INTRODUCTION:

Colorectal cancer (CRC) ranks as the world's third most prevalent form of cancer and is likewise the second most significant contributor to cancer-related fatalities globally^1-3. While population-based screening programs have helped identify more instances of early stages CRC, over 20% patients are still identified as having distant tumors and have an overall survival rate after five years of less than 15%^4-6.

Researchers earlier work indicated that Andrographolide (AG), a bioactive chemical obtained from the Chinese medicinal plant Andrographis paniculata, demonstrated anti-tumor effects in gastric and colorectal cancer (CRC) by stimulating the ferroptosis pathway. AG exhibits various significant biological properties, such as liver-protective, enhancing immune response, antioxidant, anti-inflammation, and combatting malaria effects^7,8. The AG compound has a low solubility of 3.29±0.70 µgmL^-1 in water and has a highly lipophilic nature with a log P value (2.632±0.13). Consequently, its biological absorption is very low, measuring only 2.67%^9-11. One approach to enhance solubility involves the production of nanoparticles, which involves reducing particles' size to boost surface area and hence improve a substance's ability to dissolve in a solvent. Nevertheless, the absence of this approach at extremely small sizes might lead to the clumping and clustering of particles. To mitigate this issue, a formulation in which Several pharmacological substances that are active ingredients are dispersed in a solid matrix was selected^12-14.

Solid dispersion is the organization of a number of chemical compounds within a medium that forms an inert matrix. This is achieved using a melt technique, solvent-based wetting technique, or an amalgamation of both.

Solid dispersion systems (SDS) offer several benefits, including the reduction of particle size, enhancement of the process of wetting, alteration in physical, and dispersion in molecular level, which ultimately modify the drug's solubility in water. Moreover, they are simply applied and produced, making them superior to prodrugs^15,16.Polymers can be utilized as transportation providers for the Solid-state dispersion approach to decrease the crystallinity of medicinal components and enhance their amorphous nature. The presence of an amorphous structure can decrease the point of melting of medicinal components, resulting in a reduced energy requirement for dissolving the therapeutic ingredients compared to the crystalline form with a regular lattice^{12, 17,18}.

Chitosan (CS) is a naturally occurring polysaccharide that is generated and derived by chitin analogs by a process called N-deacetylation. This kind of polymer is utilized extensively because of its biodegradable, and non-toxic properties. Chitosan exhibits hydrophilic characteristics due to the presence of an amine group that readily accepts protons. The utilization of chitosan polymer in creating an SDS enhances the surface area, prevents recrystallization, promotes wetting, facilitates solubilization, and accelerates the disintegration rate, hence leading to an improvement in drug bioavailability^19-22.

A homogeneous mixture of two or more substances in a solid state was created using the spray dry technique, in which the medication and polymers were dispersed in a solvent and then dried using spray drying. This technique effectively mitigated excessive humidity and safeguarded the medication from chemical degradation. The objective of this research is to examine the impact of increasing the quantity of chitosan on the physical properties, the ability to dissolve, and the dissolution of the AG-CS SDS, which was synthesized using the spray-drying technique. The evaluation encompassed many analyses such as morphology examination, XRD, solubility testing, dissolving rate, and assessment of toxicity against HT 29 colon cancer cell line.

MATERIAL AND METHOD:

The HT-29 human colorectal cancer cell lines were acquired from the American Type Culture Collection (ATCC). Dulbecco's modified Eagle's medium (DMEM; Gibco, Carlsbad, CA, USA) with the addition of 10% fetal bovine serum ( (Gibco, Waltham, MA), penicillin-streptomycin (Sigma, Austral. – Australia). Ferrostatin-1, obtained from Sigma-Aldrich in Missouri, USA, dimethyl sulfoxide, Andrographolide, and chitosan were purchased from Merck, India. All other reagents used in the experiment were of analytical-grade purity.

Development of a solubilization system including andrographolide and chitosan:

The formation of the AG-CS solid state dispersion combination was achieved by the wetting of a solvent process and subsequently spray drying²³. The solid dispersions were made using a different concentration of andrographolide (shown in Table 1) to chitosan. Chitosan was immersed in a solution of 50ml of acetic acid at a concentration of 0.3%. Andrographolide was dispersed in a 10ml solution of methanol. The solution with chitosan was administered into the solution containing andrographolide and agitated at a different rpm per minute (shown in Table 1) for 1hour, followed by spray drying. The process conditions were as follows: nozzle diameter of 1.0 mm, flow rate of 3 ml/min, and pressure of 2 bar.

