte, Ibbett, and Christian Schuster 2011).MCC is commonly used in pharmaceutical, food and beverage, personal care, and cosmetics industries among many others as high thickeners, dispersing agents, flow controllers, and anticaking agents (Shi et al. 2018).MCC is considered to be a material with outstanding properties, and remains the most widely used directly compressible excipient for tableting.It is also used as a strong dry binder, tablet disintegrant, an absorbent, filler or diluent, a lubricant, and antiadherent (Chaerunisaa, Sriwidodo, and Abdassah 2020).
Cordia africana Lam.(Amharic-wanza) (family: Boraginaceae) is native to Africa, and it is widely distributed in Angola, Democratic Republic of Congo, Djibouti, Eritrea, Ethiopia, South Africa, Sudan, Tanzania, and Uganda.It is a fast-growing and highly valued timber tree in Ethiopia and used for high-quality furniture, doors, windows, cabinet making, drums, beehives, joinery and interior construction, mortars, paneling, and veneering.It is also used to improve soil fertility and its importance as a (coffee) shade tree in traditional agroforestry systems.The mucilage from its fruit has also been proven as a binder in solid dosage forms such as tablets (Derero, Gailing, and Finkeldey 2011;Kassa, Ferde, and Nigatu 2020;Vidyasagar et al. 2010).
The fruits of Cordia africana Lam. are widely eaten in most parts of Ethiopia and other countries in Africa.The tree produces a lot of fruit during the dry season and in drought years as the tree has deep roots when other fruits can only be produced where there is irrigation.The seeds in the fruit are dispersed mainly by mammals and birds, generating lots of environmental waste (Tewolde-Berhan et al. 2015).Extracting cellulose and MCC as value-added products from the seeds is an effective option for economic and environmental benefits.
In this study, we aimed to extract and characterize cellulose and MCC from Cordia africana seeds.A chlorine-free bleaching was employed to extract cellulose, followed by acid hydrolysis to obtain MCC.The percentage yield, chemical composition, functionality, surface morphology, crystallinity, and thermal stability of the untreated seeds, as-extracted cellulose, and MCC were investigated.

Isolation of cellulose
First, the fruits of Cordia africana were washed, peeled, and soaked in distilled water for 24 h to remove the mucilage from the seeds with repeated washing.The seeds were ground using a locally prepared milling machine.Cellulose was then extracted from the dried and powdered seeds following a three-step chlorine-free treatment approach following a method described elsewhere with some modifications (Gabriel et al. 2020).Briefly, the powdered seeds were treated with 5% potassium hydroxide with the ratio of 1 g/10 mL.The pulps were then treated with formic acid (10%), acetic acid (20%), and hydrogen peroxide (10%) 2:1:2, in the ratio of 1 g/10 mL.Finally, the pulps were treated with 5% sodium hydroxide in 10% hydrogen peroxide at a ratio 1 g/30 mL.All the solvents were prepared in aqueous media.At each stage, the samples were heated on the water bath at 80°C, followed by continuous washing with distilled water.The final bleaching step was repeated.

Preparation of microcrystalline cellulose (MCC)
The MCC was obtained by hydrolyzing the cellulose with 100 mL 2.5 N of hydrochloric acid on a hot plate at 105°C for 15 min followed by dilution with 1000 mL distilled water and allowed to stand overnight.After this, it was filtered and washed with distilled water until it became neutral.Finally, it was kept in the oven for 30 min at 105°C to dry it (Kian et al. 2020).

Identification tests and characters
Solubility (in water, acetone, anhydrous ethanol, cuprammonium hydroxide "Cuam" solution, and so on), pH, appearance, and moisture content of the samples were determined according to the methods described in different pharmacopoeias (BP 2016;PhEur 2013;USP 2019).The pH of the untreated seeds, the cellulose, and the MCC was measured after mixing with distilled water (1 g/500 mL) after 30 min.

Chemical composition
The amount of lignin, hemicellulose content, and water-soluble components was estimated for the untreated seeds, as-obtained cellulose, and MCC according to the procedures mentioned in the Supplementary Material.

Fourier transform infrared spectroscopy (FTIR)
Perkin Elmer Fourier Transform Infrared Spectrophotometer (L1600400 Spectrum TWO DTGS, SN: 108152, LIantrisant, UK) was used to study the functional groups and chemical structure of untreated seeds, as-extracted cellulose and MCC in the frequency range of 4000-400 cm −1 .

