Characterization and safety assessment of Hydroxypropyl Musa paradisiaca starch for pharmaceutical applications

Identification test: iodine test

The formation of a bluish-violet color after the addition of iodine indicates the starch presence [17].

pH

With 25 ml of CO₂-free distilled water, 5 g of starch was mixed for 1 minute, and left for 15 minutes. The pH value was then determined using a pH meter.

Moisture content

In the oven for 3 hours at 105°C 3 g of banana starch was heated and weighed. The ratio of the final weight to the initial weight was articulated in terms of percentage.

Determination of loss on drying

One gram of starch was weighed, placed in a preheated bottle with a cap that had been heated to 105°C for 30 minutes, kept in the oven at 105°C, and dried until a constant weight was attained.

Determination of ash content

In a silica crucible, 2 g of starch powder was placed and incinerated. Using the following equation, the ash content was determined. By measuring the residue left behind after full combustion in a muffle furnace at 550°C, the ash value was determined.

$\%\mathrm{Ash}=\frac{\mathrm{((}\mathrm{Ashed}\mathrm{wt}\mathrm{)}\mathrm{–}\mathrm{(}\mathrm{Crucibler}\mathrm{wt}\mathrm{))}\mathrm{\times }\mathrm{100}}{\mathrm{((}\mathrm{Crucible}\mathrm{and}\mathrm{sample}\mathrm{wt}\mathrm{)}\mathrm{–}\mathrm{(}\mathrm{Crucible}\mathrm{wt}\mathrm{))}}$

Determination of the angle of repose (θ)

It was assessed by letting powder freely flow through a funnel. Ash content should be <1%. The addition of powder was halted as soon as the pile reached the tip of the funnel. Without disturbing the pile, a circle was drawn around it. The resultant cone’s height and diameter were measured. To determine the angle of repose, the following equation was used.

θ = tan⁻¹ h/r

Determine density, Carr’s index, and Hausner ratio

After weighing an empty measuring cylinder (100 ml), the sample was added to it to make a volume of 100 ml (V₀), and the combined weight was calculated (W₂). The sample-filled measuring cylinder was set on a motorized tapping device. The measuring cylinder was tapped 50 times with the start button engaged. Following that, the sample’s volume was determined before (V₁) and after (V₂) tapping. The Hausner ratio, Carr’s index, tapped density, and bulk density were assessed using the formulas below.

$\mathrm{Bulk}\mathrm{Density}(\rho \mathrm{b})=\frac{{\mathit{W}}_2–{\mathit{W}}_1}{{\mathit{V}}_1}$

$\mathrm{Tapped}\mathrm{Density}(\rho \mathrm{t})=\frac{{\mathit{W}}_2–{\mathit{W}}_1}{{\mathit{V}}_2}$

$\mathrm{Car}\mathrm{’}\mathrm{s}\mathrm{index}\mathrm{(\%)}=\frac{1–\mathrm{Bulk}\mathrm{Density}}{\mathrm{Tapped}\mathrm{Desnsity}}\times 100$

$\mathrm{Hausner}\mathrm{Ratio}=\frac{\mathrm{Tapped}\mathrm{Density}}{\mathrm{Bulk}\mathrm{Desnsity}}$

Determination of amylose content

The Avaro et al. [18]method was slightly modified to determine the amylose content of M. paradisiaca starch.

Determination of λmax

In a test tube containing 9 ml of 1 N sodium hydroxide and 1 ml of 95% ethanol, pure amylose (100 mg) was added. A gel was produced in the test tube after it had been heated in boiling water for 10 minutes; it was then cooled. A 1 ml of 1 N acetic acid was added to a 5 ml aliquot of the solution to adjust the pH before being transferred to a 100 ml volumetric flask. Then, to produce exactly 100 ml, 2 ml of a 0.2% iodine solution and distilled water were added. The resultant mixture was incubated for 30 minutes before being homogenized. The absorption maxima (λmax) of the samples were determined using calorimetry within the wavelength range of 400–800 nm on a UV-VIS spectrophotometer [18].

