Characterization of physicochemical properties of microcrystalline and nanocrystalline cellulose powders derived from Citrullus lanatus peels for potential pharmaceutical applications

Materials

The watermelon peel, which consists of the rind and outer flesh, was obtained from vendors at the Mushin market, Mushin, Lagos State, Nigeria. The analytical grade of sodium hydroxide (97%), hydrochloric acid (36%), sulfuric acid (98%), and sodium hypochlorite solution (4%w/v) was purchased from central drug house (CDH) Fine Chemicals (India).

Extraction of cellulose from watermelon peel

The watermelon peel was washed, cut, and sun-dried for three days. It was, further, dried in the oven at 80℃ for 90 min, ground, and passed through a sieve of 1.204 mm aperture size to obtain the watermelon peel powder. A stepwise extraction process based on Akter et al. 2015^[11] was adopted with some modifications. Watermelon peel powder of 500 g was weighed and boiled in water at 100℃ for 1 h. The mixture was filtered, and the solid mass was thoroughly washed with water. The residue was immersed in 5 L of 17.5% w/v sodium hydroxide solution and heated at 100℃ for 5 h with intermittent stirring every 15 min to solubilize the lignin, hemicellulose fraction, and other extractives. The reaction mixture was filtered, and the solid mass was washed with water until the water was neutral when tested with red litmus paper. The residue was bleached with 3.2% w/v sodium hypochlorite solution at 40℃ for 40 min. The reaction mixture was filtered and washed thoroughly with water until it was neutral to red litmus paper. The treatment of the residue with sodium hydroxide followed by sodium hypochlorite solutions was repeated, washing thoroughly with water after each treatment to obtain the native cellulose (α-cellulose). The yield of the native cellulose was calculated using Equation 1.

Synthesis of watermelon peel microcrystalline and nanocrystalline cellulose

The isolated cellulose was hydrolyzed by heating with 2 M HCl at 100°C for 1 h. The ratio of acid to cellulose was kept constant at 20:1. The reaction mixture was filtered, and the residue was washed with water until it was neutral to blue litmus paper to obtain the MCC.^[12] The MCC obtained was air-dried for 24 h and the yield was calculated using Equation 2.

Nanocrystalline cellulose was obtained from the native cellulose by adopting the method of Sainorudin et al. 2022^[13] with some modifications. Native cellulose was weighed and dispersed in 64% w/v sulfuric acid solution at a cellulose to solvent ratio of 1:20 at 25°C for 60 min with constant stirring. Water was added to the mixture at a water:mixture ratio of 100:1. The suspension obtained was centrifuged for 15 min at 10,000 rpm and the supernatant was discarded. The residue was washed with water and centrifuged after each washing for 15 min at 10,000 rpm (five cycles) to remove the excess sulfuric acid indicated by no color change to blue litmus paper. The suspension was immersed in 3.2% w/v sodium hypochlorite at 25°C at a suspension-to-sodium hypochlorite solution volume ratio of 4:1, stirred once, and allowed to stand for 10 min to attain a white color. An equal volume of water was added to the mixture, and the supernatant was decanted. This mixture was washed with water three times to obtain nanocrystalline cellulose.

The nanocrystalline cellulose suspension obtained was either subjected to freeze drying (NCC_{freeze dried}) or dried in an oven at 70°C for 40 min. The oven-dried nanocellulose sheets were blended into powder (NCC_{oven dried}) and the yield was calculated using Equation 3.

Characterization of watermelon celluloses

Statistical analysis

The measurements of the test parameters of the cellulose samples were carried out in triplicate and expressed as mean ± standard deviation. The sample means were compared using a one-way analysis of variance on Jamovi^®

Software. Significant differences (P < 0.05) were determined by the Tukey post hoc test using the same software.

Yield

The percentage yield of native cellulose obtained from watermelon peel was 18.1%, while the yield of watermelon peel MCC (Wmp-MCC), watermelon peel nanocrystalline cellulose (Wmp-NCC_{oven dried} and Wmp-NCC_{freeze dried}) from native cellulose was 56.45% and 51.03%, respectively.

Physicochemical characterization

The physicochemical properties of the cellulose samples are presented in Table 1. All samples were insoluble in water. The pH and moisture content of the cellulose samples ranged from 6.10–7.40 to 3.90–5.87, respectively. The bulk density, tapped density, Hausner’s ratio, and Carr’s index of microcrystalline and oven-dried nanocrystalline cellulose were similar but significantly different than that of the native and freeze-dried nanocrystalline cellulose. All samples had a similar angle of repose. Microcrystalline and nanocrystalline cellulose held a lesser amount of water than the native cellulose. However, the micro and nanocrystalline cellulose swell more than the native cellulose, with the highest swelling capacity observed in the nanocrystalline cellulose sample.