X-ray diffraction (XRD)
The crystalline structure of untreated seeds, as-extracted cellulose, and MCC was analyzed by using XRD-7000 X-ray diffractometer MAXima (SHIMADZU Corporation, Japan) at 40 KV.
The crystalline index (CrI) was determined using the equation proposed elsewhere (Segal et al. 1959): Where, I 200 is the maximum intensity (in arbitrary units) of the diffraction from the 200 plane, and I am is the intensity of the background scatter.Following Gaussian deconvolutions for peak separations, parameters such as d-spacings (d), apparent crystallite size or thickness for the 200 plane (τ 200 ), the proportion of crystallite interior chains for the 200 plane (X 200 ), fractional variation in the plane spacing for the 200 plane (Δd/d) 200 , and Z-values were obtained using equations described in the supplementary material (Aguayo et al. 2018;Matheus, Ornaghi Júnior, and Zattera 2014;Popescu et al. 2011).

Scanning electron microscopy (SEM)
The ESEM images of the samples were obtained using ESEM FEI/Philips XL-30 ESEM (Leuven, Belgium) at an accelerating voltage of 2.00 KV.All the samples were coated with chromium using vacuum sputter prior to SEM analysis.

Thermo gravimetric analysis (TGA)
The thermal stability of the untreated seeds, as-extracted cellulose, and MCC was determined by TGA measurements performed using DTG (Derivative thermogravimetry)-60 H (SHIMADZU Corporation, Japan).All measurements were performed under a nitrogen atmosphere with a gas flow of 60 mL/min by heating the material from room temperature to 700°C at a heating rate of 10°C/min.

Identification and composition of the plant materials
The cellulose and MCC fulfilled different parameters such as pH, solubility, and color specified in pharmacopoeia (PhEur 2013).The cellulose extracted from the seeds was off-white powder, and soluble in cuprammonium hydroxide "Cuam" solution, but insoluble in water, acetone, anhydrous ethanol, and toluene.The MCC was white and free-flowing powder.The images of the untreated seeds, and as-extracted cellulose and MCC are shown in Figure 1.The cellulose content increased from 30% (w/w) in the untreated seeds to 80.2% (w/w) in cellulose and 88.1% (w/w) in the MCC due to removal of non-cellulosic components (Table 1) (Gabriel et al. 2020).The composition of untreated seeds, as-obtained cellulose, and MCC such as cellulose content, hemicellulose, and lignin is presented in Table 1.Based on the obtained promising results with respect to various physicochemical properties of the cellulose and MCC, they can be suitably applied in different industries.The global MCC market size was valued at USD 1140.62 million in 2022, and it is projected to reach USD 2044.66 million by 2031.To meet this global need of MCC, non-wood sources are considered to be alternative sources and have a lower total production cost and a more competitive selling price.