Calibration curve of amylose

A calibration curve was generated for pure amylose over a spectrum of standard concentrations (40, 80, 120, 160, 200, 240, 280 μg/ml) in accordance with the methodology proposed by Avaro et al. [18], with minor adaptations. The quantification was conducted employing a UV-VIS spectrophotometer configured to a maximum wavelength of 620 nm to gauge the intensity of the resultant blue chromophore. Subsequently, a linear calibration curve was established, correlating amylose concentrations with corresponding absorbance measurements.

Determination of hydroxypropyl group percentage (HPG%) and degree of substitution (DS)

To obtain a transparent solution, 0.1 g of starch from the volumetric flask was combined with 25 ml of 1 N H₂SO₄ and boiled in a water bath. The volume was made up to 100 ml with distilled water after cooling to room temperature (RT). Eight milliliters of concentrated H₂SO₄ were added to 1 ml of this solution in a chilled conical flask. The flask was next heated for 20 minutes in a boiling water bath, cooled to RT, and then transferred to an ice bath. After that, 3% ninhydrin reagent (0.6 ml) prepared with 5% sodium metabisulphite was added, carefully mixed, and allowed to stand at RT for an additional hour. To measure absorbance at 590 nm in a UV spectrophotometer, volume was made up to 25 ml using concentrated H₂SO₄. Native starch was used as a standard. Aliquots of propylene glycol-containing standard aqueous solutions were used for constructing the calibration curve. DS values and HPG% were determined [19].

$\mathrm{HPG}\mathrm{(\%)}=\frac{\mathrm{Amount}\mathrm{of}\mathrm{propylene}\mathrm{glycol}\mathrm{in}\mu \mathrm{g}/\mathrm{ml}\times 0.7663\times 10}{\mathrm{weight}\mathrm{of}\mathrm{sample}}$

$\mathrm{DS}=162\times \frac{\mathrm{HPG}}{5,800-58\times \mathrm{HPG}}$

Swelling power and soluble starch content

One percent of the starch slurry was prepared, heated in a water bath that was kept at 90°C for 30 minutes while being constantly stirred, and then cooled. The suspension was centrifuged at 3,200 rpm for 10 minutes, and the supernatant was collected in an aluminum dish that had been preweighed and dried using an evaporator. For the purpose of determining soluble starch content, the dried aluminum dishes were weighed. To determine the swelling power, the weight of the wet sediment in the centrifuge tube was observed [20].

$\mathrm{Swelling}\mathrm{Power}\mathrm{(}\mathrm{\%)}=\frac{\mathrm{Weight}\mathrm{of}\mathrm{swollen}\mathrm{se}\dim \mathrm{ent}}{\mathrm{Weight}\mathrm{of}\mathrm{dry}\mathrm{starch}}\times 100$

$\mathrm{Solubility}\mathrm{(}\mathrm{\%)}=\frac{\mathrm{Weight}\mathrm{of}\mathrm{dry}\mathrm{supernantant}}{\mathrm{Weight}\mathrm{of}\mathrm{dry}\mathrm{starch}}\times 100$

Determination of viscosity

Torque applied to a rotating spindle at a fixed speed and temperature is used to evaluate the viscosity of starch solutions produced by indirect heating. The viscosity was measured using a Brookfield Viscometer. The native and hydroxyl propyl starches were combined with distilled water to prepare the starch solution (1%, 2%, 3%, and 4%). After 20 minutes of boiling, the starch solution was cooled to RT. At spindle no. 2, at 100 revolutions per minute, the time was recorded [20].

Determination of Fourier transform infrared (FTIR) spectra

NBS and HPBS samples’ FTIR spectra were acquired at RT using the KBr disc method using an FTIR spectrophotometer (PerkinElmer, Spectrum Two DTGS, UK). Wave numbers between 4,000 and 400 cm⁻¹ and a resolution of 4 cm⁻¹ were used to obtain FTIR spectra [21].

Determination of gelatinization temperature by differential scanning calorimetry (DSC) studies

In the study of the gelatinization transitions in NBS and HPBS, DSC was used. The mass of each sample was 10 mg ± 0.1 with 75% (w/w) of deionized water. For the DSC analysis, a Q100 TA Instruments calorimeter was used. The samples were placed in aluminum hermetic sealed pans and were stabilized at RT for 15 minutes, then samples were heated in a ramp at 10°C/minute using an empty pan as a reference and a nitrogen environment [22].