Particle size and percentile distribution analysis

The particle size and percentile distribution of the cellulose samples are shown in Tables 2 and 3. There was a decrease in the particle size of microcrystalline and nanocrystalline cellulose when compared with the native cellulose; the nanocrystalline cellulose samples had a mean area diameter of < 100 μm.

Structural characterization

The structures of the cellulose samples were analyzed by FTIR spectroscopy and powder X-ray diffraction. The FTIR spectra and X-ray diffractograms are presented in Figures 1 and 2, respectively. The degree of crystallinity of the cellulose samples derived rom the X-ray diffractogram is shown in Table 4. An increase in crystallinity was observed in the microcrystalline and nanocrystalline cellulose when compared with the native cellulose.

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Figure 1:

Percentile distribution of particle size in watermelon peel native cellulose, watermelon peel microcrystalline cellulose, watermelon peel oven-dried nanocrystalline cellulose (WmpNCC_{oven dried}), and watermelon peel freeze-dried nanocrystalline cellulose (d) Wmp-NCC_{freeze dried}.).

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Figure 2:

Fourier transform infrared spectra of watermelon peel native cellulose, watermelon peel microcrystalline cellulose, watermelon peel oven-dried nanocrystalline cellulose (WmpNCC_{oven dried}), and watermelon peel freeze-dried nanocrystalline cellulose (Wmp-NCC_{freeze dried}).

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Thermal analysis

The thermal properties of the cellulose samples were determined by DSC and TGA analysis. The DSC and TGA thermograms are shown in Figures 3 and 4, respectively. The weight loss and its temperature derived from the TGA are presented in Table 5.

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Figure 3:

X-ray diffraction spectra of watermelon peel native cellulose, watermelon peel microcrystalline cellulose, watermelon peel oven-dried nanocrystalline cellulose (Wmp-NCC_{oven dried}), and watermelon peel freeze-dried nanocrystalline cellulose (Wmp-NCC_{freeze dried}).

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Figure 4:

Differential scanning calorimetry thermogram of watermelon peel native cellulose, watermelon peel microcrystalline cellulose, watermelon peel oven-dried nanocrystalline cellulose (Wmp-NCC_{oven dried}), and watermelon peel freeze-dried nanocrystalline cellulose (Wmp-NCC_{freeze dried}).

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Physicochemical characterization

The pH of all cellulose samples falls within the acceptable limits of 5.0–7.5 according to the specifications in the British Pharmacopoeia 2021.^[21] All cellulose samples were insoluble in water; the nanocrystalline cellulose dispersed in water to form a milky suspension that settled with time. The ability to redisperse in a medium is a highly desired characteristic in nanocrystalline cellulose products, influencing its potential applicability.^[22]

The moisture content of pharmaceutical material affects its stability on storage and agglomeration properties, ultimately affecting the quality, safety, and efficacy of the finished pharmaceutical product. The moisture content of MCC fell within the acceptable limits of ≤5% (w/w) according to British Pharmacopoeia 2021 specifications for MCC; the oven-dried nanocrystalline had a moisture content of 3.90% while those of native cellulose and freeze-dried nanocrystalline cellulose were similar but higher than that of the oven-dried nanocrystalline cellulose at 5.77% and 5.87%, respectively [Table 1]. Excessive moisture content in cellulose can result in microbial instability during storage, swelling, and aggregation of the cellulose particles, which can lead to loss of flowability, compressibility, and reduced binding properties when used as an excipient in solid dosage forms; this, in turn, leads to non-uniformity and inconsistency in the content of the final tablet or capsule formulation.^[23]