Table 1: Formulation of solid state dispersion system

S No.	Formulations	Factor 1 A: chitosan (mg/ml)	Factor 2 B: TPP (mg/ml)	Factor 3 C: Stirrer (rpm)
1	F1	2	1	1000
2	F2	1	1	800
3	F3	2	1	800
4	F4	1	1	1000
5	F5	2	0.8	1000
6	F6	2	0.8	800
7	F7	1	0.8	1000
8	F8	1	0.8	800

TPP= tripolyphosphate

Assessment of morphology:

The particle shape as well as surface morphology were examined using scanning electron microscopy (SEM). The particles were implanted into an aluminum container and subsequently adorned with a layer of gold-palladium before examination. Photographs were captured at different levels of magnification using a voltage of 20.00 kV.

FT-IR Analysis:

The small Particle was consolidated into a pellet by blending them with potassium bromide powder and subsequently compressed using a hydraulic pump to produce a pellet as transparent. The experiment was carried out at a lambda range of 3900-550 cm⁻¹ using an FT-IR instrument.

Thermoanalytic technique:

The sample underwent thermal assessment using DTA. A quantity of 5mg of particles was deposited in a metal container for heating substances at high temperatures, which was then sealed and monitored for its thermogram. The thermogram was obtained by recording the temperature ranging from 5°C to 250°C, using a heating rate of 10°C per minute.

X-ray diffraction (XRD) studies:

The sample's crystallinity was evaluated using XRD examination. The light sources used were Ni, Cu, Kα. The current and voltage were adjusted to 40 kilovolts and 40 milliamperes, respectively. The diffractogram of the particles was contrasted to the diffraction pattern of a sample consisting only of an andrographolide compound.

Content of drug:

The concentration of AG incorporated into a solid solution was measured using a spectrometer that measures the absorption and transmission of ultraviolet and visible light using two separate beams. at a wavelength of 225nm. A precisely measured quantity of 10.0mg of the specimen was accurately the substance was measured and mixed with 5ml of methanol. The resulting solution was then purified employing a 0.4 μm filtering sheet and thereafter examined using a light wavelength of 225nm. The analysis was repeated thrice.

The amount of drug was determined with the following equations:

% of durg content = drug amount / particle weight * 100

Test for dissolution:

The dissolution test was conducted using 50ml of a solution phosphate buffer with a pH of 7.0±0.05, at a temperature of 37.0±0.5°C, and at a speed of 120rpm on a water bath. The AG-CS SDS was measured to weight 5mg AG and was then placed into the media. A 2.0ml sample was collected at time intervals of 5 15, 30, 60, 120, 180, 240, and 300 minutes. Following the process of sampling, a volume of 2.0ml of medium was introduced to maintain the sink condition. The samples were examined using a UV-visible double-beam spectrophotometer set to a wavelength of 225nm. The dissolution of andrographolide from both the andrographolide molecule and the solid dispersion system of andrographolide-chitosan was quantified. The dissolution test was conducted using three replications ²⁴.

Test for Solubility:

A solubility test was conducted using 15ml of phosphate buffer solution with a pH of 7.0±0.05. The sample was placed in a water bath equipped with a shaker, operating at a speed of 120 revolutions per minute, and maintained at an ambient temperature of 37.0±0.5°C. Samples had been taken 1ml and processed with 0.45µm Millipore filter paper. The UV-visible spectrophotometer was used to measure the absorbance at a wavelength of 225nm. The soluble test has been repeated 3 times²⁴.

Cytotoxicity:

The impact of andrographolide on the viability of colon cancer cells was determined using the MTT test, as described by Deshmukh et al²⁵. In summary, the cells (1 × 10⁵ cells per ml) were placed in a 96-well microtiter plate (100μl per well) with replications. The experiment involved adding AG at various concentrations (7.8-1000 µg/ml) over 48hours. Following incubation, 20µl of 5mg/ml MTT stock solution was added to each well and incubated for 4hours at a temperature of 37℃. The formazan crystals obtained were dissolved in DMSO and the absorbance was quantified at 570nm using a microplate reader (SpectraMax M5, Molecular Devices, USA). Cell viability is expressed as the ratio of absorbance (A570) in treated cells to absorbance in control cells (0.1% DMSO) (A570).

RESULTS AND DISCUSSION:

Morphological Studies:

The morphological studies were done by scanning electron microscopy (SEM). The observation revealed that the F2 microparticle exhibited a shape that was spherical with an even surface morphology. However, the changing concentration of andrographolide and TPP along with rpm yielded various outcomes. The presence of a 1:1 concentration of andrographolide and TPP at 800rpm resulted in the formation of surface microparticles with a smooth texture in comparison to other formulations. This can be attributed to the elevated viscosity of the aqueous combination of andrographolide and TPP (Figure 1). Moreover, the elevated viscosity of the -chitosan combination and andrographolide-TPP droplets posed challenges in their interaction and crosslinking processes. An increase in the concentration of TPP resulted in a smoother surface, which can be attributed to the increased formation of crosslinked bonds between chitosan, and TPP. This leads to the creation of a more compact surface²⁶.