¹H NMR study

NBS and HPBS samples were prepared and nuclear magnetic resonance spectroscopy (NMR) spectra were measured as described previously [23]. d1-TFA was added directly to the medium just before the NMR measurement, and the whole mixture was transferred to a 5 mm NMR tube using a Pasteur glass pipet. The tube was sealed and wrapped with Parafilm before an NMR spectrum was recorded. The spectra were recorded on Bruker DRX 400.

Surface morphology analysis

The surface morphology of NBS and HPBS was examined by scanning electron microscopy (SEM) “(Zeiss EVO LS10, Cambridge, UK)” by using the gold-sputter method. The sample was fixed in a stub and coated with gold and imaging was performed [24].

X-ray diffraction (XRD)

The XRD patterns were acquired using the Panalytical X-Per Pro equipment, employing CuKα radiation (λ = 0.15405 nm). The 2θ detector was set to vary between 4 and 60, with a step size of 0.02 per measurement. To analyze the XRD patterns, Gaussian functions were fitted, and the total crystalline areas under the peaks were calculated using the Peakfit software [24].

Animals

Healthy female Wistar rats were used to conduct acute toxicity studies. Healthy female and male Wistar rats were employed for the assessment of subacute toxicity. The rats were 6–8 weeks old, and they were stabilized for 1 week to get used to the laboratory environment. They were housed in standard cages and maintained under standard conditions at a temperature of 22°C ± 3°C with a 12-hour cycle of light and darkness. A standard diet and unlimited access to tap water were made available to them. The study protocol was approved by the Institutional Animal Ethics Committee before the commencement of experimental studies (1567/PO/Re/S/11/ CPCSEA).

Acute toxicity evaluation

With an initial dosage of 2,000 mg/kg of HPBS, the acute oral toxicity test was conducted in accordance with the experimental methodology outlined in Organization for Economic Co-Operation and Development (OECD) Guideline 423. The number of animals employed in this evaluation was in accordance with OECD recommendations from 2011 and the reduction principle, i.e., employing the minimum number of animals necessary to achieve statistical significance. The animals were divided into three groups (n = 3). One group was given a single oral dosage of HPBS (2000 mg/kg) dissolved in water. Whereas group receives water (the control group) [25].

Subacute toxicity assessment

The approach suggested in guide 407 of the OECD guidelines was employed to determine the subacute toxicity of HPBS. The animals were divided randomly as follows: group 1 served as the control, group 2 served the high dose, group 3 served the middle dose, and group 4 served as the low dose group. Animals in groups 3 and 4 were necropsied at the end of the 28-day treatment period. For up to 14 days, the animals in groups 1 and 2 were left untreated. They had necropsies at the end of the recovery period. Before necropsy, all animals were starved for the whole night, and they were all put to death by carbon dioxide inhalation. Biochemical and hematological profiles were determined. Groups 1–4 were given target dosages of 0, 1,000, 500, and 250 mg starch/kg bw/day, respectively [26,27].

Body weight and clinical observation

Each rat was weighed before the test, once a week throughout the experiment, and on the day of sacrifice. Every day, the general state of health and mortality of every animal were monitored.

Hematological analysis

On a medonic M-series hematology analyzer, the following hematological parameters were analyzed: red blood cell counts (RBCs) white blood cell counts (WBCs), hemoglobin, packed cell volume, neutrophils, eosinophils, basophils, monocytes, lymphocytes, and hematocrit.

Biochemical analysis

A cardiac puncture was used to obtain blood samples, which were subsequently centrifuged for 5 minutes at 10,000 rpm in a nonheparinized tube. On a Photometer 5010 V5+ based on an enzymatic colorimetric test, serum was separated and examined for biochemical parameters such as electrolytes (sodium, chloride, calcium, and magnesium), as well as blood creatinine, blood urea, uric acid, total cholesterol, total bilirubin, aspartate aminotransferase (AST), and alanine aminotransferase (ALT). The cholesterol oxidase/P-aminophenazone technique was used to estimate the total cholesterol. According to the International Federation of Clinical Chemistry’s recommendations, ALT and AST were measured using the kinetic approach to determine their respective activities. Based on a colorimetric test for the kinetic detection of creatinine at 25°C and 37°C without deproteinization, creatinine was measured using the Jaffe reaction. The Berthelot technique was used to determine blood urea and uric acid levels.