In this study, the microcrystalline and oven-dried nanocrystalline cellulose had comparable bulk and tapped densities, which were higher than those of native cellulose and freeze-dried nanocrystalline cellulose, which were also similar. Higher bulk and tapped density show that the powder has weaker inter-particle forces and bonds, which predispose it to less resistance in the packing and a better potential to flow and rearrange the particles during compression.^[24] Therefore, microcrystalline and oven-dried nanocrystalline cellulose may have better flow properties than the native and freeze-dried nanocrystalline. Although bulk and tapped densities are not intrinsic properties of a powder due to the ability to change based on how the material is handled, they are still critical powder properties used in the making decisions such as the size of vessels to be utilized in the production process.^[24] Hausner’s ratio, Carr’s compressibility index, and repose angle measure powders or granules’ flowability to ensure content uniformity and consistent drug levels in solid dosage formulations. Hausner’s ratios of 1.12–1.18, 1.19–1.25, 1.26–1.34, 1.35–1.45, and 1.46–1.59 indicate good, fair, passable, poor, and inferior flow, respectively; Carr’s indices of <10, 11–15, 16–20, 21–25, 26–31, and 32–37 indicate excellent, good, fair, passable, poor, and inferior powder flows, respectively, while the angles of repose of 25–30, 31–35 and 36–40, 41–45, and >46° indicate excellent, good, fair, passable, and poor flows, respectively. The oven-dried nanocrystalline cellulose had a better flow than the other types of cellulose with a Hausner’s ratio of 1.31 and Carr’s index of 23.28 [Table 1], which indicates a passable flow. The MCC (Hausner’s ratio – 1.37, Carr’s index – 26.81) exhibited a better flow than the native cellulose (Hausner’s ratio –1.49, Carr’s index –32.94) and freeze-dried nanocellulose (Hausner’s ratio –1.56, Carr’s index – 36.13), which showed an inferior flow. The improvement in the flow property of microcrystalline and oven-dried nanocellulose, when compared with the native cellulose, may be due to the acid hydrolysis treatment undergone by the powders because hydrolysis breaks long cellulose fibers to smaller particles having lesser contact area which results in increased flowability.^[25] Although freeze-dried nanocellulose exhibited very poor flow properties despite acid hydrolysis, this may be due to the higher moisture content. The result for the angle of repose was not consistent with that of Hausner’s ratio and Carr’s index; there was no statistically significant difference in the angles of repose of the cellulose powders, which fell within the range of 31.36–32.83 which indicated that all cellulose powders possessed a good flow property.

WHC refers to the ability of the cellulose polymer to hold water molecules within its structure without dissolving in it. In general, cellulose tends to interact strongly with water by hydrogen bonding.^[26] The native cellulose held more water molecules than the other cellulose while the microcrystalline and oven-dried nanocellulose held the least amount of water. The high-WHC shown by the native cellulose might be due to the presence of amorphous cellulose regions which are unstable when they interact with water compared to the other cellulose powders in which the amorphous cellulose regions have been disrupted to isolate the crystalline cellulose by subjecting to acid hydrolysis.^[27] The significant WHC shown by the freeze-dried nanocellulose powder that is comparable to native cellulose may be due to a more porous sponge-like microstructure imparted on it by the freeze-drying process; this promotes the penetration of water into the molecule leading to higher absorption kinetics and capacity. In contrast, oven drying causes shrinkage of the polymer structure while displaying a continuous matrix with a uniform surface with reduced porosity characterized by irregular pores.^[28] Hence, the oven-dried nanocrystalline and MCC exhibited lower water-retaining capacity. The acid-hydrolysis-modified cellulose samples showed a higher swelling capacity than the native cellulose despite the ability of the native polymer to retain more water. The nanocrystalline cellulose had the highest swelling capacity and swelling ratios, irrespective of the drying method employed followed by the MCC.

The ash content is an important parameter used to estimate the purity and quality of cellulose samples. The ash content values of the native and MCC fell in the acceptable limits of <0.1% (w/w) according to British Pharmacopoeia 2021 specifications while those of oven-dried and freeze-dried nanocrystalline cellulose were slightly above the upper limit at 0.113% and 0.124%, respectively [Table 1].^[21] The ash content may be affected by various factors such as the source of the cellulose, the processing method, and the presence of impurities.

Particle size analysis

A decrease in the mean volume diameter of the cellulose samples was observed on acid hydrolysis as the size of native cellulose (229.7 ± 130 μm) >MCC (196.7 ± 129.4 μm) >oven-dried nanocellulose (143.4 ± 76.07 μm) > freeze-dried nanocellulose (87.61 ± 63.75 μm) [Table 2]. This corresponds with the observed trend in the percentile distribution [Table 3], which showed that the particle sizes of the acid-hydrolyzed (microcrystalline and nanocrystalline) cellulose were smaller. Furthermore, a decrease and narrow range in PSD was observed in the nanocellulose samples [Figure 1] as shown by the progressive reduction in the degree of variability in the mean volume particle diameter depicted by the standard deviation values [Table 2] while a wide range of variability in the PSD was observed in the native cellulose and MCC [Table 3]. The specific surface area increased in the microcrystalline and nanocrystalline cellulose; this corresponds with the observed trend of decreasing particle sizes which directly predispose to higher specific surface area in the cellulose samples. The freeze-dried nanocellulose exhibited the largest specific surface area which indicates that it would have higher surface reactivity in reaction processes and increased sorption capacity.