FTIR study
The two main absorbance regions of FTIR spectra are located at high wavenumbers (2800-3500 cm −1 ) and at low wavenumbers (500-1700 cm −1 ) (Kian et al. 2017).The FTIR spectra of untreated seeds, as-extracted cellulose, and MCC are illustrated in Figure 2. The broad absorption band around 3330 cm −1 of all samples was due to stretching vibration of OH groups.The other peaks around 2890 cm −1 indicate CH stretching vibrations.The peak around 1643 cm −1 corresponds to the absorption of water molecule in the samples due to the strong interactions between the plant materials and water molecules (Abu-Thabit et al. 2020;Tarchoun, Trache, and Klapötke 2019;Gabriel et al. 2020Gabriel et al. , 2021)).The characteristic peaks related to cellulosic structure appear at 662,895,1020,1058,1115,1163,1315,1372,1425, and 1643 cm −1 .The absorption band at 895 cm −1 is attributed to CH rocking vibrations (anomeric vibration specific to βglycosidic linkage), while the absorption band at 1163 cm −1 is attributed to COC stretching (Liu et al. 2018).Untreated lignocellulosic byproducts are primarily composed of lignin, cellulose, and hemicellulose.Lignin is confirmed by the presence of multiple peaks in the range of 1500-1600 cm −1 (1527, 1543, and 1558 cm −1 ) due to the CC aromatic skeletal vibrations.Other absorption bands that correspond to lignin are the bands at 1249 cm −1 (elongation of the ether COC linkage), 1380 cm −1 (assigned to phenolic hydroxyls), and 1450 cm −1 (assigned either to aromatic ring vibrations or CH 3 of the acetyl group) (Abu-Thabit et al. 2020).The removal of hemicellulose and lignin in all celluloses and MCC was confirmed by the disappearance of the absorption peaks around 1744 cm −1 and 1533 cm −1 for CO stretching of the acetyl and uronic ester groups of hemicellulose or the ester linkage of carboxylic groups of ferulic and p-coumaric acids of lignin, respectively (Trache et al. 2014).The intense and sharp absorption bands at 2843 cm −1 and 2924 cm −1 are assigned to the CH stretching vibrations of the methoxyl groups related to the lignin component, Figure 2. The intensity of the former two bands decreased upon removal of lignin and hydrolysis of cellulose due to the presence of -CH 2 moieties in MCC samples (Abu-Thabit et al. 2020).
The peak at 1595 cm −1 is associated with an aromatic ring stretching that is strongly associated with the aromatic CO stretching band.The bands at 1316 cm −1 , 1320 cm −1 , and 1325 cm −1 are related to CH or CH 2 vibrations associated with intermolecular hydrogen bonds in the C group and the OH in plane bending vibration, respectively.The other significant peak at 1026 cm −1 , 1027 cm −1 , and 1028 cm −1 indicates that the stretching of COC pyranose ring skeletal vibration.While the peak located around 890 cm −1 is related to the β-(1-4) glycosidic linkage vibration of cellulose and MCC (Kian et al. 2017;Liu et al. 2018;Qinfeng et al. 2018).
The as-obtained cellulose and MCC displayed similar spectra indicating that they endured the cellulose extraction and MCC isolation processes, and revealed similarities in functional groups.The absorbance intensity of different peaks in the MCC FTIR spectra increased because acid hydrolysis removes the amorphous part of cellulose on the surfaces (Figure 2) showing the increase in crystallinity and removal of non-cellulosic components during acid hydrolysis process (Kian et al. 2017(Kian et al. , 2020;;Tarchoun, Trache, and Klapötke 2019).

XRD analysis
Figure 3 shows the XRD patterns of untreated seeds, as-extracted cellulose, and MCC.The asobtained samples exhibited characteristic peaks of Cellulose I at around 15°, 16°, 22°, and 34°, with assigned planes of 11̅ 0, 110, 200, and 004, respectively.Peak separations were carried out using Gaussian deconvolutions.The absence of doublet at the 200 plane indicated there was no polymorphic transition into Cellulose II (Tarchoun, Trache, and Klapötke 2019;Gabriel et al. 2022).As depicted in Table 2, the least CrI (32.36%) was observed in the untreated seeds because the cellulose is embedded in the cellulosic matrix by amorphous non-cellulosic components.Whereas the higher CrI of cellulose (68.28%) and MCC (73.19%) is due to the elimination of amorphous lignin and hemicellulose through delignification, alkali treatment, chlorine-free bleaching, and hydrolytic cleavage of glycosidic bonds, which leads to rearrangement of cellulose molecules, also confirmed by the FTIR analysis (Tarchoun, Trache, and Klapötke 2019).The subsequent increase of the CrI in MCC upon acid hydrolysis of purified cellulose was indicative of the dissolution of amorphous cellulosic domains.During the strong acid hydrolysis process, hydronium ions can penetrate the more accessible amorphous regions of cellulose and allow the hydrolytic cleavage of glycosidic bonds, which eventually releases individual crystallites (Johar, Ahmad, and Dufresne 2012).
The values for the d-spacing perpendicular to 1-10, 110, 200, and 040 planes, the proportion of crystallite interior chains, the fractional variation in the plane spacing, the crystallite   sizes perpendicular to the 200 plane, the changes in the crystallinity, and Z-values are summarized in Table 2.The increase in the crystallite sizes for the prepared MCC in 200 plane might be associated with a reduction in the corresponding amorphous region (Gabriel et al. 2020;Trache et al. 2014).