Histopathology study

The kidney, lung, heart, liver, ovaries, spleen, and testis were carefully removed from the body and fixed in a fixation mixture containing 10% buffered formalin for histological analysis. Hematoxylin and eosin-stained paraffin sections of the organ were prepared and made ready for viewing on a light microscope equipped with an Optilab viewer.

Hematological analysis

After administering HPBS for 28 days, the hematological profiles of male and female rats are shown in Table 8. Most hematological parameters were unaltered by the oral administration of the HPBS suspensions. For all parameters, the results demonstrate that none of the groups deviated substantially from the control group.

It is crucial to analyze hematological parameters to determine the pathological and physiological status of the body, as well as the toxic effects of test compounds. This is because changes in these parameters could be a sign of the toxicity of the test substances as well as infections, inflammation, leukemia, and anemia. Indicating that the HPBS had no impact on the circulating blood cells of the tested animals, there was no significant difference in WBC, hemoglobin, RBC counts, packed cell volume, neutrophils, eosinophils, basophils, monocytes, lymphocytes, and hematocrit between the treated groups and the control group.

Biochemical analysis

Following 28 days of administration of HPBS, the biochemical profiles of female and male rats are shown in Table 9. The oral administration of the HPBS altered certain metabolic markers. AST, total albumin, and ALT levels were considerably lower in female and male rats than in the control group. Blood urea and creatinine levels were slightly lower in the treatment groups than in the control group. While there was no significant change in the uric acid levels between the control and treatment groups. Total protein and albumin levels were significantly elevated in the treatment groups. Total cholesterol levels were elevated in treatment groups, especially in male rats. The levels of sodium and chloride were elevated in the treatment groups, compared to the control group. The levels of calcium and magnesium were suppressed in treatment groups, especially in male rats, in contrast to the control group.

Table 7. Body weights of rats treated with hydroxypropyl starch. [Click here to view]

Table 8. Hematological profile of rats treated with different concentrations of the HPBS for 28 days. [Click here to view]

Table 9. Biochemical profile of rats treated with different concentrations of the HPBS for 28 days. [Click here to view]

Figure 10. X-Ray diffraction of NBS and HPBS. [Click here to view]

Figure 11. Histopathological examination of various organs of rats treated with HPBS in a subacute oral toxicity study: (A–G) the heart, kidney, liver, lung, ovaries, spleen and testis respectively. [Click here to view]

Figure 12. Histopathological examination of various organs of control rats in subacute oral toxicity study: (A–G) the heart, kidney, liver, lung, ovaries, spleen, and testis, respectively. [Click here to view]

Histopathology study

Hematological and biochemical analyses are supported by histopathological investigations. When compared to the control groups (Fig. 12E–G), the histological analysis carried out in the current study revealed that animals treated with HPBS did not exhibit changes in the texture, size, shape, and color of the kidney, liver, heart, spleen, ovaries, testis, and lung. These results are consistent with the measured hematological and biochemical parameters and indicate that HPBS has no detrimental impacts on vital organs, even when repeated dosages are used.

The myocardium in the heart was depicted by cardiac striated muscle cells, which had single or double nuclei that were positioned in the center and showed transverse striations. The endocardium covered cavities and heart valves with endothelium, which was supported by a thin basal membrane. The mesothelial pavement cells that cover the epicardium were completely intact (Fig. 11A).

The lobular morphology of the kidneys was maintained, and medullary pyramids were covered with cortical tissue. The cortex displayed fine Bowman capsules and glomeruli that were distributed evenly. As with the Henle loops and collecting ducts of the medullary pyramid, the proximal and distal contorted tubules and the section of the collecting duct showed no histological peculiarities. There was no fibrosis or inflammatory response found in the interstice (Fig. 11B).

The hepatic parenchyma, which consists of the lobular center vein surrounding hepatocyte cords and sinusoid capillaries, was seen to be evenly distributed and to still have its original architecture in the liver. The hepatocyte had a polygonal form, a spherical nucleus, and a totally intact center region (Fig. 11C).

Lung tissue sections from mice in all test groups that underwent histopathological investigation revealed a normal morphological structure without any pathological alterations (Fig. 11D). The histopathological analysis of the ovaries, spleen, and testis also displayed normal architecture (Fig. 11E–G).