Graphic kurtosis is a measure of the departure of a PSD peak from normality; a graphic kurtosis value of 0.67–0.9 indicates that the PSD peak is platykurtic while <0.67 signifies very platykurtic peak distribution.^[29] The cellulose samples had graphic kurtosis values (0.0758–0.851) which indicated that the PSD had a wide range with high variability and there is an increased tendency to encounter particles that are significantly larger or smaller than the average particle diameter. This is evidenced by the bimodal distributions of the particle sizes for all the materials (except the freeze-dried nanocrystalline cellulose; [Table 3]).

The particle size, PSD, particle surface area, and morphology are critical material properties that can influence the performance of the material in pharmaceutical manufacturing processes. A wide range with high variability in the particle size and size distribution such as was observed in the Wmp-native cellulose and Wmp-MCC would influence flowability, mixing efficiency, uniformity, encapsulation, and compression in the manufacturing processes for solid oral dosage forms as well as drug dissolution, bioavailability, content uniformity, and stability amongst others. Therefore, the impact of particle sizes of pharmaceutical powders on drug product manufacturability and performance should be evaluated during pre-formulation studies to establish appropriate particle size specifications to enhance the control of drug product quality and ensure manufacturing consistency.^[30,31]

FTIR spectrophotometry

The FTIR spectra of the cellulose samples showed a broad absorption band in the range of 3600–3200cm⁻¹ [Figure 2], which is characteristic of O-H stretching, but the spectra showed a slight decrease in the transmittance for WmpNCC_{oven dried} and Wmp-NCC_{freeze dried} than that of Wmp-native cellulose and Wmp-MCC. This may be due to an increase in the number of –OH groups on the crystalline cellulose chain following acid hydrolysis. The sharp peaks observed in the spectra at 2981cm⁻¹ are characteristic stretching vibrations of –CH in the cellulose molecule which decreased in sharpness for Wmp-NCC_{oven dried} and Wmp-NCC_{freeze dried} due to changes in degree of crystallinity, molecular orientation, and degree of order of cellulose.^[32] The characteristic peak in the spectra at 1636 cm⁻¹ is the bending vibration of -OH groups due to water bound to the cellulose structure which showed an increase in the transmittance in the order – Wmp-native cellulose <Wmp-MCC <Wmp-NCC_{oven dried} <Wmp- NCC_{freeze dried}. This indicates that the concentration of these atoms and bonds was reduced with acid hydrolysis treatment, which dissolved the amorphous cellulose region on the chain to leave behind the crystalline cellulose region. The spectra peak at 1462 cm⁻¹ and 894.05–893.90 cm⁻¹ represent the bending or scissoring vibration of the –CH₂ group and – CH deformation vibration of the glycosidic linkage bond, respectively. These two peaks are collectively referred to as the crystallinity band and are associated with the crystalline region of cellulose. The cellulose samples showed the characteristic peaks of CH and CH₂ bending at the frequency band of 1375.86–1156.74 cm⁻¹ associated with amorphous regions of cellulose in which an increase in transmittance was observed in the modified cellulose samples. This indicates a reduction of the amorphous cellulose region and increased crystallinity of the cellulose with acid hydrolysis treatment.^[33] The band at 1057.28–994.62 cm⁻¹ on the spectra is indicative of stretching vibrations of C-O-C glycosidic bond and ring stretching, which showed a trend of decrease in transmittance, which indicates increased crystallinity of the cellulose with acid hydrolysis treatment which causes the cellulose chains to become more ordered and tightly packed.

Powder X-ray diffraction (PXRD)

All the diffraction patterns in Figure 3 appear the same, except for their intensities, which show differences in crystallinity. The diffraction pattern of the cellulose samples in Figure 3 showed the appearance of a doublet peak at the (002) crystalline plane, which indicates the presence of cellulose crystal polymorphs; cellulose I and cellulose II. The doublet peak also appeared on the diffractogram of Wmp-native cellulose indicating that some level of partial conversion must have occurred during the sodium hydroxide pretreatment step.^[34]

The various methods adopted did not appear to have affected the crystal packing of all the cellulose samples but had changed the particle sizes. Cellulose II typically exhibits diffraction peaks at about 12°, 20°, and 22°, which corresponded with the observed PXRD pattern, although small diffraction peaks were observed in the diffractogram at about 34°, with them all signifying the 101, 10Î, 002, and 004 crystallographic reflection plane, respectively.^[35,36] Cellulose II exhibits increased thermal stability, tensile strength, and reduced tendency to absorb moisture, which imparts it less susceptibility to the effects of humidity; it possesses excellent binding and disintegration properties in tablet formulation over cellulose I.^[34]