Thermal stability
Figure 4 and Table 3 show the thermal properties of untreated seeds, as-extracted cellulose, and MCC.The thermogravimetric analysis (TGA) and Differential thermogravimetry (DTG) curves of the samples show three weight loss regions.The first region indicates mainly the evaporation of loosely bound moisture on the surface of the samples at the region between 24°C and 150°C (Mohamad Haafiz et al. 2013).The second region indicates the dehydration, decarboxylation, depolymerization, and decomposition of glycosyl units in cellulose in the range of 250-450°C, followed by the char residue formation in the range of 450-700°C (Kian et al. 2017;Trache et al. 2016).The early decomposition of hemicelluloses begins below 400°C followed by the pyrolysis of lignin (Mohamad Haafiz et al. 2013).This result would suggest that cellulose with higher crystallinity exhibited higher thermal stability.This may be due to the absence of hemicellulose and lignin, and also the removal of the amorphous part present in the cellulose during acid hydrolysis process (Liu et al. 2018).T 50% weight loss temperature of cellulose and MCC occurred at 320.22°C and 327°C, respectively.This result is due to the thermal stability of the cellulose and MCC increased with its purity.The reason for this relatively higher thermal stability of the pure cellulose and MCC is probably the substantial removal of less stable hemicellulosic and lignin (Liu et al. 2018;Sun et al. 2004).
T 10% decomposition of untreated seeds, as-extracted cellulose, and MCC appeared at temperature of 177°C, 217°C, and 246°C.This is attributed to the high degree of crystallinity of MCC, which requires high heat energy for thermal degradation than cellulose samples.The char residues of untreated seeds, as-extracted cellulose, and MCC at 700°C were 32.24%, 27.64%, and 25.54%, much higher than those reported elsewhere.The high residual weight of untreated seeds was likely due to the char formation from flame retardant compounds (Adel et al. 2011;Kian et al. 2017).

Morphology analysis of cellulose and MCC
Figure 5 shows the SEM images of different samples, i.e. untreated seeds (5a), as-obtained cellulose (5b), and MCC (5c) extracted from the seeds.The SEM analysis showed compact surface for the raw material with natural convolution, while the extracted cellulose and MCC from the seeds showed densely entangled microfibrils and ribbons.In study reported elsewhere, SEM images of MCC samples showed a curled and soft-flat shaped with rough pits, which offers higher surface area and supports the inter-fibrillar attraction between hydroxyl groups and may result in dry binding property (Kunal et al. 2020;Misgana, Chaudhary, and Kale 2019).The removal of non-cellulosic and cementing materials such as hemicelluloses, lignin, and wax during treatment of lignocellulosic materials using different chemicals for the extraction of isolated fibrils of cellulose was also reported (Gabriel et al. 2020).After acid hydrolysis of cellulose, it will be converted into irregularly shaped compact MCC particles due to the strong hydrolysis effect of the acid at high temperature that removed lignin and the amorphous region of cellulose and formed MCC (Asif et al. 2022).It was observed in the study elsewhere that the fine powder feature of raw date palm fiber had changed to larger globular shape particles after alkaline treatment showcasing the self-assembly behavior of fibril structure (Hachaichi et al. 2021).

Conclusions
Successful extraction of cellulose and microcrystalline cellulose was conducted from unexploited Cordia africana seeds using chlorine-free isolation conditions.Removal of noncellulosic materials such as hemicellulose and lignin was confirmed by the Fourier-transform infrared spectroscopy, X-ray diffraction, Scanning electron microscopy, and Thermogravimetric analysis/Derivative thermogravimetry studies.The untreated seeds, asobtained cellulose, and MCC exhibited the typical peaks of Cellulose I β around 15°, 16°, 22°, and 34° 2θ, with assigned planes of 11̅ 0, 110, 200, and 004, respectively, and without polymorphic transformation, as confirmed by X-ray diffraction pattern, d-spacings, and Z-values.Enhanced thermal stability was observed in the as-obtained cellulose and microcrystalline cellulose.The findings suggest that cellulose and microcrystalline cellulose can be obtained from the seeds as a promising and alternative source using ecofriendly extraction method to be used in industry.
Note: (Key:-Co-0: untreated cordia seeds; Co-Cel: cellulose obtained from cordia seeds; Co-MCC: microcrystalline cellulose obtained from cordia seeds; d: the interplanar spacing of the crystal; τ 200 : average thickness of cellulose crystallites; X 200 : the proportion of crystallite interior chains for the 200 plane; Δd/d 200 : the fractional variation in the plane spacing for the 200 plane; CrI: crystallinity index/es).

Table 2 .
Properties obtained from XRD analysis of untreated seeds, as-obtained cellulose, and MCC.