Figure 1: Scanning electron microscopic image of formulation F2 andrographolide-chitosan solid dispersion system.

IR Spectrum:

The spectrum exhibited a wide range of frequencies at 3353–3284 cm−1, which can be attributed to the Stretch vibration of O-H as well as N-H groups of functions. These vibrations are a result of Chitosan particles exhibiting intramolecular bonds of hydrogen. The particular C=O frequency from andrographolide was identified at the wave number 1729.3 cm−1, along with peaks at 1729 cm−1 and 1680 cm−1. Additional peaks have been found at 1650 cm−1, 1490 cm−1, 1220 cm−1, 1240 cm−1, 980 cm−1, 1040 cm−1, and 1090 cm−1. A combination of C=O, C=C, C-O-C of lactone ring, and O-H category of alcohol in the molecular framework of AG likely causes these spikes (Figure 2).

XRD Studies:

The diffractogram of andrographolide in Figure 3 has strong intensity peaks at 2θ values of 15.4853°, 18.0840°, 23.4478°, and 24.3119°. The diffraction patterns of andrographolide- chitosan were analyzed, and it was observed that the positions of the diffraction lines (2θ) and their strengths match the data for andrographolide obtained. The diffraction lines of andrographolide-chitosan exhibit a preferential orientation of crystallites in this sample.

Figure 3: The diffractogram of andrographolide

Drug content:

The mean drug content percentages of various formulations ranged from 25.75±0.119% to 29.508±0.0% (Figure 4). The difference in drug content can likely be explained by the number of nuclei generated at the interface between the solvent and anti-solvent, as well as the impact of concentration on viscosity. The process parameters, such as temperature during mixing, mixing rate, drug concentration, solvent/anti-solvent ratio, choice, and concentration of stabilizers, all influence the degree of supersaturation and nucleation rates. These factors have the potential to generate a significant quantity of sub-micrometer particles in the final suspension, provided that the growth is prevented by stabilizers. The statistical analysis revealed a notable disparity between the formulas using a one-way ANOVA followed by Dunnett's test, with the highest concentration observed in F2.

Figure 4: Mean drug content percentages of various formulations

Solubility test:

The solubility test findings in Figure 5 indicate that the solubility of the F2 formula increased by a factor of 1.32 times more than that of the andrographolide substances. The enhanced solubility of andrographolide resulted from its dispersion inside chitosan matrices, leading to amorphization, increased surface area, and improved wetting ability of andrographolide.

Figure 5: The solubility test for different formulations and pure AG.

Release rate:

The release test was conducted on a phosphate buffer with a pH of 7.0±0.05. Figure 6 demonstrated an increase in the dissolution of andrographolide from the SD compared to the pure AG substances. The rate of dissolution andrographolide was 0.178mg/min⁻¹, while the formulation F2 had the highest dissolution rate of 0.28mg/min⁻¹ and showed 1.6 times increase in the rate of dissolution.

Figure 6: Dissolution profile of the formulation F1-F8 and plain AG.

Cytotoxicity:

To assess the impact of andrographolide on the survival of cells, colon cancer cells, specifically HT29 were cultured with or without andrographolide for 48 hours. An MTT experiment was conducted to assess the cell viability of the cell line. AG-CS, SDS induced a reduction in cellular viability in cells in a concentration-dependent manner (Figure 7).

Figure 7: MTT assays of AG-CS, SDS, where a-control, b-7.8 µg/ml, c- 15.6 µg/ml, d- 62.5µg/ml, e- 250 µg/ml, f- 500 µg/ml

CONCLUSION:

The development of solid-state dispersion approaches of andrographolide and chitosan influenced the properties of the medication, resulting in reduced crystallinity and a lower melting point. The dissolution and solubility of andrographolide were positively impacted by the formation of SDS. The dissolution rate, as well as solubility of andrographolide, exhibited an increase of 1.6-fold and 1.7-fold, respectively, in comparison to plain AG. In addition, it also exhibits anticancer activity against colon cancer cells which indicates its application in the management of colon cancer. Thus, the present study exhibits a novel method for increasing the solubility and dissolution of poorly water-soluble AG.

CONFLICT OF INTEREST:

Authors have no conflict of interest.

ACKNOWLEDGEMENT:

Authors are grateful to the IPR, GLA University, Mathura, for providing necessary facility to carryout work.

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Received on 04.11.2023 Modified on 10.12.2023

Research J. Pharm. and Tech 2024; 17(2):897-902.

DOI: 10.52711/0974-360X.2024.00139