The diffraction peak of native cellulose became sharper with acid hydrolysis indicating an increase in crystallinity which was also reflected in the crystallinity index presented in Table 4, although the nanocellulose sample showed a slightly lower crystallinity relative to Wmp-MCC. This may be due to the effect of sulfuric acid on the crystal lattice of the cellulose as the initial alkaline pretreatment disrupts the amorphous cellulose region leaving behind the crystalline cellulose, which under harsh reaction conditions may be susceptible to further digestion. The observed increase in crystallinity is expected to impart increased strength and rigidity in cellulose which increases their potential applications.^[37,38]

DSC

The DSC thermogram [Figure 4] of the cellulose samples showed the decomposition process of cellulose samples. Wmp-native cellulose and Wmp-MCC exhibited broader melting peaks while that of Wmp-NCC_{freeze dried} was sharper but unlike Wmp-NCC_{oven dried}. Only Wmp-NCC_{oven dried} exhibited a very sharp and small decomposition peak which indicates that a fast rate of transformation occurred with large enthalpy changes. Crystalline regions in polymers have ordered structures that cause restriction of molecular mobility but in the nanocellulose samples which also showed a high degree of crystallinity, their thermogram exhibited sharper peaks due to the presence of a more uniform crystalline structure such that disturbance at one crystal lattice results in disruption in all other structurally associated lattices.^[39]

Wmp-native cellulose and Wmp-NCC_{freeze dried} exhibited similar characteristic thermograms with similar peak melting temperatures, although Wmp-NCC_{freeze dried} showed a slightly higher onset temperature which indicated higher crystallinity which predisposes them to resistance to thermal degradation due to stronger intermolecular bonds in their more ordered crystalline structure.^[39] Therefore, the Wmp-native cellulose and Wmp-NCC_{freeze dried} had the highest thermal stability among the cellulose samples. On the other hand, Wmp-NCC_{oven dried} exhibited the lowest thermal stability. This may be due to the possible effect of the oven drying method employed which may have resulted in some degree of loss of its crystalline structure. This resulted in a lower onset temperature at 197°C as well as low peak melting temperatures at 200.67°C [Figure 4]. The thermal characteristics exhibited indicate weaker intermolecular bonds compared to Wmp-native cellulose and Wmp-NCC_{freeze dried} which predisposes to less resistance in the packing and rearrangement of the particles which are the same factor that resulted in having a higher bulk and tapped density.^[24]

The thermal stability demonstrated by Wmp-native cellulose and Wmp-NCC_{freeze dried} allows for their ability to withstand the elevated temperatures involved in tablet manufacturing processes like granulation, drying, and compression without significant degradation or loss of functionality which is an important requirement for the structural stability and integrity of the tablet formulation.

TGA

The TGA thermogram [Figure 5] of the cellulose samples showed that they exhibited different thermal properties, but they followed a similar degradation pattern. All cellulose samples were stable at temperatures below 200°C; however, the Wmp-native cellulose and Wmp-NCC_{freeze dried} were observed to have higher thermal stability than the Wmp-MCC and Wmp-NCC_{oven dried} which corresponds with the observation in the DSC thermal analysis data, even though the temperature of onset of decomposition varied when compared to those observed in the DSC analysis. At the end of the decomposition process, there was no trend in the weight of residue obtained at 694.9°C [Table 5] but Wmp-NCC_{freeze dried} had the least residual weight at 14.89%.

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Figure 5:

Thermogravimetric analysis thermogram of watermelon peel native cellulose, watermelon peel microcrystalline cellulose, watermelon peel oven-dried nanocrystalline cellulose (WmpNCC_{oven dried}), and watermelon peel freeze dried nanocrystalline cellulose (Wmp-NCC_{freeze dried}).

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The weight loss leading up to T_5% as the temperature of the sample increases corresponds to the evaporation of absorbed water.^[40] The weight loss that occurred leading up to T_5% in the Wmp-NCC_{freeze dried} (4.85%) and Wmp-NCC_{oven dried} (5.66%) corresponds with the physicochemical characteristic that they exhibited in being redispersible in water because they contain residual moisture of about 4% by weight which is similar to the values observed on the TGA thermogram.^[22] The redispersibility of Wmp-NCC_{freeze dried} in water and the high thermal stability it exhibited shows that it has potential for use in tablet manufacturing processes involving high-temperature input such as hot melt extrusion where it would be resistant to degradation and when incorporated into a drug matrix could promote drug solubility and more efficient release of active pharmaceutical ingredient.